Handbook of Microbiological Culture Media Dehydrated Culture Media ■ Culture Media Ingredients ■ Additives ■ Supplements ■ Reagents

Home , Microbiological culture

■ Edition No. 10 ■ TABLE OF CONTENTS

Scharlau Microbiology Introduction I

Scharlau Microbiology Quality Policy II

Safety (Regulations, Hazard Categories, Risk and Safety Phrases, Disposal) V

Guidelines for correct manipulation in Microbiology XIII

Dehydrated Culture Media 1-176

Culture Media Ingredients 177-204

Additives 205-213

Supplements 215-229

Reagents 231-244

Index of products in this Manual 245-251 Scharlau Microbiology is part of the Scharlau Science Group

Introduction If you work in Spain please contact:

It is a pleasure to introduce to you this edition no. 8 of SCHARLAB, S.L. our Handbook of Microbiological Culture Media, revised Tel.: 902 20 18 98 and extended, in a new and more attractive format. Fax: 900 50 29 35 E-mail: [email protected] The range of products has been increased, reaffirming the Post: Gato Pérez, 33. Pol. Ind. Mas d’en Cisa Scharlau Microbiology brand as an attractive alternative 08181 Sentmenat, Barcelona, Spain to other manufacturers of culture media for Microbiology. You will find new products, according to the standards Customers outside of Spain please contact: of the European Pharmacopoeia, USP and other official organisms. SCHARLAU CHEMIE, S.A. In this edition, we have separated additives, reagents Tel.: +34 93 715 18 11 and selective supplements into different chapters, mak- Fax: +34 93 715 31 75 ing it easier to locate each product. E-mail: [email protected] We have also included additional safety information and Post: Ctra. de Polinyà a Sentmenat, Km. 8,2 R+S sentences, following the EU guidelines 2001/59/ 08181 Sentmenat, Barcelona, Spain CE. Or ask for the address of our distributors in any of the following countries:

The company ALBANIA GUINEA BISSAU QATAR ANGOLA HAITI REUNION ISLAND ARGENTINA HONG-KONG ROMANIA In 1979, Dr. José Sancho Valls, Professor at the Uni- AUSTRALIA HUNGARY RUSSIA versity of Barcelona, decided to start manufacturing AUSTRIA ICELAND RWANDA BAHRAIN INDIA SERBIA & MONTENEGRO dehydrated culture media. A partnership was formed BANGLADESH INDONESIA SAUDI ARABIA with a local importer of research chemicals and the BARBADOS IRAN SENEGAL BELGIUM IRAQ SINGAPORE products sold on the local market under ADSA=MICRO BENIN IRELAND SLOVAKIA brand name. In 1992 the company was integrated into BOLIVIA ISRAEL SLOVENIA BOSNIA & HERZEGOVINA ITALY SOUTH AFRICA the Scharlau group. Thus we look back to more than 25 BOTSWANA JORDAN SRI-LANKA BRUNEI KENYA SUDAN years of experience in the manufacture of culture media. BULGARIA KOREA SULTANATE OF OMAN CHAD KUWAIT SWEDEN CHILE LATVIA SWITZERLAND At present, a wide rage of products is offered under the CHINA P.R. LEBANON SYRIA Scharlau brand, from standard laboratory chemicals COLOMBIA LIBYA TAIWAN COSTA RICA LITHUANIA TANZANIA (inorganic salts, acids, buffers, volumetric solution, IVORY COAST MADAGASCAR THAILAND solvents, etc.), to high purity specialized solvents for CROATIA MALAYSIA THE NETHERLANDS CUBA MALTA TOGO HPLC and environmental analysis, up to culture media CYPRUS MAURITANIA TONGA for microbiology. CZECH REPUBLIC MAURITIUS TRINIDAD & TOBAGO DENMARK MOLDOVA TUNISIA The technical supervision remains in the hands of Pro- DOMINICAN REPUBLIC MOROCCO TURKEY fessor Sancho. Through his wide scientiﬁc knowledge, ECUADOR MOZAMBIQUE UGANDA EGYPT MYANMAR UKRAINE we can offer good technical support. EL SALVADOR NEW CALEDONIA UNITED ARAB EMIRATES ERITREA NEW ZEALAND VENEZUELA ESTONIA NIGERIA VIETNAM ETHIOPIA NORWAY YEMEN FINLAND PAKISTAN ZAMBIA Contact Scharlau FRANCE PANAMA ZIMBAWE FRENCH POLYNESIA PERU GERMANY PHILIPPINES Don’t hesitate. Contact Scharlau for any requirement of GHANA POLAND GREECE PORTUGAL culture media for microbiology. The international presence of Scharlau Microbiology grows day by day. A wider network of distributors and a larger portfolio help to better penetrate the markets. Scharlau Chemie, S.A., which was born 53 years ago to manufacture exclusively organic chemicals for research laboratories, has been increasing its products and processes to become a global manufacturer of laboratory chemicals and culture media for microbiology.

We stock over 700 different microbiological references in our new 4.000 m2 warehouse in Sentmenat (Barcelona).

I Scharlau Microbiology Quality Policy

www.scharlau.com Environmental care

Please use our website for the latest news and MSDS: One of Scharlau Chemie’s target regarding environmen- www.scharlau.com. tal issues was achieved in 2003, when we received ISO Our website has been redesigned recently to offer you a 14001 approval. Scharlau Chemie has always designed much more modern and intuitive interface. its processes to minimize environmental impacts on the COA come with each bottle, which is why we don´t pub- rural area where it is located for about 30 years. lish them on the web.

As a contribution to the packaging residue minimization targets, Scharlau has replaced all its packing materials to avoid the use of expanded polystyrene, which is dif- The factory ﬁcult to recycle and expensive to dispose of. All internal and external elements of our packaging are made from The microbiological division of Scharlau Chemie, Schar- recycled cardboard and are fully biodegradable and lau Microbiology, manufactures a wide range of culture recyclable. media, reagents, stains, supplements and additives.

25 years experience in the production of dehydrated culture media allows us to develop fast new formulations to keep up with the latest developments in microbiology.

New production facilities have been inaugurated in the year 2002, thus enabling us to further improve our processes and expand production capacity. The new factory has been designed following the most latest directives to assure the maximum quality of the ﬁnal product.

More than 700 references are available from Scharlau Microbiology, ready to be delivered from our new 4000 m2 warehouse in Sentmenat, Barcelona. The strategic situation, within less than 1000 km from most European capitals, makes it possible to quickly restock our distributors. Barcelona, being one of the busiest ports in Europe, boasts excellent world wide shipping connections.

II Scharlau Microbiology Quality Policy

Quality as standard

Our commitment to quality has always been very important to keep our customers trust.

Scharlau Microbiology purchases raw materials and transforms them into microbiological culture media, using various processes and strict analytical controls. Our target is the production of products of the highest possible quality. To achieve this, we have always worked under internal quality standards, even before ISO regulations were adopted. This fact not only allows us to manufacture products of the highest quality, but also to maintain our quality at a constant level.

The quality concept has evolved and nowadays, quality is not only understood as an attribute of the product, but a feature which has to be present in everything done in the company. It is useless to have an excellent product, if delivery times or technical assistance are not adequate. In Scharlau Microbiology we work under these premises, injecting quality into all our services, from the development of a product designed to cover real customer requirements, to our after sales assistance.

Since 1997 Scharlau Microbiology is ISO 9002 accred- ited through a German TUV.

Certiﬁcate of Analysis

Every pack is sent together with its Certificate of Analysis, which guarantees the quality of our products. Together with the Certificate of Analysis we supply a second certificate to assure absence of Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease in our raw materials of animal origin.

Each certiﬁcate includes the following information: Physical-chemical speciﬁcations: 1. Of the medium before reconstitution: Colour Appearance Particle size / homogenization 2. Of the reconstituted medium ready to use: Clarity Precipitates Gelifying or solidifying strength

Growth control Results obtained with control strains under optimal conditions.

III Scharlau Microbiology Quality Policy

Quality in our labels We have changed the design of our label to make it Expiry date and batch number more useful and modern. Good Laboratory Practices (GLP) which are followed Product name and directions appear now in several by many of our customers, indicate that all products in languages with medium speciﬁcation and formula in the laboratory must be labelled showing batch number, English. Our labels are printed according to CE regula- expiry date and other additional data (date of reception, tions for dangerous goods. opening date, opened by...) Our labels contain batch number and expiry date and also a speciﬁc area is reserved for the user to write down additional data.

IV Safety Regulations

Material Safety Data Sheets

MSDS include physical and chemical data, handling recommendations, toxicological information and considerations relative to the environment, disposal, storage and transport of each product. In some cases, MSDS are delivered with the products but it is better to get them in advance. We edit MSDS of our products on CD-ROM and they are also available from our web: www.scharlau.com > MSDS

Storage and Transport

Storage and transportation of dangerous goods is sub- jected to special regulations due to the speciﬁc risks they involve. All parts handling dangerous goods must adopt the appropriate safety measures to avoid damages.

Safe storage Dangerous goods must be stored under special safety conditions to avoid health or environmental injuries. It is always important to keep in mind chemical in compatibili- ties and to separate the products to avoid its mixture in case of accident. Every substance can be classified into a group of substances having similar hazards, that can be stored together. The same security measures will be applied to all the substances belonging to the same classification. If a substance has several hazard degrees, it should be classified as per its highest one: Explosive > Oxidising > Flammable > Toxic > Corrosive > Harmful

There are three basic criteria that should be kept in mind to guarantee a safe storage of chemicals in the laboratories: • Stock of dangerous products must be limited to the minimum quantity needed. As a general rule, purchases should never exceed the quantity needed for one year, and preferably should cover a period of between 3 and 6 months. • Products must be stored in groups depending on its chemical compatibility, as shown below:

EXPLOSIVES OXIDIZING FLAMMABLE TOXIC CORROSIVES HARMFUL EXPLOSIVES YES NO NO NO NO NO OXIDIZING NO YES NO NO NO NO FLAMMABLE NO NO YES NO YES* YES TOXIC NO NO NO YES YES YES CORROSIVES NO NO YES* YES YES YES HARMFUL NO NO YES YES YES YES *COULD BE STORED TOGETHER IF CORROSIVE PRODUCTS ARE CONTAINED IN NON FRAGILE BOTTLES

• Some products, that are specially dangerous, should be isolated from the rest. Carcinogenic or high toxicity substances should be stored in speciﬁc cabinets conveniently marked and closed by key.

Special attention must be paid to the safe storage of ﬂammable liquids, due to the big amounts that are usually needed in the laboratories. They should be stored in security cabinets and security drums.

Storage regulations of dangerous goods indicate minimum distances that should be kept between different classes and special storage conditions for each class. Our new warehouse was built following this and other EC regulations regarding safety and risk prevention.

V Safety. Regulations

Safe transportation By air Dangerous goods can be transported outside manufac- IATA (International Air Transport Association) / ICAO turing sites only if they are packed, labelled and deliv- (International Civil Aviation Organization) ered in safe conditions. IATA regulations for transport of dangerous goods include all requirements from ICAO as well as additional • Packaging must be officially approved to contain dan- technical instructions. All data in this catalogue refer to gerous goods. Strict tests are performed to evaluate the 43rd edition of “Dangerous goods regulation” from physical resistance to break. IATA. • Packaging must be labelled according to specific You will also find in this catalogue the packing instruc- regulations for transport of dangerous goods. tions for passenger aircraft (PAX) and cargo aircraft • Goods must be accompanied by several documents (CAO). The “Packing Instruction” indicates the condi- describing its hazard and how to proceed in case of tions for the packaging, as well as the quantity author- damage during loading, transporting or downloading ized on board the aircraft. If transport of the substance operations. is forbidden, an “F” is printed instead of the PAX or CAO data. Dangerous goods cannot be transported by unauthor- The packages that comply PAX conditions, can be ized transport means. There are different international always transported on cargo aircraft. regulations that are applied to the transport by road, IATA issues an ID number that is an identification air or sea. The following classes apply to all means of number for hazardous goods. It is only used in cases transport: where there is no UN number.

By sea IMDG (International Maritime CLASS DESCRIPTION Code for Dangerous Goods) 1 Explosive substances 2 Gases has been drawn by the IMO (In- 3 Flammable liquids ternational Maritime Organiza- 4.1 Flammable solids tion of the UN) and is the regu- 4.2 Spontaneously flammable solids lation applied to any transport of 4.3 Substances that develop flammable gases in contact with water dangerous goods by see. 5.1 Substances that promote combustion (oxidants) 5.2 Organic peroxides 6.1 Toxic substances Other transport / security data 6.2 Substances that induce vomiting or infection included in this catalogue: 7 Radioactive substances UN number 8 Corrosive substances The Committee of Experts of 9 Various hazardous substances the United Nations (UN) issues recommendations for all freight forwarders on the assessment Transport regulations of hazardous goods for transport purposes. They are numbered consecutively. These UN numbers are given to each product so that everyone knows how to handle By road / railway it, no matter if it is going to be sent by road, air or sea. ADR (Accord européen rélatif au transport international In case there is no UN number, IATA gives an ID number des merchandises Dangereuses par Route) for air transport of hazardous goods. This European agreement on the international transport of dangerous goods by road changed last July 1st EC index number (EC = European Community) 2001 and all data in this catalogue have been updated This number is issued by the EC commission for the according to this new version. classification, packaging and marking of dangerous sub- The former ADR 1999 can be used until the end of stances. The marking with hazard symbols, risk warn- 2002, but, from January 1st 2003, only ADR 2001 will ings (R phrases) and safety precautions (S phrases), as be applied. used in this catalogue, is linked with this number. RID (Reglement International concernant le transport des marchandises Dangereuses par chemin de fer) Regulates the international transport of dangerous goods by railway. The new ADR2001 includes all requirements described in RID and could be used instead.

VI Safety. Hazard categories

E: EXPLOSIVE T: TOXIC Are considered all products and preparatio- Are considered all products and preparations, ns, if they can explode through ignition or if if they lead to death or to acute or cronical they are more sensitive than dinitrobenzene health injuries when inhaled, swallowed or towards blows absorbed through the skin in small quantities. and friction. Precautions: All contact with the human Precautions: Avoid impact, knocks, friction, body must be avoided, as severe or even sparks, fire and heat. lethal damage to health cannot be excluded. Particular attention is drawn to the carcinogenic, teratogenic or mutagenic risks associated with certain substances. O: OXIDISING Are considered all products and preparations, which not being flammable, can produce and enhance fires in contact with flammable Xn: HARMFUL products. Are considered all products and prepara- Precautions: Avoid all contact with flamma- tions, that if inhaled, swallowed or absorbed ble substances. Risk of ignition! The substan- through the skin can cause death or serious ce promotes fires once started and impedes acute or chronic effects. fire fighting. Precautions: Avoid contact with the human body, including the inhalation of vapours. Injury to health is possible with improper use. With some substances, carcinogenic, tera- F+: EXTREMELY FLAMMABLE togenic or mutagenic action cannot be fully Are considered all products and preparatio- excluded, as well as possible sensitization. ns, with a flash point below 0°C and a boiling point of 35°C or below. Precautions: Keep away from naked flames, sparks and sources of heat. C: CORROSIVE Are considered all products and preparations, if they can destroy skin by contact. Precautions: Take special measures to F: FLAMMABLE protect the eyes, skin and clothes. Do not Are considered all products and preparatio- inhale vapours. ns, if: a) They heat up and finally start burning in contact with air at normal temperature without any external energy supply Xi: IRRITANT b) They can start burning in solid condition Are considered all products and prepara- ater short contact with a source of ignition tions, if they can produce irritation in a short, and continue burning after the source has prolonged or repetitive contact with the skin been taken away, or the respiratory tract. c) They have a flash point below 21°C in Precautions: Avoid contact with eyes and liquid condition, skin, do not inhale vapours. d) They form in gaseous condition an explosive mixture with air under normal pressure, e) They create in contact with water or wet air highly flammable gases. N: DANGEROUS FOR THE Precautions: Keep away from naked flames, ENVIRONMENT sparks and sources of heat. for the Environment can be considered those products and substances, which can have detrimental effects upon the ecosystem of water, soil or air, climate, fauna, flora or T+: VERY TOXIC microorganisms in such a way, that they con- are considered all products and preparations, stitute an immediate or future danger for the if they lead to death or to acute or cronical environment. health Precautions: According to the kind of haz- injuries when inhaled, swallowed or absorbed ard do not dispose to wastewater, soil or through the skin in very small quantities. environment. Observe special disposal regu- Precautions: All contact with the human lations! body must be avoided, as severe or even lethal damage to health cannot be excluded. Particular attention is drawn to the carcinogenic, teratogenic or mutagenic risks associated with certain substances.

VII Safety. Risk and Safety Phrases

R: Risk Phrases 58 May cause long-term adverse eflects in the environment. 1 Explosive when dry. 59 Dangerous for the ozone layer. 2 Risk of explosion by shock, friction, fire or other 60 May impair fertility. sources of ignition. 61 May cause harm to the unborn child. 3 Extreme risk of explosion by shock, friction, fire or 62 Possible risk of impaired fertility. other sources of ignition. 63 Possible risks of harm to the unborn child. 4 Forms very sensitive explosive metallic 64 May cause harm to breastfed babies compounds. 65 Harmful: may cause lung damage if swallowed. 5 Heating may cause an explosion. 67 Vapours may cause drowsiness and dizziness. 6 Explosive with or without contact with air. 66 Repeated esposure may cause skin dryness or 7 May cause fire. cracking. 8 Contact with combustible material may cause fire. 68 Possible risk of irreversible effects. 9 Explosive when mixed with combustible material. 10 Flammable. Combination of particulars risks 11 Highly flammable. 12 Extremely flammable. 14/15 Reacts violently with water, liberating extremely 13 Extremely flammable liquefied gas. flammable gases. 14 Reacts violently with water. 15/29 Contact with water liberates toxic, extremely 15 Contact with water liberates extremely flammable flammable gas. gases. 20/21 Harmful by inhalation and in contact with skin. 16 Explosive when mixed with oxidising substances. 20/21/22 Harmful by inhalation, in contact with skin and if 17 Spontaneously flammable in air. swallowed. 18 In use, may form flammable/explosive vapour-air 20/22 Harmful by inhalation and if swallowed. mixture. 21/22 Harmful in contact with skin and if swallowed. 19 May form explosive peroxides. 23/24 Toxic by inhalation and in contact with skin. 20 Harmful by inhalation. 23/24/25 Toxic by inhalation, in contact with skin and if 21 Harmful in contact with skin. swallowed. 22 Harmful if swallowed. 23/25 Toxic by inhalation and if swallowed. 23 Toxic by inhalation. 24/25 Toxic in contact with skin and if swallowed. 24 Toxic in contact with skin. 26/27 Very toxic by inhalation and in contact with skin. 25 Toxic if swallowed. 26/27/28 Very toxic by inhalation, in contact with skin and if 26 Very toxic by inhalation. swallowed. 27 Very toxic in contact with skin. 26/28 Very toxic by inhalation and if swallowed. 28 Very toxic if swallowed. 27/28 Very toxic in contact with skin and if swallowed 29 Contact with water liberates toxic gas. 36/37 Irritating to eyes and respiratory system. 30 Can become highly flammable in use. 36/37/38 Irritating to eyes, respiratory system and skin. 31 Contact with acids liberates toxic gas. 36/38 Irritating to eyes and skin 32 Contact with acids liberates very toxic gas. 37/38 Irritating to respiratory system and skin 33 Danger of cumulative effects. 39/23 Toxic: danger of very serious irreversible effects 34 Causes burns. through inhalation. 35 Causes severe burns. 39/23/24 Toxic: danger of very serious irreversible effects 36 Irritating to eyes. through inhalation and in 37 Irritating to respiratory system. contact with skin. 38 Irritating to skin. 39/23/24/25 Toxic: danger of very serious irreversible 39 Danger of very serious irreversible effects effects through inhalation, in contact with 40 Limited evidence of a carcinogenic effect. skin and if swallowed. 41 Risk of serious damage to eyes. 39/23/25 Toxic danger of very serious irreversible effects 42 May cause sensitisation by inhalation. through inhalation and if swallowed. 43 May cause sensitisation by skin contact. 39/24 Toxic: danger of very serious irreversible effects in 44 Risk of explosion if heated under confinement. contact with skin. 45 May cause cancer. 39/24/25 Toxic: danger of very serious irreversible effects in 46 May cause heritable genetic damage. contact with skin and if swallowed. 47 May cause birth defects. 39/25 Toxic: danger of very serious irreversible effects if 48 Danger of serious damage to health by prolonged swallowed. exposure. 39/26 Very toxic: danger of very serious irreversible 49 May cause cancer by inhalation. effects through inhalation. 50 Very toxic to aquatic organisms. 39/26/27 Very toxic: danger of very serious irreversible 51 Toxic to aquatic organisms. effects through inhalation and in contact with skin. 52 Harmful to aquatic organisms. 39/26/27/28 Very toxic: danger ol very serious 53 May cause long-term adverse effects in the irreversible effects through inhalation, aquatic environment. in contact with skin and if swallowed. 54 Toxic to flora. 39/26/28 Very toxic: danger of very serious irreversible 55 Toxic to fauna. effects through inhalation and if swallowed. 56 Toxic to soil organisms. 39/27 Very toxic: danger of very serious irreversible 57 Toxic to bees. effects in contact with skin.

VIII Safety. Risk and Safety Phrases

39/27/28 Very toxic: danger of very serious irreversible 68/20 Harmful: possible risk of irreversible effects effects in contact with skin and if swallowed. through inhalation. 39/28 Very toxic: danger of very serious irreversible 68/21 Harmful: possible risk of irreversible effects in effects if swallowed. contact with skin. 40/20 Harmful: possible risk of irreversible effects 68/22 Harmful: possible risk of irreversible effects if through inhalation. swallowed. 40/20/21 Harmful: possible risk of irreversible effects 68/20/21 Harmful: possible risk of irreversible effects through inhalation and in contact with skin. through inhalation and in contact with skin. 40/20/21/22 Harmful: possible risk of irreversible 68/20/22 Harmful: possible risk of irreversible effects effects through inhalation, in contact through inhalation and if swallowed. with skin and if swallowed. 68/21/22 Harmful: possible risk of irreversible effects in 40/20/22 Harmful: possible risk of irreversible effects contact with skin and if swallowed. through inhalation and if swallowed. 68/20/21/22 Harmful: possible risk of irreversible 40/21 Harmful: possible risk of irreversible effects in effects through inhalation, in contact contact with skin. with skin and if swallowed. 40/21/22 Harmful: possible risk of irreversible effects in contact with skin and if swallowed. S: Safety phrases 40/22 Harmful possible risk of irreversible effects if swallowed. 1 Keep locked up. 42/43 May cause sensitisation by inhalation and skin 2 Keep out of reach of children. contact. 3 Keep in a cool place. 48/20 Harmful: danger of serious damage to health by 4 Keep away from living quarters. prolonged exposure. 5 Keep contents under _ (appropriate liquid to be 48/20/21 Harmful: danger of serious damage to health by specified by the manufacturer) prolonged exposure 5.3 Keep contents under paraffin oil. through inhalation and in contact with skin. 6 Keep under (inert gas to be specified by the 48/20/21/22 Harmful: danger of serious damage to manufacturer) health by prolonged exposure 7 Keep container tightly closed. through inhalation, in contact with skin 8 Keep container dry. and if swallowed. 9 Keep container in a well ventilated place. 48/20/22 Harmful: danger of serious damage to health by 12 Do not keep the container sealed. prolonged exposure through inhalation and if 13 Keep away from food, drink and animal feeding stuffs. swallowed. 14 Keep away from _ (incompatible materials to be 48/21 Harmful: danger of serious damage to health by indicated by the manufacturer) prolonged exposure in contact with skin. 14.1 Keep away from alkalis. 48/21/22 Harmful: danger of serious damage to health by 14.2 Keep away from oxidizing and acidic substances prolonged exposure in contact with skin and if as well as heavy metal compounds. swallowed. 14.9 Keep away from flammable organic substances. 48/22 Harmful: danger of serious damage to health by 15 Keep away from heat. prolonged exposure if swallowed. 16 Keep away from sources of ignition - No Smoking. 48/23 Toxic: danger of serious damage to health by 17 Keep away from combustible material. prolonged exposure through inhalation. 18 Handle and open container with care. 48/23/24 Toxic: danger of serious damage to health by 20 When using do not eat or drink. prolonged exposure through 21 When using do not smoke. inhalation and in contact with skin. 22 Do not breathe dust. 48/23/24/25 Toxic: danger of serious damage to 23 Do not breathe gas/fumes/vapour/sray health by prolonged exposure through (appropiate wording to be inhalation, in contact with skin and if specified by the manufacturer). swallowed. 23.2 Do not breathe vapour. 48/23/25 Toxic: danger of serious damage to health by 24 Avoid contact with skin. prolonged exposure through 25 Avoid contact with eyes. inhalation and if swallowed. 26 In case of contact with eyes, rinse immediately 48/24 Toxic: danger of serious damage to health by with plenty of water and seek medical advice. prolonged exposure in contact with skin. 27 Take off immediately all contaminated clothing. 48/24/25 Toxic: danger of serious damage to health by 28 After contact with skin, wash immediately with prolonged exposure in plenty of _ (to be specified by the manufacturer). contact with skin and if swallowed. 28.1 After contact with skin, wash immediately with 48/25 Toxic: danger of serious damage to health by plenty of water. prolonged exposure if swallowed. 28.2 After contact with skin, wash immediately with 50/53 Very toxic to aquatic organisms, may cause long- soap and water. term adverse effects in 28.3 After contact with skin, wash immediately with the aquatic environment. soap and water, if possible 52/53 Harmful to acquatic organisms, may cause long- also with polyethylene glycol 400. term adverse effects in the acquatic environment. 28.6 After contact with skin, wash immediately with 51/53 Toxic to acquatic organisms, may cause long-term polyethylene glycol 400 effects in the aquatic environment. (then rinse with plenty of water).

IX Safety. Risk and Safety Phrases

29 Do not empty into drains. 61 Avoid release to the environment.Refer to special 30 Never add water to this product. instructions/Safety data sheets. 33 Take precautionary measures against static 62 If swallowed, do not induce vomiting: seek discharges. medical advice immediately 34 Avoid shock and friction. and show this container or label. 35 This material and its container must be disposed 63 In case of accident by inhalation: remove casualty of in a safe way. to fresh air and keep at rest. 36 Wear suitable protective clothing. 64 If swallowed, rinse mouth with water (only if the 37 Wear suitable gloves. person is conscious). 38 In case of insufficient ventilation, wear suitable respiratory equipment. Combination of safety precautions 39 Wear eye/face protection. 40 To clean the floor and all objects contaminated by 1/2 Keep locked up and out of reach of children. this material use _ (to 3/7 Keep container tightly closed in a cool place. be specified by the manufacturer). 3/7/9 Keep container tightly closed, in a cool well 41 In case of fire and/or explosion do not breathe ventilated place. fumes. 3/9 Keep in a cool well ventilated place 42 During fumigation/spraying wear suitable 3/9/14 Keep in a cool, well ventilated place away from _ respiratory equipment (incompatible materials (appropriate wording to be specified) to be indicated by the manufacturer) 43 In case of fire, use _ (indicate in the space the 3/9/14.1 Keep in a cool, well ventilated place away from precise type of fire-lighting alkalis. equipment. If water increases the risk add - Never 3/9/14/49 Keep only in the original container in a cool, well- use water) ventilated place away from ... (incompatible 43.1 In case of fire, use water. materials to be indicated by the manufacturer). 43.3 In case of fire, use powder extinguisher. Never 3/9/49 Keep only in the original container in a cool, well- use water. ventilated place. 43.6 In case of fire, use sand. Never use water. 3/14 Keep in a cool place away from ... (incompatible 43.7 In case of fire, use metal-fire powder. Never use materials to be indicated by the manufacturer). water. 7/8 Keep container tightly closed and dry. 43.8 In case of fire, use sand, carbon dioxide or 7/9 Keep container tightly closed and in a well- powder extinguisher. Never use water. ventilated place. 44 If you feel unwell, seek medical advice (show the 20/21 When using do not eat, drink or smoke. label where possible) 24/25 Avoid contact with skin and eyes. 45 In case of accident or if you feel unwell, seek 27/28 After contact with skin, take off immediately all medical advice immediately contaminated clothing, and wash immediately with (show the label where possible) plenty of ... (to be specified by the manufacturer). 46 If swallowed seek medical advice immediately 29/35 Do not empty into drains; dispose of this material and show this container or label and its container in a safe way. 47 Keep at temperature not exceeding _ ºC (to be 36/37 Wear suitable protective clothing and gloves. specified by the manufacturer) 36/37/39 Wear suitable protective clothing, gloves and eye/ 48 Keep wetted with _ (appropriate material to be face protection. specified by the manufacturer) 36/39 Wear suitable protective clothing and eye/face 49 Keep only in the original container. protection. 50 Do not mix with _ (to be specified by the 37/39 Wear suitable gloves and eye/face protection. manufacturer) 47/49 Keep only in the original container at temperature 50.1 Do not mix with acids. not exceeding _ ºC (to be specified by the 51 Use only in well ventilated areas. manufacturer). 52 Not recommended for interior use on large surface areas. 53 Avoid exposure - obtain special instruction before use. 54 Obtain the consent of pollution control authorities before discharging to wastewater treatment plants. 55 Treat using the best available techniques before discharge into drains or the aquatic environment. 56 Dispose of this material and its containes at hazardous or special waste collection point. 57 Use appropriate containment to avoid environmental contamination. 58 To be disposed of as hazardous waste. 59 Refer to manufacturer/supplier for information on recovery/recycling 60 This material and its container must be disposed of as hazardous waste.

X Safety. Disposal

Disposal of Laboratory Waste Hazardous chemicals In this handbook risk phrases (R-phrases), safety Avoid contamination of the water phrases (S- phrases) and hazard symbols are added in Elimination of residue through the drain is strictly forbid- accordance with the EU Directive (67/548) and (83/467). den. You have to bear in mind, that many chemicals The absence of R- and/or S-phrases or hazard symbols shall not be reduced by the waste water plants and shall does not mean that those substances are harmless. contaminate the environment. The normal safety precautions for handling chemicals should always be observed. Residue in the laboratory We highly recommend naming a person in charge of the residue of your company. This person should be in- General Waste - formed of the laws and regulations regulating the waste Disposal procedures of residue in your own country. The disposal methods outlined below are intended as a In order to hand over the residue to a treatment plant, guideline for the technician. We do not assume respon- it is necessary to organize the collection and storage sibility of their use. Careful consideration must be given previously. to the chemical and physical properties of the substance. In addition, local laws and regulations may preclude the Collection of residue in the laboratory use of these methods which were primarily designed The material of the residue containers has to comply for the quantities we list. Observe all Federal, State and with the following conditions: local laws concerning health and pollution. 1. One has to be able to close the containers hermeti- All procedures require the use of goggles or face shield, cally and the material has to be resistant to the con- gloves and apron. Gas masks or self- contained breath- tents. Plastic containers should not form toxic fumes ing units may also be required. when disposed of through burning. 2. Generally, plastic containers are being used. 1. Neutralize the bases with diluted sulphuric acid, then 3. Corrosive products should be collected in metal flush neutral solution down the drain with an abun- drums with inner plastic lining, like our Combi drum dance of water. (Ref. 055-0C0025). 2. Treat the aldehydes or peracids with an excess of 4. Inflammable or oxidizing products should be collect- sodium bisulfite solution. Use starch iodide paper to ed in metal or plastic drums resistant to the solvent. ensure complete reduction of any peracid. Flush the 5. For products that produce gases or vapours, special resulting solution down the drain with an abundance containers with a security valve are required, in order of water. to avoid the danger of explosions. 3. The compound should be burned. Ideally, all hydro- carbons and related solvents should be burned in an If the collection containers are handed over to a trans- approved presence of sodium carbonate and cal- port agency, they have to carry the UN number that cium hydroxide (slaked lime). The solid or the liquid approves them for transport by road. absorbed on vermiculite should be wrapped in paper and burned in an approved incinerator. 4. Bury in an approved chemical and hazardous-waste sand fill site, well away from streams, rivers or wells. 5. Azides and azo-compounds should be treated with dilute ceric ammonium nitrate solution with cooling. Solutions may be discarded upon completion of the reaction. Mixtures should be separated and the organic portion burned. 6. Treat with an alcohol such as ethanol or butanol in an appropriate solvent. The chemical reaction may be vigorous and/or exotermic. Provisions must be made for venting of large volumes of highly flammable hydrogen and/or hydrocarbon gases. Neutralize the solution with aqueous acid. Filter off any solid residue for disposal as hazardous waste. Burn residual organic materials in an incinerator equipped with an afterburner and scrubber.

XI Safety. Disposal

7. Neutralize the base with dry sodium bisulfate add water slowly and ﬂush the neutral solution down the drain with an excess of water. 8. Carefully mix the acidic compound with dry sodium bicarbonate. Dilute slowly and ﬂush down the drain with an excess of water. 9. Oxidize the mercaptan or nitrite with a weak aqueous solution (up to 15%) of sodium or calcium hypoclorite. Vigorous stirring may be required to ensure completion of the reaction. Neutralize the resulting mixture, then discard down the drain with an excess of water. 10. Flush down the drain with an excess of water. 11. Bury in a site approved for disposal of poisonous or hazardous wastes. 12. Refer to our Material Safety Data Sheet before handling or disposing.

Caution regarding hazardous components Some media contain components that are toxic or carcinogenic. Appropriate safety precautions must be taken when using media with such components.

Basic fuchsin and acid fuchsin are carcinogens and caution must be used in handling media with these compounds to avoid dangerous exposure that could lead to the development of malignancies . Thalium salts, sodium azide, sodium biselenite and cyanide are among the toxic components found in some media. These compounds are poisonous and steps must be taken to avoid ingestion, inhalation, and skin contact. Azides also react with many metals, especially copper, to form explosive metal-azides. The disposal of azides must avoid contact with copper or achieve sufﬁcient dilution to avoid the formation of such hazardous explosive compounds. Media with sulphur containing compounds may result in the formation of hydrogen sulphide which is a toxic gas. Care must be used to ensure proper ventilation. Media with human blood or human blood components must be handled with great caution to avoid exposure to human immunodeﬁciency virus and other pathogens that contaminate some blood supplies. Proper handling and disposal procedures must be followed with blood-containing as well as other media that are used to cultivate microorganisms.

XII Guidelines for correct manipulation in Microbiology

Following instructions provide the correct guidelines for D. Administrative procedures the manipulations in microbiology. Every microbiological D.1. Systems to confirm and assure the effectiveness of laboratory is responsible for their proper implementation. the biological security code carried out by quali- fied personnel. A. Personal techniques D.2. Training programmes regarding the security during A.1. Hygiene is the basic element and need for the safe or at work, adapted to the level required by every manipulation. maninpulator / technician. A.2. All manipulated microorganisms must be presumed D.3. Adequate medical surveillance and facilities . potentially pathogenic, regardless of their nature. D.4. Procedures for the verification and maintenance The following procedures or techniques must of the ventilation system and the equipment in therefore be avoided : oral pipetting, application general. of cosmetics ,eating, drinking or smoking in the D.5. Emergency procedures and systems for use in case laboratory. Wrappings and labels in the labortory of any accident. should be self-adhesive. D.6. Strategies to restrain the freak entries and exits, to A.3. Manipulation of the microorganisms should be maintain the security standards of laboratory and conducted in such a way that will not provoke the plant. any environmental contamination or hazard. For D.7. Procedures for the transportation, postage and example, sprays should always be avoided and reception of the biological material (postage pipettes must be submerged immediately in the regulations and related things). disinfectant after use. D.8. Training of personnel to act as security officers. A.4. Manipulators (technicians) must be aware of the D.9. Systems to eliminate any potentially hazardous risks and should know how to prevent them. residues. A.5. Manipulators must make sure that both, the user and the equipment itself are perfectly safe from References any potential risk. Report of Committee of Enquiry into Smallpox (1974) Outbreak in March and April 1973. Cmnd.5626, Her B. Equipment in general Majesty’s Stationery Office. B.1. A standard equipment for the sterilization of any Report of the Working Party on the Laboratory Use of potentially hazardous material must be available Dangerous Pathogens. (The Godber Report) (1975). (autoclave, jars with disinfectant for the pipettes, Cmnd. 6054, Her Majesty’s Stationery Office. dry heat ovens etc). Report of the Working Party on the Experimental Manip- B.2. Protective clothes must be used exclusively in the ulation of the Genetic Composition of Micro-organisms. working area, and should never be worn while in (1975) Cmnd. 5880, Her Majesty’s Stationery Office. public areas. PUBLIC HEALTH LABORATORY SERVICE (1980) B.3.Toilets and other facilities must be available close Safety Precautions-Notes for Guidance. Colindale, to the working areas, and separate from those of MILLER, B.M. (1987) Laboratory Safety: Principles and use to the general public. Practices. ASM. Whasington, D.C. B.4. Any device which may probably produce the sprays HARTREE, E., U. BROTH (1977) Safety in Biological must be always handled in microbiological secu- Laboratories. Biochem. Soc. Spec. Pub. nº5. The Bio- rity cabins like Laminar Air Flow . chemical Society. London B.5. In case of any accident, all necessary substances COLLINS, C.H., E.G. HARTLEY, R. PILSWORT (1976) and equipments , either to minimize or neutralize The Prevention of Laboratory Adquired Infection. PHLS the risks must be readily available. Monograph nº6. Her Magesty Stationery Office. SMITH, J.A. (1996) Laboratory Safety in Clinical Microbi- C. Basic characteristics of the ology. Cumitech nº29. ASM. Whasington, D.C. laboratory WHO (1983) Laboratory Biosafety Manual. Geneva. C.1. The laboratory or plant should not be located in a passage or corridor so that any outside person The appearance and quality of the prepared medium would always remain away from it. usually depends on the method used for rehydration C.2. It must be equiped with an appropiate ventillation and storage. It is therefore very important to follow the system, mechanically regulated if possible, so as recommendations stated below. to create a drift of flowing air from the areas of low risk to the contaminated areas, and then going outside, through the HEPA filters. C.3. The laboratory must be built of easily washable, waterproof and disinfectant-resistant surfaces.

XIII Guidelines for correct manipulation in Microbiology

Rehydration container. Use a container with a capacity of approxi- Directions for the individual reconstitution are detailed mately 2 to 3 times more the volume of the medium, to on the container of every media and must be strictly allow plenty of space for manipulation. followed. Sometimes, It is necessary to boil the medium for more than 1 minute. In such cases, the volume of evaporated The water used for reconstitution purposes must be water must be restored. distilled or deionized, of «pharmaceutical grade». The solution or suspension must be completely homogene- Media not containing the agar or any solidifying agents ous and the exposure to heat if necessary, should be (broths and Nutrient Solutions) tend to dissolve eas- minimal. ily and rapidly in preheated water, however sometimes boiling is necessary to attain the complete dissolution. In any case, it is always helpful to heat the water previ- These media have a tendency to form a concentrated ously to 50-60°C, to help dissolve the media. syrup or suspension which settles at the bottom of the container. This thick suspension usually remains at the For the preparation of the culture media the most recom- bottom, risking the destruction of the medium contents mended technique is the following: by hydrolysis, caramelization and pH drift when the heat Add necessary amount of powder to half the volume is applied. Therefore, thorough mixing should be as- of water needed. The water should have already been sured before heating. warmed to 50-60° C. Stir the mixture thoroughly until the powder is dissolved. Use rest of the water to dissolve the Media containing solidifying agents (Agars and Fluid powder sticking to the walls or sides of the container and Media) must be preheated before sterilization. Media continue stirring and heating if necessary. Heat must containing agar must be brought to the boiling, since be applied directly and smoothly. Constant agitation will agar is insoluble in water below 98-100° C. It is useful to prevent the components from sticking to the walls of the let the agar soak in preheated water for 5 to 10 minutes before heating, in order to allow it to swell and to secure its solubility and uniform distribution and dissolution.

A highly recommended procedure can be practised by removing the rehydrated medium from the source of heat when the mixture begins to boil, letting it stand for a while and then quickly bringing it to the boiling again. This may be repeated 2 or 3 times with constant agitation. The complete dissolution of the agar will be indicated by the absence of granules in the container. Dissolution of culture media directly during sterilization in the autoclave is a frequent and totally incorrect practice. It alters the medium’s qualities and frequently causes the uneven dissolution of the agar into layers with different concentration gradients, pH drifts and browning.

Nowadays, heating the rehydrated medium in a microwave oven in order to melt the agar

XIV Guidelines for correct manipulation in Microbiology or dissolve the medium components is a very common dium is introduced in the autoclave , and to let the tem- procedure. In this case also, it is recommended to let perature drop to 70-80°C before removing the medium the agar-medium to soak for some time before using so as to avoid severe temperature fluctuations. Although the microwave and use the containers suitable for the the use of cold water to cool media is widespread, it is volume of medium to be prepared. Using a microwave not recommended for media containing agar since it with 800 W for 4 minutes are enough to achieve the total causes flakes and cloud formation. dissolution or melting of agar. However, it must be noted that since this is a static procedure, a concentration gra- dient will appear which will create a stratification. Thus, it may be necessary to shake the rehydrated medium vigorously to homogenize the solidifying agent before using or sterilizing it in the autoclave, in the same way as if it were an autosterile medium. Microwave heating can never be adopted as a substitute for autoclave sterilization.

Dehydrated culture media usually maintain their features if reconstituted properly. Nevertheless, veriﬁcation is encouraged since some of them (colour, pH, etc) may slightly change subject to the conditions of reconstitution and the type of water used.

These recommendations are particularly important when referring to ﬂuid media, since such media contain small amounts of solidifying agent and are therefore very delicate. Cooling becomes as important as heating, as any violent process may induce the formation of ﬂakes and clouds. A slow and smooth cooling process is recommended.

Media containing phosphate buffers together with glucose or other carbohydrates may darken if overheated as a concentrated solution. Precipitates may also appear when using poor quality water.

Special attention must be paid to all media containing agar at a pH under 5, since it hydrolyzes and looses the ability to form gels. In those cases, unnecessary heating must be avoided.

Sterilization The indications given on every container must be followed, while bearing in mind that they refer to quantities of up to 1 litre. As for bigger volumes, autoclaving and heat penetration conditions of the medium will have to be taken into consideration.

In all the cases autoclaving must be monitored regularly by the manometers and thermometers, as well as the even distribution of heat. Nowadays this can be done mechanically (thermocouples), chemically (thermal indicators) or biologically (thermoresistent spores). Considering that overheating is one of the main causes of culture media alteration, it is only natural to prevent, whenever possible, the treatment of large quantities of media and the prolonged exposure to heat. For large autoclaves it is recommended to preheat before the me-

XV Guidelines for correct manipulation in Microbiology

Containers used for sterilization purposes must have a and can even allow direct growth of microorganisms in large head space for air to allow foaming. If screw-cap them. Under such circumstances they must not be used. containers are used,the closures must be a half turned Although they usually have a dry and powdery appear- or screwed to allow inner and outer pressure balancing. ance, the media may occasionally appear moist, oily but All containers should be chemically inert to prevent al- never stiffened, without there being any alteration in their teration of the pH of contents. Borosilicate glass contain- composition (as e.g.in MRS, Tributyrin Agar, etc). ers are highly recommended. In any case, storage of dehydrated media is not indefi- Media containing carbohydrates darken a little after heat nite. It is not recommended to store large quantities sterilization, therefore, sterilization should be carried out but to keep small amounts in stock, so as to maintain a at a temperature below 120°C if possible. rotation of ready prepared dehydrated media. Register- Should autoclave not be available, sterilization can be ing the reception dates on the containers will avoid the done in a pressure vessel (at 100°C for 30 minutes). In accumulation of older media. such case it is useful to resterilize the medium during three days, if the medium allows the treatment. Storage of ready Some media containing selective substances do not prepared culture media need sterilization. The preparation process is quicker Although it is best to prepare the media as you required, since they are ready for use after dissolution. Since it is common to store them ready prepared and sterilized these media are usually not very stable and preparation in order to spare preparation time. is brief and easy, the exact quantities required should be Under these circumstances the biggest drawback is the prepared. dehydration and every precaution taken to avoid it will prolong the medium’s useful life, which is usually be- Additions to the medium (if any) after sterilization must tween 4 and 6 weeks. We therefore recommend the use be done aseptically, that is, additives should have been of hermetically sealed screw-capped containers (either previously sterilized. Additives are generally thermolabi- bottles or tubes) instead of those plugged with cotton les if not they would be part of the dehydrated medium’s wool. A moderate refrigeration (4°C) usually prolongs composition. It is therefore necessary to allow the medi- the life of the medium but it should also be noted that a um to cool to 50-60°C before their addition, so as not to refrigerated environment is very dry and consequently harm the additive while permitting its correct distribution. renders dehydration. In some cases, as regards in Once the addition is over, reheating must be avoided. thioglycolate media and almost all media recommended Distribution into final containers must be completed for anaerobes, they are best stored at room temperature before sterilizing, to avoid any manipulation. Since it is for the less penetration of air. Direct light must always rarely possible to do so, manipulation should be done be avoided, particulary when it is specifically indicated in safety cabinets instead. The sterile cabinets or rooms on the label of the medium. are not be exposed to any radiations and so, no any Solid media for the plates are best stored in their original kind of highly active reagent could affect the medium’s containers than in the poured plates. Nevertheless, components. Excessive steam condensation can be poured plates may also be prepared and stored if the eluded by distributing the medium at temperatures close indications below are followed: to solidification, ranging from 45 to 50°C. a) Refrigerate the media immediately after solidification while incubating «controls» to check the sterility of Storage of dehydrated media the batch. Dehydrated culture media should be stored safely, pro- b) If the media are to be stored for a longer period, tected from humidity or moisture, light and heat, which plates should be individually sealed with a waterproof are the most frequent causes of their alteration. seal and, if possible, stored in single containers. SCHARLAU media are supplied in opaque, waterproof, c) If storage is to last more than a few days, the plates screw-cap plastic bottles, with an internal relief which should be wrapped in plastic bags to avoid dehydra- eliminates the need for an intermediate seal. Never- tion of the media. theless a hermetic seal is assured by accurately fitting the cap and keeping both its sides and the border of its Since condensation water will inevitably appear, we sug- mouth clean. gest to store the poured plates inverted. Contamination with condensation water can be avoided by pouring the It is important to open the bottles in dry atmosphere molten medium on the plates as cold as possible. and close them immediately after their use. Refrigera- Media which have been stored in refrigeration should tion is not necessary even if low temperatures prolong be allowed to warm up gradually. It is recommended to the medium’s effectivity. The nature of the containers keep them at room temperature for a few hours before of SCHARLAU make culture media are suitable for inoculation, allowing misted containers to clear and prolonged storage in cool, dry places. If the media are avoiding the delayed initiation of growth (or eventually its moist they become stiff and hard, lose their properties total failure).

XVI Guidelines for correct manipulation in Microbiology

Stored ready prepared media which may involve a high General Medium: Medium that supports the growth of level of dehydration should never be used. Dehydra- a very wide range of microorganisms without tion is indicated by excess condensation of water or special nutrient needs. by a decrease in volume of liquid media; an increase of Membrane Filter (MF): Media used when the Mem- concentration or precipitation of fluid and liquid media; brane Filter Technique is employed. or retraction, cracking and precipitation of solid media. Plates with very dry surfaces or those which appear to Membrane Filter Media have changed colour should not be used either. Utilization of the Membrane filter technquies has offered some undoubted advantages to the Microbiology which Remelting solid media has certain specific problems. Membrane filtration allows Although most solid culture media can be stored ready to examine big samples (in volume of liquid) with very prepared and sterilized, and can be remelted and poured low microorganisms concentration or load, to separate into plates whenever necessary, this procedure should the microorganisms from the culture medium and even not be applied to media which do not need sterilization the exchange of the microorganisms between two media or those with a pH 5 or less, since it would alter their without affecting their growth. properties. Moreover, the MPN technique is more precise in these Remelting is done in a boiling water bath or by fluent media and also, the membrane with the colonial growth steam autoclaving for 30 minutes. Do not apply direct may be stored and filed. heat. However, it must be emphasized that remelted The membrane filtration system is accepted by most media are prone to precipitation and darkening if held in pharmacopeias as an alternative or the single method to a molten state for more than 1 hour and at temperatures examine antimicrobial or with strong inhibitors sub- ranging from 45 to 65°C. Such media may also undergo stances, since a proper wash of the filter remove all the nutritional impairment to the desired microorganisms. substances that interfere with the microbial growth. It is therefore recommended to avoid remelting of solid On other hand, the wide range of filters and qualities media whenever possible. available allows the examination of any product, since you may find membranes resistant to almost all the dis- One of the quickest and most recommended remelt- solvents. ing methods for culture media is the use of microwave ovens. In this case all the precautions exposed must be Technique carefully observed and practised in the media which are Essentially, the technique consists of filtering the sample labile and consequently radiation and minimum intensity through a filter with a suitable porosity (0,22 microns should be dosed for the shortest time intervals. for bacteria and 0,45 microns for fungi) with pressure A usual and erroneous practice consists of imposing or suction, in such a way that the microorganisms are strong radiation intensities for short periods, which re- retained on the membrane. sults in partial remelting, sudden boiling and overflowing If the fluid that has been filtered contains inhibitors, wash of the medium and an overall alteration of its properties. the membrane several times with rinse liquid to remove them. Membrane is removed aseptically and it is taken Differentiation of culture media and placed on the culture medium. SCHARLAU names for culture media are based on their For sterility controls, the membrane is incubated directly final consistency or appearance. Thus, a medium is in the classical media. For these tests refer: Thioglyco- named as: late Broth (Ref. 2-186), Thioglicolate USP Fluid Medium Agar: Solid media with an agar content of 1% or more. (Ref. 3-187), Tryptone Soy Broth (Ref. 2-200), Sabour- Fluid Medium (FM): Semisolid media with an agar con- aud USP Broth (Ref. 2-165). tent of less than 1%. Broth: Liquid culture media with undefined organic com- Should an enumeration of colonies be desired, incubate ponents (peptones, organ and tissue extracts, the filter on a solid medium or on a pad soaked in liquid etc...) medium, but beware that the lower surface touches the Nutrient Solution: Culture media with defined chemical culture medium and that there are no air bubbles or air composition. gap in between. Selective Medium: Medium that allows the growth of a Usually, general media may be used for these tech- biotype or a few biotypes between those that are niques. However, there are several culture media spe- present in the inocule. cifically developed for these techniques, specially in the Differential Medium: Medium that allows to distinguish water Microbiology. or differentiate between similar culture types. Enrichment Medium: Medium that aids the growth of a defined biotype over the rest in the inocule.

XVII

Dehydrated Culture Media Culture media manufacture process ﬂow chart

2 Acetamide Medium

Ref. 03-428 Xn Some authors suggest the use of this nutrient solution as a previous enrichment medium before the isolation me- R-40 dium, especially if you are studing very polluted samples Specification S-36/37 Liquid medium for the enrichment and confirmative test with companion flora. of presence of Pseudomonas aeruginosa in water acc. EN 12780:2002 and ISO 16266 standard. Technique Medium is inoculated with a couple of loops from the Formula (in g/L) culture or inoculum to be assayed and is incubated at Acetamide ...... 2,0000 32-35°C for 24-48 hours before proceeding for the isola- Magnesium sulfate, anhydrous ...... 0,2000 tion medium. Sodium chloride ...... 0,2000 Ferrous sulfate ...... 0,0005 To confirm Pseudomonas aeruginosa, inoculate a loop Monopotassium phosphate ...... 1,0000 of culture inoculum in Asparagine Broth (Ref. 02-271) Sodium molybdate ...... 0,0050 and incubate at 35-37°C for 24 hours. After this period, Final pH 7,0 ± 0,5 pour 1 or 2 drops of Nessler’s Reagent (Ref. 06-084) on to the culture and observe for ammonia production: a change in colour to yellow indicates ammonia production Directions and thus the presence of Pseudomonas aeruginosa . Dissolve 3,4 g of powder in 1 L of distilled water. Heat only if necessary. Sterilize in the autoclave at 121°C for 15 minutes. Prepared medium may be opalescent and References with precipitate. The prepared medium remain active for EN 12780 Standard (2002) Water Quality-Detection and 3 months if it is stored in the dark in a cool place. enumeration of Ps.aeruginosa by membrane filtration. CEN. Brussels. DIN Standard 3841. Deutsche Einheitsverfahren zür Description Wasser, Abwasser und Schlammuntersuchung Mikro- This nutrient solution has acetamide as the unique biologische Verfahren: Nachweiss von Pseudomonas carbon and nitrogen source, and it allows the growth of aeruginosa (K8). only those microorganisms that are able to use aceta- KELLY, N.M., C.T. KEANZ (1983) Acetamide Broth for mide. In water and in almost all the food materials, these Isolation of Pseudomonas aeruginosa from patients with microorganisms are the non fermenting gramnegatives, cystic fibrosis. J. Clin. Microbiol. 17:159-163. thus Pseudomonas aeruginosa is the only one that can ISO 16266 (2006). Water Quality. Detection and enu- liberate ammonia by deaminating acetamide. meration of Pseudomonas aeruginosa. Method by membrane filtration.

Ref. 03-428 Acetamide Medium. Left: control; center: Pseudomonas aeruginosa ATCC 27853; right: Pseu- domonas aeruginosa ATCC 9027.

3 Actinomycete Media

Actinomycete Agar Directions Dissolve 57 g of powder into1 L of distilled water. Heat if Ref. 01-003 necessary. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. Specification Should a fluid medium be required, incorporate 7 g of Agar and follow the directions for the solid medium. After Solid culture medium for actinomycete maintenance and the sterilization, cool it quickly but gently to avoid agar propagation according to Ajello et al. formulation. flocculation.

Formula (in g/L) Description Meat peptone ...... 10,00 Basically Pine and Watson formulated this medium for Casein peptone ...... 4,00 the maintenance of pathogenic actinomycytes which was Heart extract ...... 10,00 then further modified by Ajello et al. Those media do not Yeast extract ...... 5,00 possess any inhibitory agents, and so are not recom- Dextrose ...... 5,00 mended for the isolation, but their slight reducing power Potassium phosphate ...... 15,00 and their high nutritive capacity make them suitable for Sodium chloride ...... 5,00 facultative anaerobes also. Starch ...... 1,00 Ammonium sulfate ...... 1,00 Cysteine ...... 1,00 Technique Magnesium sulfate ...... 0,20 If growth conditions require strict anaerobiosis, the incu- Calcium chloride ...... 0,01 bation must be done in special flasks, but if the condi- Agar ...... 20,00 tions are only moderate or not strict anaerobic, a good Final pH 7,0 ± 0,2 anaerobiosis can be obtained by sealing the flasks with Sealing Anaerobic Agar (Ref. 01-174) or with the classic Directions alkaline pyrogallate caps. Suspend 77 g of powder into 1 L distilled water and let it Best results are obtained using an Anaerobic Jar with a soak. Bring to boiling with constant stirring and distribute CO mixture generator. into suitable containers. Sterilize by autoclaving at 121°C 2 for 15 minutes. The use of a liquid medium allows freezing of the cultures after their growth and the use also as a massive Actinomycete Broth production system for cellular preparations. In the fluid format, the Actinomycete Broth reaches a high reduction Ref. 02-003 potential allowing an easy growth to strict anaerobics.

Specification There is only one difference between solid and broth Liquid culture medium, able to form a fluid medium, for formulation which is the addition of agar 20,0 g/L to the the pathogenic actinomycete maintenance and propaga- solid medium as a solidifying agent, providing the suf- tion, according to Ajello et al. formulation. ficient strength to support the surface growth.

Formula (in g/L) Meat peptone ...... 10,00 References Casein peptone ...... 4,00 AJELLO, GEORG, KAPLAN & KAUFFMAN (1963) CDC Heart extract ...... 10,00 Lab Manual for Medical Mycology, Publication nº994 US Yeast extract ...... 5,00 Govt. Prmtmg. Ofﬁce. Washington. Dextrose ...... 5,00 LENNETTE, SPAULDING & TROULANT (1974) Manual Potassium phosphate ...... 15,00 of Clinical Microbiology. ASM 2d. Ed. Washington. Sodium chloride ...... 5,00 PINE, L., A.HOWELL, S.J.WATSON (1960) Studies on Starch ...... 1,00 the morphological characters of Actynomyces bovis. J. Ammonium sulfate ...... 1,00 Gen. Microbiol. 23:403-424. Cysteine ...... 1,00 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Magnesium sulfate ...... 0,20 biological Media. CRC Press. London. Calcium chloride ...... 0,01 Final pH 7,0 ± 0,2

4 AFP Agar (Aspergillus ﬂavus/parasiticus Agar)

Ref. 01-405 T Technique

R-45 The sample is inoculate on the surface of the AFP Agar Specification S-45-53 ad the plates are incubated at 30ºC for 42-48 hours. Selective solid medium for the isolation and enumeration Count the fungal colonies that shows the typical yellow- of Aspergillus flavus and Aspergillus parasiticus. orange pigmentation on the reverse. Express the results as “Aspergillus flavus/parasiticus colonies per g or mL of Formula (in g/L) sample”. Peptone ...... 10,000 In this conditions Aspergillus orizae can produce a very Yeast extract ...... 20,000 similar pigmentation than induce error in the counting. Ammoniun Iron Citrate ...... 0,500 Also, Aspergillus niger can be present but its pigment is Dichloran ...... 0,002 pale yellow and never turns to orange and after 48 hours Chloramphenicol ...... 0,100 of incubation begin to produce the typical black conidi- Agar ...... 15,000 ophora. Final pH 6,3 ± 0,2

Directions Suspend 45,6 g of powder in 1 L of distilled water and References KING D.A. A.D. HOCKING & J.I. PITT (1979) Dichloran- bring to the boil. Distribute in suitable containers and rose bengal médium fot enumeration and isolation of sterilize in autoclave at 121ºC for 15 minutes. moulds from foods. Appl. Environm. Microbiol 37:959- 964 Description VANDERZANT, C. & D.F. SPLITTSTOESSER (1992) AFP Agar is a modiﬁcation of the original medium devel- Compendium of methods for the microbiological exami- oped by King, Hocking & Pitt for the enumeration and nation of foods. APHA. Washington. isolation of moulds in foods. The inclusion of ammonium ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- iron citrate and the high temperature (30ºC) of incubation biological Media. CRC Press. London enhances the quickly typical pigmentation in Aspergillus ﬂavus and A. parasiticus.

The elimination of glucose from the formula, with the low value of pH and the presence of chloramphenicol inhibits the bacterial growth and the dichloran repress the spreading of the fungal colonies.

Ref. 01-405 AFP Agar. Left: control; right: Aspergillus ssp.

5 Algae Media

Algae Agar Formula (in g/L) Sodium nitrate ...... 1,000 Ref. 01-007 Dipotassium phosphate ...... 0,250 Magnesium sulfate ...... 0,513 Speciﬁcation Ammonium chloride ...... 0,050 Calcium chloride ...... 0,058 Solid culture medium for the isolation and cultivation of Ferrous chloride ...... 0,003 algae from soil, water and waste water. Final pH 7,0 ± 0,2

Formula (in g/L) Directions Sodium nitrate ...... 1,000 Dissolve 1,87 g of powder in 1 L of distilled water and Dipotassium phosphate ...... 0,250 distribute into suitable containers. Sterilize in the auto- Magnesium sulfate ...... 0,513 clave at 121°C for 15 minutes. Ammonium chloride ...... 0,050 Calcium chloride ...... 0,058 Ferrous chloride ...... 0,003 Description Agar ...... 15,000 This liquid medium is suitable for algae and cyanobac- Final pH 7,0 ± 0,2 teria cultivation, and it is especially adapted for inocule preparation and algicide biotesting, as per Fitzgerald’s Directions technique. Due to the less content of energy source, fungi and Suspend 17 g of powder into 1 L of distilled water and let bacteria do not grow or grow badly unlike algae, which it soak . Bring to boiling with constant stirring. Distribute obtain all the necessary nutrients, except the energy into appropriate containers and sterilize in the autoclave source. at 121°C for 15 minutes. Technique Description Fitzgerald’s procedure for the algicide ability verification The balanced nutrient medium composition provide all of chemical products is: necessary nutrients for the good growth of the algae , but it does not support the good growth of fungi and a) Inoculum preparation bacteria which can not grow well or have difficulties in Prepare the Algae Broth and distribute 20 mL. each in the medium. the 50 mL capacity conical flasks. Sterilize and keep It is also suitable for algicide testing, however it is essen- them cool until usage. tially recommended for the algae-pattern maintenance Regularly, one of the conical flasks is inoculated with a and cultivation or for the isolation of water contaminants. couple of loops from Chlorella emersonii culture from slanted Agar (Ref 1-007) and it is incubated at room Technique temperature until good growth is observed. For the maintenance of algal strains it is recommended This culture can be used as biotest inoculum, but for to incubate at room temperature, under a suitable only up to 30 days. light source (natural, fluorescent tube or incandescent lamp) until a good growth is obtained (within one to two b) Biotest weeks). 1- Samples In these conditions, and without gel dehydration, cultures Prepare one litre of pure distilled water and 1 litre of can be maintained up to two months. distilled water containing the inhibitor. Add 120 mg of Sodium nitrate and 2,5 g of Di-potassium phosphate to Algae Broth each sample. 2- Technique of the Test Ref. 02-007 Prepare a double series of 50 mL capacity conical flasks and add 5, 12.5 and 25 mL respectively of water algicidal Specification mixture, and then refill with pure water to get 25 mL in each conical flask. Nutritive solution for algae and cyanobacteria, suitable Add only 25 mL of pure water in one or two conical for water algicide biotesting. flasks to use them for control purposes. All the conical flasks are inoculated with the same volume of inoculum, the necessary amount to get an algae concentration about 300.000 cells/mL in each flask. As a practice (not exactly) this concentration produces a slight greenish tinge. If necessary, adjust the inoculum by counting or photocolorimetry.

6 Algae Media

Incubate the inoculated flasks at room temperature under an homogeneous and standardized light (i.e. 20 W fluorescent light). Countings of all the flasks is carried out daily with a glob- ule-cell count (Thoma, Neubauer type or similar). The test is said to be over when the control conical flasks have an average concentration greater than 5x106 cells/mL, comparing the other flasks to them. 3-Interpretation Inhibitor concentration in the flasks with equal growth to the control is considered non toxic or ineffective. If algal concentration is maintained or remains same as at the starting of experiment, it is considered algistatic. Concentrations that have reduced starting population are considered as algicidic, with the different effectivity ratio depending on the amount of loss.

References CLESCERI, L., A.E. GREENBERG, A.D. EATON (1998) Standard Methods for Examination of Water and Waste- water. APHA-AWWA-WEF. Washington, D.C. Ref. 01-007 Algae Agar. Chlorella sp. ALLEN, (1952) Arch. Microbiol. 17:34 FITZGERALD (1962) Water and Sewage Works. 109:361.

Antibiotic Media

While performing the antibiotic assays, the only meth- the bibliography mentioned at the end of this publication, odology accepted universally is the microbiological especially 21CFR for the details about the preparation of methodology. National pharmacopeia provides different samples and the extraction of antibiotics for each phar- directives to adapt to new compounds. Nowadays, the maceutical preparation. European Pharmacopeia (1996), following a path to The reference substances used in the assays are sub- standardize all the countries in the EU, provides several stances whose activity has been precisely determined rules and recommendations but it does not impose strict with references to the corresponding international stand- criteria, since they could interfere with the local legisla- ard or international reference preparation. tion. Instead of this, the USP 25/NF 20 (2002) incorpo- rates more concrete guidelines about the antibiotics that Microorganisms are accepted for human consumption and their assay methodology. However, many times the USP refers Strains used in the antibiotics assay as well as the to the US/FDA, which is the more detailed publication preparation methods and inocula are resumed in the about the antibiotics assay. Table I. Strains may be obtained from the following and Kirshbaun and Arret, in 1959, published a report with all other collections: the variations of this methodology. The success of their first attempt, and the discovery of new antibiotics made ATCC: American Type Culture Collection, 12301 Park- the same authors revise their work and , in 1967, they lawn Drive, Rockville, Maryland 20852-176. USA. published a new report detailing 57 official methods, and Fax +1 301-231-5826 in 1971 last edition they detailed 83 official methods and CIP/CNCM: Collection de l’Institut Pasteur / Collection 10 non official methods. The XIX edition of the USP con- Nationale de Cultures de Microorganismes, sider this last 1971 edition as its information source. Institut Pasteur, 25 rue du Docteur Roux, 75724 The present article simply pretends to resume the most PARIS CEDEX 15. France. Fax +33 143 06 98 important characteristics of this methodology, following 35 the guidelines of the current pharmacopeia. There are CECT: Colección Española de Cultivos Tipo, Dpto Micro- no detailed directions to perform the assay because it biologia, Fac. Ciencias Biológicas, Dr. Moliner is considered that this is going to be read by microbiol- 50, 46100 Burjasot (Valencia) Spain. Fax +34 ogists who are familiar with the basic techniques.If you 63 86 43 72 are interested in this subject, we would ask you to read

7 Antibiotic Media

NCTC:National Collection of Type Cultures, PHLS Method #2 Central Public Health Laboratory, 61 Colindale Process is the same as the Method #1 but the Avenue, LONDON NW9 5HT England. standardization is performed in the following way: NCIB: The National Collections of Industrial and Marine Centrifuge and collect the pellet. Sediment is sus- Bacteria Ltd., 23 St Machar Drove, ABERDEEN pended with 50-70 mL of Ringer 1/4 Solution (Ref. AB2 1RY, Scotland. U.K. Fax +4402 24 487 06-073) and heat up to 70°C for 30 minutes. Refrig- 658 erate the spore suspension obtained in this way. NCYC: National Collection of Yeast Cultures. Institute Method #3 of Food Research. Norwich Laboratory. Colney Proceed as per Method #2 performing the thermal Lane. NORWICH NR4 7UA. U.K. shock before the centrifugation. Wash the spore suspension 3 consecutive times with distilled water Preparation methods (25-50 mL each time) and centrifuge. Reconstitute Method #1 the final suspension with 50-70 mL of sterile distilled Strains to be assayed are mantained in slanted tubes water and store it in refrigeration. of Antibiotic Medium 1 (Ref. 01-009), subculturing Method #4 to fresh tubes every week. The growth on a slanted Let the fungi grow for 6-8 weeks at room temperature tube, incubated at 32-37° for 24 hours, is washed (20-25°C) in several 3 L Erlenmeyer flasks, contain- with 3 mL of Ringer Solution (Ref. 06-073), and the ing each one about 200 mL of Antibiotic Medium 22. resulting suspension is used to inoculate a Roux After six weeks, test the sporulation regularly and, flask containing 250 mL of sterile and controlled when it reaches 80%, collect the spores of the aerial Antibiotic Medium 1 (Ref. 01-009). micelium with a sterile spatula or any other suitable For the maintenance of antibiotic resistent strains instrument. The collected spores are suspended in prepare Antibiotic Medium 1 (Ref. 01-009) with the 20 mL of Ringer 1/4 Solution (Ref. 06-073) and store addition of proper amounts of antibiotic, as described refrigerated. in the Table II. The growth obtained in the Roux flask after a 24 hours incubation at 32-37°C is washed and collected with 50 mL of Ringer Solution (Ref. 06-073) and it is stored in refrigeration (4-6°C)

Table I. Strains (Test organisms) for the Antibiotic Assays # Strain Genus & species Strain and Type culture collection # Method American Collection European Collections

A Staphylococcus aureus ATCC 6538P CIP 53.156 NCTC 7447 1 B Staphylococcus aureus ATCC 29737 1 C Staphylococcus aureus ATCC 9144 CIP 53.134 NCIB 6751 1 D Staphylococcus epidermidis ATCC 12228 CIP 68.21 NCIB 8853 1 E Micrococcus luteus ATCC 9341 CIP 53.45 NCTC 8340 1 F Micrococcus luteus (ﬂavus) ATCC 10240 CIP 53.160 NCTC 7743 1 G Enterococcus hirae ATCC 10541 CIP 58.55 NCIB 8192 5 H Bacillus pumilus CIP 76.18 NCTC 8241 2 I Bacillus cereus ATCC 11778 CECT 193 NCTC 10320 3 J Bacillus subtilis ATCC 6633 CIP 52.62 NCTC 10400 2 K Bacillus subtilis CIP 1.83 NCTC 8236 2 L Klebsiella pneumoniae ATCC 10031 CIP 53.153 NCTC 7427 1 M Escherichia coli ATCC 10536 CIP 54.127 NCIB 8879 1 N Escherichia coli ATCC 9637 CIP 2.83 NCIB 8666 1 O Bordetella bronchiseptica ATCC 4617 CIP 53.157 NCTC 8344 1 P Saccharomyces cerevisiae ATCC 9763 CIP 1432.83 NCYC 87 61 Q Saccharomyces cerevisiae ATCC 2601 CECT 1324 NCYC 853 6 R Pseudomonas aeruginosa ATCC 25619 NCIB 10817 1 S Mycobacterium smegmatis ATCC 607 NCTC 7017 7 T Candida tropicalis CIP 1433.83 NCYC 1393 6 1 For candicidine, use method #1

8 Antibiotic Media

Method #5 ardization it is necessary to test every batch of inoculum With the help of a loop inoculate a flask containing to determine the exact volume that must be added to 100 mL of sterile and controlled Antibiotic Medium 3 each assay medium. (Ref. 02-011). from a slant culture. Incubate for 16-18 If the inoculum suspensions are prepared as described hours. in the above sections, they will be homogeneous enough Method #6 to determine the volumes that are going to be added, Follow the directions stated in the Method #1 but in- with tests, without any other previous standardization cubate the slanted subcultures at 30°C for 24 hours, step. and the Roux flasks at 30°C for 48 hours. Diffusion agar Assay Method #7 Prepare a series of flasks with 100 mL of sterilized, Organisms are maintained on Antibiotic Medium 36 melted and cooled Seed Layer Medium (Ref. 01-009) in slanted tubes, subculturing them each week with and add to each flask a different volume of micro- an incubation at 37°C for 48 hours. organisms suspension, as per the volume pattern Collect the growth of a recent subculture with 3 mL suggested in Table IV. of Ringer 1/4 Solution (Ref. 06-073) and use it to Prepare Petri plates with the inoculated medium inoculate a 500 mL Erlenmeyer flask containing 100 according to the specificacions of each antibiotic and mL of Antibiotic Medium 34. Add asseptically to the put a steel or glass cylinder or a disc with the refer- flask 50 g of sterile glass balls and incubate at 27°C ence concentration of antibiotic. Incubate the plates for 5 days with constant stirring (120 rpm and 3,5 cm and measure the inhibition zones. The volume of radius). Keep the suspension in refrigeration. suspension that produces optimum zones, in definition as well as in diameter, is the one that is adopted Inoculum Determination for the inoculum. In Table IV the optimum inhibition For many years the official US/FDA methods provided zones are shown that are supposed to appear in the specific instructions for the inoculum standardization. Af- different assays. ter 1985 the USA Federal Regulations and the USP XIX Turbidimetric Assay adopted the criterion suggested by Kirshbaum, Kramer Proceed in the same way as in the previous case but and Gorth that recommends to dilute the microorgan- instead of inoculating over the melted medium, inoc- isms suspension until it provides a 25% transmitance at ulate in the flasks containing 100 mL of the suitable 580 nm in a 13 mm tube. However, even with this stand- liquid medium, using the volumes pattern specified in

Table II. Buffer solutions and diluents # Buffer1 Formula (in g/L of distilled water)

1 Phosphate buffer 1% at pH 6,0±0,05 K2HPO4 ...... 2,000 g

KH2PO4 ...... 8,000 g

3 Phosphate buffer 0,1M at pH 7,9±0,1 K2HPO4 ...... 16,730 g

KH2PO4 ...... 0,523 g

4 Phosphate buffer 0,1M at pH 4,5±0,05 KH2PO4 ...... 13,600 g

6 Phosphate buffer 10% at pH 6,0±0,05 K2HPO4 ...... 20,000 g

KH2PO4 ...... 80,000 g

10 Phosphate buffer 0,2M at pH 10,5±0,1 K2HPO4 ...... 35,000 g KOH 10N ...... 2,000 mL

16 Phosphate buffer 0,1N at pH 7,0 K2HPO4 ...... 13,600 g

KH2PO4 ...... 4,000 g

17 Phosphate-methanol buffer at pH 6 Methanol ...... 50,000 mL Buffer #1 ...... q.s.

18 Sodium carbonate solution 1% Sodium carbonate ...... 10,000 g

1 May require adjustment with phosphoric acid 18 N or KOH 10N before or after sterilization.

9 Antibiotic Media

Table III . With these volumes of inoculated medium The components of the culture media accomplishes the continue as if you were performing an assay, using speciﬁcations of the several pharmacopeia, however the just the highest and lowest concentrations in relation technician is responsible for the end use of dehydrated to the pattern. Incubate for 3-4 hours and read the culture media or any other variation in it, as long as they absorbances. With all these data you will be able to still have the same characteristics described for the establish the right inoculum that provides a better above media. pH of the medium must be checked when answer between the high and low point, and so adopt it is completely reconstituted and at 25°C. it for the assay. Antibiotic Medium 1 Strain maintenance (Eur. Phar. Antibiotic Medium A Assay strains are maintained by vegetative propagation in slants and in duplicate. One of the duplicates is at pH 6,6) used only for the next subculture, and the other one is used for all the other operations. Medium, if there is no Ref. 01-009 other directive, is Antibiotic Medium 1 (Ref. 01-009) with antibiotics or other additive if necessary. Details may be Speciﬁcation obtained in Table II. Nonetheless, other media may be Antibiotic Medium 1 or Seed Layer is used for the anti- used if considered necessary. biotic assays by the diffusion method in agar, that may be performed in several ways (cylinder, punched-hole or Buffer Solutions and diluents paper disc method).

Composition of the most common buffer solutions and Formula (in g/L) diluents is given in Table II. Dilutions of pure chemical Peptone ...... 6,0 products as well as organic diluents have been om- Casein Peptone ...... 4,0 mited. In anycase, the compounds must be according Yeast extract ...... 3,0 to the purity standards of the corresponding pharmaco- Beef extract ...... 1,5 peia. Water must always be distilled and be of reagent Dextrose ...... 1,0 grade. All buffer solutions and diluents have to be sterile. Agar ...... 15,0 Sometimes a little pH readjustment is necessary after Final pH 6,6 ± 0,2 the sterilization. Directions Culture Media Add 30,5 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Steri- The composition of the several SCHARLAU Microbiol- lize in the autoclave at 121°C for 15 minutes. ogy Antibiotic Media is given below. Media still have the nomenclature according to the US/FDA, which uses the one described by Grove and Randall (media 1 to 13) and Kirshbaum and Arret (media 18 to 21). The Euro- pean Pharmacopeia names the media with alphabetical letters, and the corresponding Scharlau media are as follows:

Medium A ...... Antibiotic Medium 1 and 11 Medium B ...... Antibiotic Medium 10 Medium C ...... Antibiotic Medium C Medium D ...... Antibiotic Medium D Medium E ...... Antibiotic Medium E Medium F...... Antibiotic Medium 19 Medium G ...... Antibiotic Medium 35

Note: Medium D is very similar to Antibiotic Medium 3 but with the addition of 2 g/L of potassium nitrate in it.

SCHARLAU media described following are prepared with the ingredients speciﬁed in the USP 23/NF 18, Eur. Pharm. 3 and 21 CFR for these components, providing an absolute reproducubility and reliability.

10 Antibiotic Media

Antibiotic Medium 2 Formula (in g/L) Peptone ...... 6,0 Ref. 01-010 Yeast extract ...... 3,0 Meat extract ...... 1,5 Speciﬁcation Dextrose ...... 1,0 Agar ...... 15,0 Antibiotic Medium 2 or Basal Layer is used for the anti- Final pH 6,5 ± 0,1 biotic assays by the diffusion method in agar, that may be performed in several ways (cylinder, punched-hole or paper disc method). Directions Add 26,5 g of medium to 1 L of distilled water. Heat to Formula (in g/L) the boiling and dispense into suitable containers. Steri- lize in the autoclave at 121°C for 15 minutes. Peptone ...... 6,0 Yeast extract ...... 3,0 Meat extract ...... 1,5 Antibiotic Medium 5 Agar ...... 15,0 Final pH 6,5 ± 0,2 Ref. 01-013

Directions Specification Add 25,5 g of medium to 1 L of distilled water. Heat to Antibiotic Medium 5 is used in the antibiotic assays by the boiling and dispense into suitable containers. Steri- the diffusion method in agar, which may be performed lize in the autoclave at 121°C for 15 minutes. in several ways (cylinder, punched-hole or paper disc method). Antibiotic Medium 3 Formula (in g/L) Ref. 02-011 Peptone ...... 6,0 Yeast extract ...... 3,0 Specification Meat extract ...... 1,5 Agar ...... 15,0 Antibiotic medium 3 or Antibiotic Assay Broth may be Final pH 7,9 ± 0,1 used in the inoculum preparation, serial dilutions or turbidimetric antibiotic assays. Directions Formula (in g/L) Add 25,5 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Steri- Peptone ...... 5,00 lize in the autoclave at 121°C for 15 minutes. Yeast extract ...... 1,50 Meat extract ...... 1,50 Sodium chloride ...... 3,50 Antibiotic Medium 8 Dextrose ...... 1,00 Monopotassium phosphate ...... 1,32 Ref. 01-014 Dipotassium phosphate ...... 3,68 Final pH 7 ± 0,05 Specification Antibiotic Medium 8 is used in the antibiotic assays by Directions the diffusion method in agar, which may be performed Add 17,5 g of medium to 1 L of distilled water. Heat to in several ways (cylinder, punched-hole or paper disc the boiling and dispense into suitable containers. Steri- method). lize in the autoclave at 121°C for 15 minutes. Formula (in g/L) Antibiotic Medium 4 Peptone ...... 6,0 Yeast extract ...... 3,0 Ref. 01-012 Meat extract ...... 1,5 Agar ...... 15,0 Specification Final pH 5,9 ± 0,1 Antibiotic Medium 4 is used in the antibiotic assays by the diffusion method in agar, which may be performed Directions in several ways (cylinder, punched-hole or paper disc Add 25,5 g of medium to 1 L of distilled water. Heat to method) the boiling and dispense into suitable containers. Steri- lize in the autoclave at 121°C for 15 minutes.

11 Antibiotic Media

Antibiotic Medium 9 Antibiotic Medium 11 (Eur. Phar. Antibiotic Medium A Ref. 01-015 at pH 8,0)

Specification Ref. 01-017 Antibiotic Medium 9 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc Specification method). Antibiotic Medium 11 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc Formula (in g/L) method) Casein peptone ...... 17,0 Soy peptone ...... 3,0 Sodium chloride ...... 5,0 Formula (in g/L) Dipotassium phosphate ...... 2,5 Peptone ...... 6,0 Dextrose ...... 2,5 Casein peptone ...... 4,0 Agar ...... 20,0 Yeast extract ...... 3,0 Final pH 7,2 ± 0,2 Meat extract ...... 1,5 Dextrose ...... 1,0 Agar ...... 15,0 Directions Final pH 8,0± 0,2 Add 50 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Directions Add 30,5 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Steri- Antibiotic Medium 10 lize in the autoclave at 121°C for 15 minutes. (Eur. Phar. Antibiotic Medium B) Antibiotic Medium 13 Ref. 01-016 Ref. 02-544 Specification Antibiotic Medium 10 is used in the antibiotic assays by Specification the diffusion method in agar, which may be performed Antibiotic Medium 13 is used in the turbidimetric antibi- in several ways (cylinder, punched-hole or paper disc otic assays. method). Formula (in g/L) Formula (in g/L) Peptone ...... 10,0 Casein peptone ...... 17,0 Dextrose ...... 20,0 Soy peptone ...... 3,0 Final pH 5,5 ± 0,2 Sodium chloride ...... 5,0 Dipotassium phosphate ...... 2,5 Dextrose ...... 2,5 Directions Add 30 g of medium to 1 L of distilled water. Heat to the Agar ...... 12,0 boiling and dispense into suitable containers. Sterilize in Final pH 7,2 ± 0,2 the autoclave at 121°C for 15 minutes. Directions Add 42 g of medium to 1 L of distilled water. After disso- Antibiotic Medium 19 lution, add 10 mL of Polysorbate 80. Heat to the boiling (Eur. Phar. Antibiotic Medium F) and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Ref. 01-434

Speciﬁcation Antibiotic Medium 19 is used in the antibiotic assays by the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method)

12 Antibiotic Media

Formula (in g/L) Directions Peptone ...... 9,4 Add 33 g of medium to 1 L of distilled water.Add 10 mL Yeast extract ...... 4,7 of glycerol. Heat to the boiling and dispense into suitable Beef extract ...... 2,4 containers. Sterilize in the autoclave at 121°C for 15 Sodium chloride ...... 10,0 minutes. Dextrose ...... 10,0 Agar ...... 23,5 Antibiotic Medium 35 Base Final pH 6,0 ± 0,1 (Eur. Phar. Antibiotic Medium G) Directions Add 60 g of medium to 1 L of distilled water. Heat to the Ref. 01-545 boiling and dispense into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes. Speciﬁcation Antibiotic Medium 35 is used in the antibiotic assays by Antibiotic Medium 32 the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method). Ref. 01-069

This medium is sold by Scharlay Microbiology under the Formula (in g/L) Peptone ...... 10,0 name Sporulating AK Agar. Meat extract ...... 10,0 Sodium chloride ...... 3,0 Speciﬁcation Agar ...... 15,0 Antibiotic Medium 32 is used in the antibiotic assays by Final pH 7,0 ± 0,1 the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method). Directions Add 38 g of medium to 1 L of distilled water containing 10 mL glycerol. Heat to the boiling and dispense into Formula (in g/L) suitable containers. Sterilize in the autoclave at 121°C Peptone ...... 6,000 for 15 minutes. Casein peptone ...... 4,000 Yeast extract ...... 3,000 Meat extract ...... 1,500 Antibiotic Medium 36 Dextrose ...... 1,000 Ref. 01-200 MnSO4.H20 ...... 0,003 Agar ...... 15,000 Final pH 6,5 ± 0,2 Sharlau Microbiology sold this medium as Tryptic Soy Agar (TSA). Directions Add 30,503 g of medium to 1 L of distilled water. Heat to Speciﬁcation the boiling and dispense into suitable containers. Steri- Antibiotic Medium 36 is used in the antibiotic assays by lize in the autoclave at 121°C for 15 minutes. the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc Antibiotic Medium 34 Base method)

Ref. 02-545 Formula (in g/L) Casein peptone ...... 15,0 Soy peptone ...... 5,0 Speciﬁcation Sodium chloride ...... 5,0 Antibiotic Medium 34 is used in the turbidimetric antibi- Agar ...... 15,0 otic assays. Final pH 7,3 ± 0,1

Formula (in g/L) Directions Peptone ...... 10,0 Add 40 g of medium to 1 L of distilled water.Heat to the Meat extract ...... 10,0 boiling and dispense into suitable containers. Sterilize in Sodium chloride ...... 3,0 the autoclave at 121°C for 15 minutes. Final pH 7,0 ± 0,1

13 Antibiotic Media

Antibiotic Medium 39 Formula (in g/L) Casein Peptone ...... 9,00 Ref. 02-547 Yeast extract ...... 5,00 Sodium citrate ...... 10,00 Speciﬁcation Dextrose ...... 20,00 Monopotassium phosphate ...... 1,00 Antibiotic Medium 39 may be used in the inoculum Dipotassium phosphate ...... 1,00 preparation, serial dilutions or turbidimetric antibiotic Final pH 6,8 ± 0,1 assays. Directions Formula (in g/L) Add 46g of medium to 1 L of distilled water. Heat to the Peptone ...... 5,00 boiling and dispense into suitable containers. Sterilize in Yeast extract ...... 1,50 the autoclave at 121°C for 15 minutes. Meat extract ...... 1,50 Sodium chloride ...... 3,50 Dextrose ...... 1,00 Antibiotic Medium C Monopotassium phosphate ...... 1,32 (Eur. Phar. Antibiotic Medium C) Dipotassium phosphate ...... 3,68 Final pH 7,9 ± 0,1 Ref. 02-601

Directions Specification Add 17,5 g of medium to 1 L of distilled water. Heat to Antibiotic Medium C may be used for the inoculum prepa- the boiling and dispense into suitable containers. Steri- ration, serial dilutions or turbidimetric antibiotic assays. lize in the autoclave at 121°C for 15 minutes. Formula (in g/L) Antibiotic Medium 40 Peptone ...... 6,00 Yeast extract ...... 3,00 Ref. 01-546 Beef extract ...... 1,50 Sodium chloride ...... 3,50 Specification Dextrose ...... 1,00 Antibiotic Medium 40 is used in the antibiotic assays by Monopotassium phosphate ...... 1,32 the diffusion method in agar, which may be performed Dipotassium phosphate ...... 3,68 in several ways (cylinder, punched-hole or paper disc Final pH 7,0 ± 0,2 metho Directions Formula (in g/L) Add 20 g of medium to 1 L of distilled water. Heat to the Polypeptone ...... 5,0 boiling and dispense into suitable containers. Sterilize Dextrose ...... 10,0 in the autoclave at 121°C for 15 minutes. If pH 8,0 is Yeast extract ...... 20,0 desired, adjust with NaOH1N. Polysorbate 80 ...... 0,1 Monopotassium phosphate ...... 2,0 Antibiotic Medium D Agar ...... 12,0 (Eur. Phar. Antibiotic Medium D) Final pH 6,8 ± 0,1 Ref. 02-549 Directions Add 49 g of medium to 1 L of distilled water. Heat to the boiling and dispense into suitable containers. Sterilize in Specification the autoclave at 121°C for 15 minutes. Antibiotic Medium D may be used for the inoculum preparation, serial dilutions or turbidimetric antibiotic assays. Antibiotic Medium 41 Formula (in g/L) Heart extract ...... 1,50 Ref. 02-548 Yeast extract ...... 1,50 Casein peptone ...... 5,00 Specification Sodium chloride ...... 3,50 Antibiotic Medium 41 is used in the antibiotic assays by Dextrose ...... 1,00 the turbidimetric method. Monopotassium phosphate ...... 1,32 Dipotassium phosphate ...... 3,68 Potassium nitrate ...... 2,00 Final pH 7,0 ± 0,2

14 Antibiotic Media

Directions Formula (in g/L) Add 19,5 g of medium to 1 L of distilled water. Heat to Peptone ...... 5,00 the boiling and dispense into suitable containers. Steri- Meat extract ...... 3,00 lize in the autoclave at 121°C for 15 minutes. Disodium phosphate ...... 26,90 Agar ...... 12,00 Antibiotic Medium E Final pH 7,9 ± 0,2 (Eur. Phar. Antibiotic Medium E) Directions Add 46,9 g of medium to 1 L of distilled water. Heat to Ref. 01-430 the boiling and dispense into containers. Sterilize in the autoclave at 121°C for 15 minutes. Precipitates ap- Speciﬁcation pearing after sterilization do not interfere in its normal Antibiotic Medium E is used in the antibiotic assays by operation. the diffusion method in agar, which may be performed in several ways (cylinder, punched-hole or paper disc method).

Table III. Speciﬁcations for the antibiotic master standard solutions

Drying cond. Initial solvent Master soln Days (cool) Final solvent

Amikacin 6 Distilled water 1,0 mg 15 Distilled water Amphotericin B1 1 Dimethylsulfoxide 1,0 mg 1 10 Bacitracin 1 1 100,0 ui 7 1 Bleomycin 7 16 2,0 ui 15 16 Carbenicillin None 1 1,0 mg 15 1 Cephalothin 1 1 1,0 mg 5 1 Cycloserine 1 Distilled water 1,0 mg 30 1 Chloramphenicol None Ethanol 1,0 mg 30 1 Chlortetracycline None HCl 0,01N 1,0 mg 4 4 Cloxacillin None 1 1,0 mg 7 1 Colistimethate Na 1 Distilled water 1,0 mg 1 6 Colistin 1 Distilled water 1,0 mg 15 6 Demeclocycline None HCl 0,01N 1,0 mg 5 4 Dihydrostreptomycin 5 3 1,0 mg 30 3 Doxycycline None HCl 0,01N 1,0 mg 5 Distilled water Erythromycin 1 Methanol 1,0 mg 14 3 Framicetine 3 Distilled water 1,0 mg 7 3 Gentamicin 3 3 1,0 mg 30 3 Gramicidin 1 Ethanol 1,0 mg 30 Ethanol

Kanamycin SO4 None 3 1,0 mg 30 3 Methacycline None HCl 0,01 N 1,0 mg 15 4 Methylmicin None 3 1,0 mg 7 3 Nafcillin None 1 1,0 mg 2 1 Natamycin 4 Dimethylsulfoxide 1,0 mg 1 10 Neomycin 1 3 1,0 mg 15 3 Novobiocin 5 Absolute Ethanol 1,0 mg 5 6 Nystatin 4 Dimethylformamide 1000,0 ui 1 6 Oxytetracycline None HCl 0,1N 1,0 mg 4 4 Paromomycin 1 3 1,0 mg 21 3 Penicillin G None 1 1000,0 ui 2 1 Polymyxin B 1 Distilled water 10000,0 ui 15 6 Rifampin None Methanol 1,0 mg 1 1 Rolitetracycline None Distilled water 1,0 mg 1 Distilled water Sisomycin None 3 1,0 mg 15 3 Spectinomycin 6 Distilled water 1,0 mg 30 Distilled water Spiramycin 1 Methanol 1,0 mg 7 3 Streptomycin 1 3 1,0 mg 30 3 Tetracycline None HCl 0,1N 1,0 mg 1 4 Ticarcillin None 1 1,0 mg 1 1 Thiostreptone None Dimethylsulfoxide 1,0 ui 1 Dimethylsulfoxide Tobramycin 3 Distilled water 1,0 mg 15 16 Troleandomycin None Isopropanol 1,0 mg 1 Distilled water Vancomycin 1 Distilled water 0,4 mg 7 4

1 Use amber glass or work under red light

15 Antibiotic Media

Reference Standard Diffusion Method in Agar (Plate assay) Each antibiotic must be assayed with a proper reference standard or master standard pattern. Patterns may Once we decide the assay conditions (tabulated in be obtained from the USP NF Convention Inc. or other Table IV), we can prepare the plates as per the following organisms, such as: method . European Pharmacopeia Secretariat, B.P. 907, F-67029 Strasbourg. CEDEX 1. Fax +33 388 41 20 36. e-mail:10 [email protected] Plate preparation ..Pour basal medium, melted and cooled to 50°C, into USP-NF Reference Standards, 12601 Twinbrook Park- each plate, and let it solidify horizontally. Using 10 cm. way, Rockville. Md. 20852, USA diameter plates, suggested volumes are 21 mL for each British Pharmacopoeia Comission Laboratory, Govern- basal layer and 4 mL for the seed layer. Nonetheless, in ment Buildings, Block 2, Honeypot Lane, Stan- the case of amphotericin and nystatin a single seed more, Middlesex, HA7 1A4. England. layer of 8 mL is used, without any basal layer. Another exception to this rule is bleomycin, for which a 10 mL of However, you may use domestic or working standards basal layer and a 6 mL of seed layer is used. In practise, if they have been compared to a reference pattern. In when the standard 9 cm diameter plastic plates are used Table III you may find details about the reference stand- it is advisable to use the volumes of about 15 mL for the ard: dessication conditions, initial diluent to obtain the basal layer and 4 mL for the seed layer, in order to avoid mother solution, activity period,etc... the contact between the cylinders and the plate cover. Following are described the dessication methods for the While the basal layer solidifies, adjust the temperature standards. US/FDA numeration have been maintained. for the medium of seed layer to 45-48°C, and inoculate with the suggested (or tested) volume of inoculum US/FDA Method #1 suspension. Homogenize by gentle stirring and carefully In vacuum oven, dry the pattern or master reference pour the medium over the basal layer in 4 mL volumes, standard at 60°C for 3 hours, at pressure 5 mm Hg. except for the cases mentioned above. Be very careful in this operation, since the final results may be affected US/FDA Method #3 if the layer is not very homogeneous, and forms bubbles In vacuum oven, dry the pattern or master reference or clots. Once the agar solidifies, place the cylinders or standard at 40°C for 3 hours, at pressure 5 mm Hg. make the holes. Put or make 6 cylinders or holes per plate, centered in the plate at 60° angle from each other. US/FDA Method #4 In vacuum oven, dry the pattern or master reference standard at 40°C for 2 hours, at pressure 5 mm Hg. Reference pattern preparation and standard curve US/FDA Method #5 Master pattern is prepared according to the specifica- In vacuum oven, dry the pattern or master reference tions in Table IV, starting from the stock solution and standard at 100°C for 4 hours, at pressure 5 mm Hg. using the adequate diluent until the desired concentration is obtained. Reference level is always the medium US/FDA Method #6 concentration among the 5 concentrations used to obtain In vacuum oven, dry the pattern or master reference the standard curve (see Table IV). standard at 45°C for 3 hours, at pressure 5 mm Hg . Assay technique US/FDA Method #7 To obtain the curve, 12 plates are used, three for each In vacuum oven, dry the pattern or master reference level, except for the reference standard, which is in- standard at 35°C for 4 hours, at pressure 5 mm Hg . cluded in all. In each batch of three plates, 3 alternate holes or cylinders are filled with the reference concentration and the other three are filled with the corresponding concentration. By proceeding in this way we can obtain 30 inhibition halos or inhibition zones corresponding to the reference concentration and 9 halos for each one of the other assay standards. Three plates, with 6 holes or cylinders, are used in each test. Holes or cylinders are filled alternatively with the reference level and the diluted sample. Plates are incubated at appropriate temperature (see Table IV) for 16-18 hours. After this period, inhibition halo readings are performed using the proper measuring instrument.

16 Antibiotic Media

Table IV. Agar diffusion test speciﬁcations

Medium Final concentrations: assay levels for the standard curve in Assay Maintenance Inoculum Incubation IU/mL or in mcg/mL Reference Antibiotic strain Medium (mL/100 mL Basal Seed Temperat. halo (Table I) (A.M. nº) medium) strain strain (°C) Ref. (mm)

Amphotericin B P 19 1,00 - 19 30 0,640 0,800 1,000 1,250 1,560 16-18 Bacitracin F 1 0,30 9 10 32-35 0,640 0,800 1,000 1,250 1,560 16-18 Bleomycin S 36 1,00 35 35 32-35 0,010 0,020 0,040 0,080 0,160 18-20 Carbenicillin R 1 0,50 9 10 37 12,800 16,000 20,000 25,000 31,200 16-18 Cephalothin A 1 0,10 2 1 32-35 0,640 0,800 1,000 1,250 1,560 14-16 Cloxacillin A 1 0,10 2 1 32-35 3,200 4,000 5,000 6,250 7,810 18-20 Colistimethate sodium O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 13-15 Colistin O 1 0,10 9 10 37 0,640 0,800 1,000 1,250 1,560 15-17 Dihydrostreptomycin J 1 0,05 5 5 37 0,640 0,800 1,000 1,250 1,560 14-16 Doxycyclin I 1 0,40 8 8 30 0,640 0,800 1,000 1,250 1,560 18-20 Erythromycin E 1 1,50 11 11 32-35 0,640 0,800 1,000 1,250 1,560 17-19 Framicetin J,H 1 0,40 1 1 35-37 0,640 0,800 1,000 1,250 1,560 14-16 Gentamicin D 1 0,03 11 11 37 0,640 0,800 1,000 1,250 1,560 15-17 Nafcillin A 1 0,30 2 1 32-35 1,280 1,600 2,000 2,500 3,120 14-16 Natamycin P 19 1,00 19 19 28-30 3,200 4,000 5,000 6,250 7,810 17-19 Neomycin A,H,J 1 0,40 11 11 32-35 6,400 8,000 10,000 12,500 15,600 16-18 Netilmycin D 1 0,25 11 11 32-35 0,064 0,080 0,100 0,125 0,156 14-16 Novobiocin D 1 4,00 2 1 35 0,320 0,400 0,500 0,625 0,781 16-18 Nystatin Q 19 1,00 - 19 30 12,800 16,000 20,000 25,000 31,200 16-18 Paromomycin D 1 2,00 11 11 37 0,640 0,800 1,000 1,250 1,560 19-21 Penicillin G A 1 1,00 2 1 32-35 0,640 0,800 1,000 1,250 1,560 17-19 Polymyxin O 1 0,10 9 10 37 6,400 8,000 10,000 12,500 15,600 15-17 Rifamycin J 1 0,10 2 2 30 3,200 4,000 5,000 6,250 7,810 15-17 Sisomycin D 1 0,03 11 11 32-35 0,064 0,080 0,100 0,125 0,156 15-17 Spiramycin J 1 0,20 1 1 35-37 0,640 0,800 1,000 1,250 1,560 15-17 Ticarcillin B 1 0,40 8 8 32-35 3,200 4,000 5,000 6,250 7,810 17-19 Vancomycin J 1 0,20 8 8 37 6,400 8,000 10,000 12,500 15,600 16-18

Potency calculation To extrapolate from the standard curve, use the average To estimate the potency of the test sample, calculate of the 36 diameters of the reference level and the sev- average of the inhibition halo diameters and the refer- eral batches of 9 halos obtained in each assay level. The ence and sample levels of the three plates, and perform average of the 36 halos of reference is the correction the corrections related to the correction point. Corrected value of the standard curve, and thus we can achieve value of the problem is projected on the standard curve each point as the function of the average value of the and thus the theoritical concentration is obtained. Theo- reference values of the corresponding standard. ritical concentraion multiplied by the dilution factor gives Values obtained in this way are plotted over a semiloga- the real concentration of antibiotic in the sample. rithmic graph paper, drawing the antibiotic concentration (in mcg/mL) on X axis, and the halo or zone diameters Turbidimetric assay on the Y axis. Standard curve is drawn by linking the five points or by tracing a straight line between the high Reference pattern preparation and and low point, which are calculated using the following statistical formulae: standard curve Once the assay conditions are determined as per Table 3a+2b+c-e 3e+2d+c-a III, prepare the pattern according to the specifications in A = B = Table V. At the time of assay experiment, take an aliq- 5 5 uote of mother solution and prepare appropriate dilutions to obtain the desired concentrations. Reference level is where: the medium concentration between the five proposed for A: Halo diameter, calculated for the highest standard the standard curve. concentration. B: Halo diameter, calculated for the lowest pattern concentration. c: Reference halo diameter, average of the 36 halos of the pattern plates. a,b,d,e: Corrected average values of the other pattern assay levels,from the highest to the lowest order.

17 Antibiotic Media

Table V. Turbidimetric assay speciﬁcations

Final concentrations: assay levels for the pattern curve in IU/mL or Assay strain Inoculum Assay Incubation mcg/mL Antibiotic (Table I) (mL/100 mL medium tempera- medium) ture Ref. (°C)

Amikacin A 0,50 3 32 6,000 8,000 10,000 12,500 15,600 Candicidin P 0,20 13 29 0,030 0,042 0,060 0,084 0,120 Capreomycin L 0,05 3 37 64,000 80,000 100,000 125,000 156,000 Chloramphenicol M 0,70 3 32 2,000 2,240 2,500 2,800 3,120 Chlortetracycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094 Cycloserine A 0,40 3 32 32,000 40,000 50,000 62,500 78,100 Demeclocycline A 0,10 3 32 0,064 0,080 0,100 0,125 0,156 Dihydrostreptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500 Doxycyclin A 0,10 3 32 0,064 0,080 0,100 0,125 0,156 Gramicidin G 1,00 3 37 0,028 0,034 0,040 0,048 0,057 Kanamycin B 0,20 3 32 8,000 8,900 10,000 11,200 12,500 Methacycline A 0,10 3 32 0,038 0,048 0,060 0,075 0,094 Neomycin L 2,00 39 37 0,640 0,800 1,000 1,250 1,560 Oxytetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390 Rolitetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390 Spectinomycin M 0,10 3 32 24,000 26,800 30,000 33,500 37,500 Streptomycin L 0,10 3 37 24,000 26,800 30,000 33,500 37,500 Tetracycline A 0,10 3 32 0,160 0,200 0,250 0,312 0,390 Thiostreptone G 0,20 3 37 0,400 0,600 0,800 1,000 1,250 Tobramycin A 0,15 41 32 2,000 2,236 2,500 2,795 3,125 Troleandomycin L 0,10 3 32 16,000 20,000 25,000 31,200 39,000

Assay Technique where: The assay is performed in the following way: A: Absorbance value, calculated for the highest value of the pattern curve. Prepare 3 tubes and put 1 mL (or 0,1 mL if the antibiotic B: Absorbance value, calculated for the lowest value of is gramicidin or tyrothricin) of every assay level of stand- the pattern curve. ard and sample. Thus, 15 tubes will be employed for the a,b,c,d,e: Average values for each value in the triplicate pattern and 3 tubes for each problem. standard curve, from the highest to the lowest Add asseptically 9 mL of inoculated assay broth to concentration. each tube, and put them into a boiling water bath for 2-4 hours. The exact incubation time is determined by Potency estimation is performed by taking the average observing the growth inside the tube, taking the standard value of each assay level in the pattern curve over a tube as the reference. semilogarithmic paper. Draw the absorbance over the Tubes are removed from the boiling water bath and logarithmic scale and concentration over the abscissa. 0,5 mL of 12% formaldehyde is added in each tube in If the calculated values are used, the result is a straight order to stop the growth. Growth in the form of turbidity line, but a curve may be drawn by linking the ﬁve points. is measured by a Spectrophotometer at the wavelength The absorbance average value of the test sample is pro- 530 nm. The zero is ﬁxed by the device and 100% trans- jected over the standard curve, obtaining in this way the mission is provided by a blank containing only the sterile theoritical concentration which, multiplied by the dilution medium and formaldehyde at the same proportion or factor, gives the real potency of the test sample. concentration as that of the assay. Cross Contamination Potency calculation with Penicillin Statistical values corresponding to the highest and lowest concentrations are determined according to the To perform this assay all the instrumentation and even following formulae: the environment must be free of penicillin. Plates are prepared following the general method, using 10 mL of 3a+2b+c-e 3e+2d+c-a A = B = Antibiotic Medium 1 (Ref. 01-009) in the basal layer and 5 5 4 mL of Antibiotic Medium 4 in seed layer and inoculating the C strain of microorganism (see Table I).

18 Antibiotic Media

Reference standard is prepared according to the stand- with the pattern reference level. Fill another 2 holes or ard for penicillin, proposed in Table II, but taking the cylinders in each plate with the untreated sample, and mother solution of 100 units and the final concentrations the last 2 holes or cylinders in each plate with the treated at 0,0005;0,0125; 0,0050; 0,100 and 0,200 penicillin sample. units. Reference is the one with final concentration 0,05 Plates are incubated at 30°C for overnight. Presence units (see Table III). of inhibition halos in the untreated sample, and the absence of holes in the treated sample are a sign of Sample is prepared by dissolving 1 g in 18 mL of distilled penicillin contamination. Should a qualitative estimation water. From this initial solution, take 9 mL into a separat- of penicillin be desired, measure the halos and proceed ing funnel, and add 20 mL of amyl acetate. Add then 1 as in the normal penicillin assay. mL of Solution #11 (see Table II) and stir it vigorously. Let it rest and once the two layers are separated, take Special lab requirements the aqueous layer to another separating funnel. Check the pH of the solution and if it is higher than 3, readjust The instruments used in all these assays must be to 2,5 with HCl. Extract again with amyl acetate and washed carefully before and after each test, in order to discard the aqueous layer. remove all the traces of antibiotic. Mix the two parts of amyl acetate, and wash them with To sterilize lab commodities, cover them well and use a 10 mL of Solution #2 (see Table II). Discard the aqueous hot air oven at 200-220°C for 2 hours. Volumetric flasks , layer. Extract penicillin from the amyl acetate with 10 mL pipets, ... must be washed carefully. of phosphate buffer pH 6 (Solution #1, Table II). Penicillin presence is determined in this solution. Use 15 plates to perform the assay: three for each standard curve assay, except for the reference standard, which is present in all of them. Place 6 holes or cylinders in each plate and fill them alternatively with the reference solution and the corresponding assay level. In this way, you will obtain 45 inhibition halos for the reference level and 9 halos more for each one of the other levels. A part of sample (2-5 mL) is treated with 0,1 mL of peni- cillinase and incubated at 37°C for 1 hour. Use 3 plates for each sample, and fill 2 holes or cylinders in each one

Plate external Ø 9 mm Cylinder ext. Ø 6 mm Cylinder int. Ø 8 mm

Figure 1. Holes, discs or cylinders at 90° Figure 2. Holes, discs or cylinders at 60°

19 Antibiotic Media

Scharlau Chemie may supply all the necessary instru- Colourimeter / Spectrophotometer ments to perform antibiotic assays, which is detailed It must be able to read at 530 nm, have its own zero below: adjustment and 100% transmission can be determined with a sterile medium solution at the same Volumetric material conditions of assay. If it is possible, use guaranteed, checked and class A glass. References Cylinders ARRET, B. DIANA, P. JOHNSON y A. KIRSHBAUM Cylinders must be made of stainless steel. Exterior (1971) Outline of Details for Microbiological Assays diameter: 8 mm, interior diameter: 6 mm, height: 8-10 of Antibiotics: Second Revision. J. PHARM, Sci. mm. 60,11,1689-1694. Holes EUROPEAN PHARMACOPEIA (1997) 3rd Ed. 2.7 Bio- Holes should be made with a suitable punching logical Assays. 2.7.2 Microbiological Assay of Antibiotics. instrument of the cylinder dimension. Council of Europe. Strasbourg. Discs SANCHO, GUINEA, PARES. (1980) Microbiología Use normalized paper ﬁlter discs, 6 mm diameter. Analítica Básica. Ed. JIMS. Barcelona, Plates U.S. PHARMACOPEIA XIX (1975) Antibiotic Assays. Use plastic or glass plates. If glass plates are em- U.S./F.D.A.: 21 CFR (1976 and following) 436.100 and ployed, they must be washed and sterilized. following. Tubes U.S. PHARMACOPEIA XX/National Formulary XV All the tubes used in one assay must be equal and (1980) Antibiotic Assays. uniform in their dimensions. U.S. PHARMACOPEIA 23/National Formulary 18 (1995) Biological Tests and Assays. {81} Antibiotics Microbial Assays. U.S. PHARMACOPEIA 25/National Formulary 20 (2002) Biological Tests and Assays. {81} Antibiotics Microbial Assays. EUROPEAN PHARMACOPEIA, Supplement (2002), 4th Ed.,Council of Europe, Strasbourg,

20 APT Media

APT Agar Directions Dissolve 46,2 g of powder into 1 L of distilled water, Ref. 01-026 heating up slightly if necessary. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 Specification minutes. Solid medium for general purposes, especially designed for the cultivation of the heterofermentative lactic acid Description bacteria that causes meat greening. These two general purpose media (APT= All Purpose with Tween), originally formulated by Evans and Niven, Formula (in g/L) have been used successfully for the isolation and cultivation of lactic acid bacteria that alter the food quality Casein Peptone ...... 12,500 and composition (especially meat) and require a high Yeast Extract ...... 7,500 thiamine level. For this reason, ignoring what peptone Sodium Chloride ...... 5,000 and yeast extract could provide, the medium has been Dipotassium Phosphate ...... 5,000 complemented with an extra amount of thiamine. Trisodium Citrate ...... 5,000 Both versions, solid and liquid, have demonstrated their Dextrose ...... 10,000 efficacy for detection of lactobacilli that produce meat Magnesium Sulfate ...... 0,800 greening. Moreover, if media are supplemented with Manganous Chloride ...... 0,140 5% fruit juices, as APHA states, they are converted into Iron Sulfate ...... 0,040 irreplaceable prospection media for any kind of food Thiamine HCl ...... 0,001 biomodifier. Polysorbate 80 ...... 0,200 However, without the inclusion of any inhibitory agent in Agar ...... 15,000 the formulation signifies that the media have no selective Final pH 6,7 ± 0,2 ability, and thus they can support the growth of almost all the microbial types. Directions Suspend 61,2 g of powder into 1 L of distilled water and Technique let it soak . Heat to boiling with constant stirring. Distrib- Usual detection technique for the bacteria those causes ute in suitable containers and sterilize in the autoclave at the greening is as mentioned below: 121°C for 15 minutes. Products to be examined are crushed carefully in Tryp- tone Water (Ref. 03-156), and with the same diluent a dilution bank is prepared. From each dilution, APT Agar APT Broth plates are inoculated in mass and in triplicate, and they are incubated at 32°C for 48 hours. After the incubation Ref. 02-026 period, colonies are counted using usual technique, and different types are selected. Every type is inoculated Specification in APT Broth, and is incubated at 32°C for 24 hours or Liquid version of the medium with the same name. It more. From these pure cultures, streak on Frankfurt is especially designed for the cultivation of lactic acid sausages slices, and incubate those slices and also the bacteria. one without the culture as a Control, in a humid room or atmosphere to verify its greening capacity. Final Formula (in g/L) identification is done by morphological and biochemical characteristics. Casein Peptone ...... 12,500 Yeast Extract ...... 7,500 Sodium Chloride ...... 5,000 References Dipotassium Phosphate ...... 5,000 EVANS, J.B. & C.F. NIVEN (1951) Nutrition of the het- Trisodium Citrate ...... 5,000 erofermentative lactobacilli that cause greening of cured Dextrose ...... 10,000 meat products J.Bact. 62:599 Magnesium Sulfate ...... 0,800 DEIBEL, R.H, J.B. EVANS & C.F. NIVEN (1957) Micro- Manganous Chloride ...... 0,140 biological assay for the thiamin using Lactobacillus Iron Sulfate ...... 0,040 viridescens. J. Bact. 74:818-821 Thiamine HCl ...... 0,001 DOWNES, F.P., K. ITO (2002) Compendium of methods Polysorbate 80 ...... 0,200 for the microbiological examination of food. 4rd. ed. Final pH 6,7 ± 0,2 APHA. Washington.

21 Asparagine Broth

Ref. 02-271 Description Asparagine medium is recommended for the microbio- Specification logical analysis of bottled water. This is an excellent Liquid medium for the presumptive assay and enumera- enrichment medium for Pseudomonas aeruginosa, since tion of Pseudomonas aeruginosa in bottled water by it is composed of a mineral base and the only carbon MPN method. source is asparagine. It may also be used in the multiple tube technique in microbiological analysis of recreational Formula (in g/L) waters and as a presumptive test medium for the differentiation of non fermentative gramnegative bacteria. Asparagine ...... 2,0 Technique Dipotassium phosphate ...... 1,0 Some standards suggests the viable enumeration by Monopotassium phosphate ...... 10,0 MPN method with 5 tubes per series, inoculating 10 mL, Magnesium sulfate ...... 0,5 1 mL and 0,1 mL. All the tubes are incubated at 37°C Final pH 7,0 ± 0,2 for 48 hours. Growth, with or without pigmentation, is estimated as a presumptive evidence of presence of Directions Pseudomonas aeruginosa. Enumeration is carried out Dissolve 13,5 g of powder in 1 L of distilled water con- with MPN tables for 5 tubes (See MPN chapter in this taining 8 mL of glycerol. Sterilize by filtration and dis- book). tribute in tubes (10 mL/tube). To obtain broth of double Confirmation is performed subculturing a loop of each strength, dissolve 27 g of powder in 1 L of distilled water tube in Acetamide Medium (Ref. 03-428). containing 16 mL of glycerol. References PASCUAL ANDERSON, M.R. (1992) Microbiología Alimentaria. Diaz de Santos. Madrid.

22 Azide Dextrose Media

Azide Dextrose Broth Directions

Xn Dissolve 35,6 g in 1 L of distilled water. Heat if neces- Ref. 02-343 sary to help dissolution. Divide into 10 mL portions and R-22-32-52/53 pour into tubes. Sterilize by autoclaving at 121°C for 15 S-7-46-61 Specification minutes. As for the double strength medium, dissolve 71,2 g/L and proceed as indicated above. Liquid and selective medium for the detection of streptococci in water, waste water and milk. Description Formula (in g/L) The Azide Dextrose Broth acc. to Rothe has been widely used since 1948 for the detection of fecal streptococci. It Meat extract ...... 4,5 usually provides higher positive results than other similar Tryptose ...... 15,0 media. Its efficiency is due to the Sodium azide, which Dextrose ...... 7,5 is both selective for enterococci and inhibitor of the ac- Sodium chloride ...... 7,5 companying flora through interference of the electron Sodium azide ...... 0,2 transport chain. Final pH 7,2 ± 0,2 This medium is also used for the primary enrichment of food samples, particularly frozen vegetables. Directions Dissolve 35 g of powder in 1 L of distilled water, heat if Technique necessary. Distribute into tubes and sterilize in the auto- Streptometry in Water clave at 121°C for 15 minutes. To make double strength Add 10 mL of water to be examined to each one of three broth, dissolve 70 g of powder in 1 L of distilled water. tubes containing 10 mL of double strength medium. Add 1 mL of sample to another three tubes containing Description 10 mL of medium each, of single strength. Then add 0.1 Azide Dextrose Broth is formulated according to the mL of water to each one of three tubes containing 10 “Standard Methods for the Examination of Water and mL of medium of single strength. Incubate at 37° C and Wastewater”. examine after 24 hours and 48 hours. All tubes which Azide concentration in the medium restrains the growth show turbidity due to growth will be considered as PRE- of gramnegative bacteria and lets a good growth of en- SUMPTIVLY POSITIVE and will have to be confirmed on terococci. This medium composition is very similar to the EVA Broth (Ref.2-028). All tubes which result positive on Azide Dextrose Broth acc. to Rothe (Ref. 02-027), but this second testing should be considered for the count without phosphate buffering. Therefore, it is advisable to of the Most Probable Number (MPN). use this medium with samples with a high salts concen- When considering other type of samples, dilute them in tration, to avoid any precipitation in the medium. Ringer solution 1/4 or peptone water and then inoculate the tubes as done before. Azide Dextrose Broth acc. to In case of the highly contaminated samples, dilutions Rothe (Rothe Azide Broth) should be done before inoculation.

Xn Ref. 02-027 References R-22-32-52/53 APHA-AWWA-WPCF (1980) Standard Methods for the S-7-46-61 Examination of Water and Wastewater. 15th. Ed. APHA Speciﬁcation Inc., Washington, D.C. Medium for the detection and enumeration of entero- CLESCERI, L., A.E. GREENBERG, A.E. EATON (1998) cocci in water. Standard Methods for the Examination of Water and Wastewater. APHA-AWWA-WEA. Washington. Formula (in g/L) GUINEA, SANCHO y PARÉS. (1979) Análisis Meat peptone ...... 10,0 Microbiológico de Aguas: Aspectos Aplicados. Ed. Casein peptone ...... 10,0 Omega,Barcelona,. Dextrose ...... 5,0 ROTHE (1948) Illinois State Health Department. Sodium chloride ...... 5,0 DOWNES, F.C. & K.ITO (2001) Compendium of Meth- Dipotassium hydrogen phosphate ...... 2,7 ods for the Microbiological Examination of Food. 4th ed. Potassium dihydrogen phosphate ...... 2,7 APHA. Washington. Sodium azide ...... 0,2 Final pH 7.0 ± 0,2

23 Bacillus cereus Media

Bacillus cereus Agar (MYP Agar) Sometimes,the confusion with other colonies of grampositive bacilli is possible, and hence to identity this, confirmation has to be performed verifying the glucose Ref. 01-262 fermentation, gelatin degradation and nitrate reduction, which are positive tests for Bacillus cereus. Specification Selective solid medium, according to Mossel, for the References isolation and identification of Bacillus cereus from food ATLAS, R.M. & L.C. PARKS (1993) Handbook of samples, acc. ISO 7932 (2003) and ISO 21871:2006. Microbiological Media. CRC Press. Londres. CORRY, J.E.L., G.D.W. CURTIS & R.M. BAIRD. (2003) Handbook of Culture Media for Food Microbiology. Elsevier Formula (in g/L) Sci. B.V. Amsterdam. The Netherlands. Peptone ...... 10,000 DOWNES, F.P. & K. ITO (2001) Compendium of methods Mannitol ...... 10,000 for the microbiological examination of foods. 4th ed. APHA. Sodium chloride ...... 10,000 Washington DC. USA. Meat extract ...... 1,000 FIL-IDF 181:1998 Provisional Int. Standard. Dried Milk Phenol red ...... 0,025 Products. Enumeration of Bacillus cereus.- Most probable Agar ...... 15,000 number technique Final pH 7,2 ± 0,2 ISO 7932 Standard (2004) 3rd ed. Microbiology of food and animal feeding stuffs. Horizontal method for the Directions enumeration of presumptive Bacillus cereus. Colony count Suspend 46 g of powder in 900 mL of distilled water. technique at 30ºC. Let it soak and bring to boil. Sterilize in the autoclave at ISO 21871 Standard (2006) Microbiology of food 121°C for 15 minutes. Let it cool to 50°C and then add and animal feeding stuffs.- Horizontal method for the 100 mL of Egg’s Yolk Sterile Emulsion (Ref. 06-016) and determination of low numbers of presumptive Bacillus 100 mg/L of Polymyxin (Ref. 06-021CASE). Homogenize cereus.- Most probable number technique and detection well and distribute into plates. Do not reheat or remelt method. the complete medium. MOSSEL, D.A.A., KOOPMAN. M.J. y JONGERIUS, E. (1967) Enumeration of Bacillus cereus in foods. Appl. Description Microbiol. 15:650-653. Mossel’s formulation is developed to detect and enu- PASCUAL ANDERSON, Mª.Rª (1992) Microbiología merate B.cereus in any kind of food, since it lets a good Alimentaria. Díaz de Santos, S.A. Madrid. differentiation and selection of these microorganisms. Polymyxin addition inhibits most of accompanying bacteria, but it does not affect the growth of B.cereus. Bacillus cereus Selective Agar (PEMBA) This bacteria do not ferment mannitol and thus there is no change in the indicator around the colonies. On other Ref. 01-487 hand, due to lecithinase activity of B.cereus it produces a halo or zone of white precipitate around the colonies. A count of B.cereus over 100.000 cells/g of the food Specification sample is considered to be hazardous, since the ac- Selective solid medium for the enumeration of Bacillus cumulated phosphoril-choline may cause intoxication cereus in food, according to ISO 21871. symptoms in children. For this reason, besides common isolation and identification, a viable enumeration must be Formula (in g/L) performed to evaluate the real population of cells. Peptone ...... 1,00 Technique Mannitol ...... 10,00 According to the authors, dehydrated or dry samples Sodium chloride ...... 2,00 must follow a recovery process in the following way: 20 Magnesium sulfate ...... 0,20 g of sample is mixed with 90 mL of Tryptone Water (Ref. Disodium phosphate ...... 2,50 03-156) for a minimum period of 1 hour, at room tem- Potassium phosphate ...... 0,25 perature. Afterwards, add 90 mL more of Tryptone Water Brom thymol blue ...... 0,12 and homogenize it. A dilution of 1:10 should be obtained. Sodium pyruvate ...... 10,00 If necessary, a decimal dilution bank using Tryptone wa- Agar ...... 14,00 ter as the diluent can be prepared. With a Drigalsky loop Final pH 7,2 ± 0,2 (Ref. 5-010), spread aliquotes of 0,1 mL over the surface of the agar plates and let the agar medium absorb those aliquots. Incubate the plates at 30°C for 18-24 hours to Directions allow spore germination before giving definite results, Suspend 40 g of powder in 950 mL of distilled water. Let which are referred as B. cereus colonies per gram of it soak and bring to the boiling. Distribute into suitable sample. containers and sterilize by autoclaving at 121ºC for 15 Suspected colonies show the following appearance: minutes. Let it cool to 50ºC and then add 50 mL/L of irregular borders, pink colour and even purple in the Egg’s Yolk Sterile Emulsion (Ref. 06-016) and Polymyxin center, and with a halo of white precipitate. Colonies B Sulfate (Ref. 06-021CASE) to reach a 100 U/mL con- with yellow halos must be discarded surely. centration. Homogenize and pour into plates.

24 Bacillus cereus Media

Description determination of low numbers of presumptive Bacillus Bacillus Cereus Selective Agar is formulated according cereus.- Most probable number technique and detection to the Food Analysis Nordic Committee (NMLK) stand- method. ards. This standard uses this medium and Blood Agar Base (Ref. 01-352) simultaneously for the detection and enumeration of B. cereus in any type of food. This me- Ref. 01-262 Bacillus cereus Agar dium can also be used to conﬁrm doubtful colonies, and in this last case Polymyxin addition may be ommitted.

Technique NMLK proposes the simultaneous use of Bacillus cereus Selective Agar and Blood Agar Base (Ref. 01-352). Both media are inoculated by surface streaking with 0,1 mL aliquotes which are spread with a Drigalsky loop. Both series of plates are incubated at 30ºC for 24 hours. Typical B.cereus colonies over Blood Agar are big, irreg- Control ular, dirty white or grey-like colour with a hemolysis halo surrounding them. In B.cereus Selective Agar, colonies are blue, surrounded by a clear zone of egg yolk digestion (lecithinase positive). If there is an equal amount of typical or similar colonies in both the media, later conﬁrmation is not necessary.

References ISO 21871 Standard (2006) Microbiology of food and animal feeding stuffs.- Horizontal method for the Bacillus cereus ATCC 10876

Baird Parker Agar Base (Eur. Phar. Medium O)

Ref. 01-030 accompanying bacteria is usually suppressed by the high concentration in lithium, glycine and pyruvate. Lithium Speciﬁcation and glycine enhances the growth of staphylococci. Even Solid and selective culture medium for the screening of if its high selectivity does not affect staphylococci it may staphylococci from a variety of samples, acc. to pharma- occasionally permit the growth of some Bacillus species, copeias and ISO standard. yeast and very rarely, Proteus. The growth of Proteus species can be suppressed by adding 50 mg/l of sul- Formula (in g/L) phamethazine. Tryptone ...... 10,0 The presence of tellurite and egg’s yolk, which must Sodium pyruvate ...... 10,0 always be added to the medium after sterilization, allows Glycine ...... 12,0 the differentiation of presumptly pathogenic staphylococ- Meat extract ...... 5,0 cal colonies. A high correlation has been found between Lithium chloride ...... 5,0 the coagulase test and the presence of clearing zones Yeast extract ...... 1,0 of lypolysis in this medium, which is due to the staphylo- Agar ...... 17,0 coccal lecithinase. On the other hand, studies show that Final pH 7,0 ± 0,2 almost 100% of coagulase-positive staphylococci are capable of reducing tellurite, which produces black colo- Directions nies, whereas other staphylococci can not always do so. Suspend 60 g in 950 mL of distilled water. Allow it to When using sterile reagents other than SCHARLAU soak and then bring to the boil with constant stirring. MICROBIOLOGY, the complete medium may be ob- Sterilize by autoclaving at 121° C for 15 minutes. Cool to tained by adding 50 mL sterile egg’s yolk and 10 mL of 50° C and add 50 mL of Egg’s Yolk Tellurite Sterile Emul- 1% potassium tellurite solution. Plates should be used sion (Ref. 06-026). Homogenize on the same day of preparation or within 48 hours, to and distribute into plates. Once prepared, the medium avoid the loss of deﬁnition in the precipitated zones . The must not be reheated nor sterilized again. basal medium, without the yolk or the tellurite, is perfectly stable and therefore can be repeatedly melted. Description The Baird Parker Agar Base is specially recommended Technique for the detection and enumeration of staphylococci in The inoculation is done by spreading 0,5 mL of sample food and other material, since it allows a good differen- over each plate with a Drigalski loop (Ref. 5-010). After tiation of coagulase-positive strains. The growth of the 18-24 hours of incubation at 35° C, select the colonies

25 Baird Parker Agar Base (Eur. Phar. Medium O) which are black, shiny and convex with regular margins References surrounded by a zone of clearing. These can be pre- ATLAS R.M. & L.C. PARKS (1993) Handbook of Micro- sumptly identified as coagulase-positive Staphylococcus biological Media. CRC Press. Londres aureus. BAIRD-PARKER, A.C. (1962) An improved diagnostic Colonial appearance after 24 h. at 35°C: and selective médium for isolating coagulase-positive Staphylococcus aureus: Black, shiny, convex, regular staphylococci. J. Appl. Bact. 25:12. margins, 1.0-1.5 mm diameter, surrounded by a DOWNES, F.P. & K. ITO (2001) Compendium of Meth- clearing zone of lipolysis 2-5 mm in width. May ods for the Microbiological Examination of Foods. 4rd produce wide opaque precipitate zones extending ed. APHA. Washington. USA into the cleared medium after 48 hours. EUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl. Other species of Staphylococcus: Black, usually dully, 5.6 § 2.6.13 Microbiological examination of non-sterile with regular margins. Sometimes they are brown products. EDQM Council of Europe. Strasbourg. with zones of clearing but these present wide ISO 5944:2001 Standard. Milk and Milk based products opaque zones. – Detection of coagulase positive staphylococci – MPN Micrococcus spp: Brown, very small and without clear- Technique. Geneva. ing. ISO 6888-1:1999 Standard.Microbiology of food and Bacillus spp: Various shades of brown, big. May produce animal feeding stuffs – Horizontal method for the enu- clearing after 48 hours. meration of coagulase-positive staphylococci – Part 1 Yeasts: White, big and smooth. Technique using Baird-Parker Agar médium. Geneva. The egg’s yolk emulsion can be prepared by mixing ISO 22718:2006 Standard. Cosmetics – Detection of a fresh egg’s yolk with an equivalent quantity (w/v) of Staphylococcus aureus. saline solution. Sterilize by filtration and aseptically add to the medium. This reagents´s reference, already steri- FIL-IDF 60:2001 Standard. Latí et produits à base de lized, is SCHARLAU 6-016. lait – Détection des staphylocoques à coagulase positive – Technique du nombre le plus probable. Brussels. The potassium tellurite solution is prepared by dissolv- USP 29 – NF 25 (2006) <61> Microbial Limit Tests. US ing 3,5 g potassium tellurite in 100 mL distilled water. Phamacopoeial Conv. Inc. Rockville. Md, USA Sterilize by filtration. This sterile reagent’s SCHARLAU ZANGERL, P. & H. ASPERGER. (2003) Media used MICROBIOLOGY reference is 6-011. in the detection and enumeration of Staphylococcus Although these solutions can be mixed to be added aureus in Handbook of Culture Media for Food Microbiol- to the Baird Parker Agar Base forming the commonly ogy Corry et als. Eds. Elsevier Sci. BV Ámsterdam known Egg Yolk Tellurite Sterile Emulsion (Ref. 06-026), they are also stable as the separate supplement and can be used in many other culture media.

Bile Esculin Media

Bile Esculin Agar Description This medium formulation is based on the modification by Ref. 01-265 Facklam and Moody of the original formulation by Swan to verify the esculin hydrolysing capacity of streptococci Specification and their resistance to bile salts which inhibit gram positive bacteria. Solid culture medium for the identification of probiotic In fact, this medium can substitute KAA Confirmative streptococci in food samples. Agar (Ref. 01-263), but it has not the same selectivity. Hence, it is used just as a substrate to verify the two as- Formula (in g/L) says simultaneously in the biochemical tests that identify Meat extract ...... 3,0 enterococci. Peptone ...... 5,0 Bile ...... 40,0 Technique Ferric citrate ...... 0,5 Assay is performed by inoculating the surface of a slant Esculin ...... 1,0 with the pure culture that is going to be verified. After the Agar ...... 15,0 24 hours incubation at 35°C, it might produce translucid Final pH 7,0 ± 0,2 colonies, surrounded by black halos or zones, due to esculin hydrolysis. Resistance to bile salts is indicated Directions by the growth. Suspend 64,5 g of powder in 1 L of distilled water and let it soak. Heat to boiling and distribute into containers. References Sterilize in the autoclave at 121°C for 15 minutes. LEUCHNER, R.G.K., J.BEW, K.J.DOMIG, & W.KNEIFEL. (2002) A collaborative study of a method for enumeration of probiotic enterococci in animal feed J. Appl. Microbiol. 93:781-786 26 Bile Esculin Media

DEIBEL, R.H., HARTMAN, P.A. (1976) The enterococci, Description Compendium of Methods for the Microbiological Exami- The Bile Esculin Azide Medium is a modification of the nation of Food. APHA. classical Bile Esculin proposed by Isenberg, Goldberg FACKLAM, R.R., MOODY, M.D. (1970) Presumptive and Sampson in 1970, with a reduction in the amount of identification of group D streptococci: The bile-esculin bile and the addition of the sodium azide. Brodsky and test. Appl. Microbiol. 20:245. Schieman shown that this medium, also know as Pfizer PASCUAL ANDERSON, MªRª. (1992) Microbiología Enterococci Selective gave best results with the filtration Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN. technique. SWAN,A. (1954), The use of bile-esculin medium and The actual formulation is according the ISO Standard Maxted’s technique of Lancefield grouping in the identifi- 7899-2:2000 for the second step in the confirmation and cation of enterococci. J.Clin.Pathol. 7:160. enumeration of enterococci in water by the membrane FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. filtration method. The colonies previously selected in A. APHA International. Gaitherburg, VA the Slanetz-Bartley Agar (Ref. 01-579 + 6-023) must ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- be confirmed by a short incubation on the Bile Esculin biological Media. CRC Prss. London. Azide Medium that permits the verification of the esculin DOWNES, F.P. & K. ITO (2001) Compendium of Meth- hydrolysis in a selective environment. ods for the Microbiological examination of Foods. APHA. Washington Technique After an incubation of 24-48 hours on Slanetz Bartley Bile Esculin Azide Agar Agar (Ref. 01-579 + 6-023), the membrane filter that

Xn show typical colonies is transferred, with sterile forceps Ref. 01-592 and upright position, to a pre-warmed plate of Bile R-22-32-52/53 Esculin Azide Agar. After two hours of incubation at 44 ± S-7-46-61 Specification 0,5ºC the membrane filter is inspected. All the typical colonies that show a brown to black colour in the surround- Solid medium for the enterococci confirmative test in ing medium are considered as positives and enumerate water by the membrane filtration method according to as intestinal enterococci. ISO 7899-2. A heterogeneous distribution of the colonies or the presence of abundant and different micro organisms can Formula (in g/L) interfere with the differentiation of positive colonies. Tryptone ...... 17,00 Peptone ...... 3,00 Yeast extract ...... 5,00 References ISO Standard 7899-2 (2000) Water Quality. Detection Bile ...... 10,00 and enumeration of intestinal enterococci. Part 2: Mem- Sodium chloride ...... 5,00 brane filtration method. Esculin ...... 1,00 ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- Ammonium-ferric citrate ...... 0,50 biological Media. CRC Press Boca Raton. Fla. Sodium azide ...... 0,15 ISENBERG, H.D., D. GOLDBERG & J. SAMPSON Agar ...... 15,00 (1970) Laboratory studies with a selective enterococcus Final pH 7,2 ± 0,2 medium. Appl. Microbiol. 20:433 BRODSKY M.H. & D.A. SCHIEMANN (1976) Evalua- Directions tion of Pfizer Selective Enterococcus and KF media for Suspend 56.6 g of powder in 1 L distilled water and bring recovery of fecal streptococci from water by membrane to the boil. Distribute in suitable containers and sterilize filtration. Appl. Environ. Microbiol. 31:695-699 in autoclave at 121ºC for 15 minutes. Cool to 50-60ºC and pour plates to 3-5 mm thickness. These plates can be stocked at 2-8ºC until two weeks.

Ref. 01-592 Bile Esculin Azide Agar. Entero- coccus faecalis ATCC 29212

27 Blood Media

Blood Agar Base Directions Suspend 42,5 g in 950 mL of distilled water and bring to Ref. 01-352 boil. Distribute into flasks and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 45-50ºC and aseptically Specification add 7% of sterile defibrinated blood. Mix gently and pour into plates. General purpose medium for the isolation and cultivation Note: Blood and medium should be mixed in a big flask of microorganisms. to assure proper blood oxidation and mixing.

Formula (in g/L) Description Meat extract ...... 10,0 Blood Agar Base No. 2 allows a maximum recovery of Tryptone ...... 10,0 weak organisms without altering or interfering in their Sodium chloride ...... 5,0 hemolytic reactions. Compared to other Blood Agar Agar ...... 15,0 bases, this one shows an equal or higher stimulatory Final pH 7,3 ± 0,2 growth ability, however it specially helps the formation of pigment in the chromogenic bacteria. Directions Suspend 40 g of powder in 950 mL. of distilled water and References let it soak. Heat to the boiling and distribute into contain- ATLAS, R.M. and L.C. PARKS (1993) Handbook of ers. Sterilize by autoclaving at 121°C for 15 minutes. Let Microbiological Media. CRC Press, London. it cool to 45-50°C and then add deﬁbrinated blood in a CASMAN, E. (1947) A noninfusion blood agar base for proportion about 7% or the desired enrichment. neisseriae, pneumococci and streptococci. Am. J. Clin Path. 17:281-289 Description FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. Blood Agar Base may be used for the cultivation of non A. APHA International. Gaitherburg, VA fastidious microorganisms, since it has a balanced nutri- DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ent base . ods for the Microbiological examination of Foods. APHA. For the fastidious microorganisms, it is advisable to add Washington special enrichments, such as ascitic liquid, egg yolk, ISO 7932 Standard (2003). Microbiology of food and etc.. animal feeding stuffs. Horizontal Methods for the enu- This medium, with the addition of blood, is very suitable meration of presumptive Bacillus cereus. Colony count for studies in hemolytic activity, but for the isolation of tecnique at 30°C. pathogens Blood Agar Base Columbia type (Ref. 01- 034) is more suitable. Blood Agar Base (Columbia) References ATLAS, R.M. and L.C. PARKS (1993) Handbook of Ref. 01-034 Microbiological Media. CRC Press, London. Speciﬁcation Blood Agar Base No. 2 Medium especially rich in peptone, appropriate for blood addition or to prepare Chocolate Agar.

Ref. 01-505 Formula (in g/L) Casein Peptone ...... 12,0 Speciﬁcation Meat peptone ...... 11,0 Improved base with high nutrient properties especially Starch ...... 1,5 adapted for the growth of very fastidious Sodium Chloride ...... 5,0 microorganisms, acc. ISO 7932 (2003). Agar ...... 15,0 Final pH 7,3 ± 0,2 Formula (in g/L) Proteose peptone ...... 15,0 Directions Liver extract ...... 2,5 Add 44,5 g of powder to 950 mL of distilled water and Yeast extract ...... 5,0 bring it to the boil. Distribute into suitable containers and Sodium chloride ...... 5,0 sterilize at 121°C for 15 minutes. To obtain Blood Agar Agar ...... 15,0 cool it to 45-50°C and aseptically add sterile deﬁbrinated Final pH 7,0 ± 0,2 blood at 5% proportion.

Description Blood Agar Base contains an equilibrated mixture of meat and casein peptones, being suitable for preparing selective and as diagnostic media with the addition of

28 Blood Media blood or inhibitors. As it is presented, without additions, it Formula (in g/L) is also an excellent general culture medium. Tryptose ...... 10,00 Generally, Blood Agar base contains a casein peptone, Meat extract ...... 3,00 that aids big size colonies formation, or a meat petone, Sodium chloride ...... 5,00 that provides a well defined hemolysis halos or zones. Sodium azide ...... 0,20 Blood Agar Base is prepared according to the Columbia Agar ...... 15,00 University formulation, and meets the two conditions Final pH 7,2 ± 0,2 mentioned above. Directions Technique Suspend 33 g of powder in 1 L of distilled water and Some applications for this base are: bring to the boil. Distribute in suitable containers and Base Agar without either enrichment and inhibitors: sterilize in autoclave at 121ºC for 15 minutes. Cool to 45- This medium supports growth of normal micro- 50ºC and aseptically add 5% of defibrinated blood. Pour organisms as enterobacteriaand othyers more in sterile plates. strengh as Pasteurella, Brucella and Clostridium perfringens. Description Clostridium Selective Base Agar: Should a selective This medium is a nutrient base (Tryptose and Meat clostridium medium be desired, add 240 mg/L extract) with a suitable osmotic value (Sodium chlo- Sodium Azide and 180 mg/l Neomycin before the ride) that includes a selective agent (Sodium azide) to sterilization, or alternatively the contents of SC suppressing the growth of gram-negative bacteria. The Inhibitor container (Ref. 06-012CASE). addition of 5% defibrinated blood supplies growth factors Blood Agar: Aseptically add to the sterile medium 5% for the fastidious microorganisms and is used for the sterile defibrinated sheep blood and cool it to determination of the haemolytic patterns. 45°C. This way, medium is enriched and allows the determination of typical hemolytic reactions necessary for the identification of enterococci, Technique The plates are inoculated by the surface striking and staphylococci and other microorganisms. stabbing the agar several times to deposit inoculum be- Selective Gram-positive cocci Blood Agar: Simulta- neath the agar surface, to show the hemolytic reaction of neously at the time blood addition, also add 10 both oxygen-stable and oxygen-labile streptolysins. After mg/L of colistine and 15 mg/L of Nalidíxic Acid, an incubation of 18-24 and 48 ours in a suitable environ- or, the contents of a CP Inhibitor container (Ref. ment the hemolytic reactions are displayed: 06-013CASE), to obtain an excellent selective Alpha-( )-haemolysis is the reduction of haemo- medium for gram-positive cocci. α globin to met-haemoglobin that sows a greenish Note: Some authors recommend Blood Agar Base as decolourisation of the medium surrounding the the maintenance medium for Campylobacter. colony. Beta-(β)-haemolysis is the total lysis of erythro- References cytes that produce a clear zone surrounding the CASMAN, E. (1947) A non infusion blood agar base for colony. neiseriae, pneumococci and streptococci. Am. J. Clin. Gamma-(γ)-haemolysis means no haemolysis Path. 17:281-289. and there is no change in the medium ATLAS, R.M. and L.C. PARKS (1993) Handbook of Alpha-prime-(α’)-haemolysis is a small zone of Microbiological Media. CRC Press, London. complete haemolysis that is surrounded by area of partial lysis around the colony. Blood Azide Agar Base In the haemolysis studies must be in mind that the haemolytic reactions are affected by several conditions: Xn Ref. 01-242 atmosphere (aerobic, anaerobic or CO2 enriched) of in- R-22-32-52/53 cubation, the composition of the culture media (presence S-7-46-61 Specification of sugars or growth factors) and the source of the blood (horse rabbit, sheep, human…) Selective basal medium for staphylococci and streptococci isolation. With blood added can be used for the determination of haemolytic reactions. References ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press. London ISENBERG, H.D. (1992) Clinical microbiology procedures handbook. Vol. I ASM. Washington DC. PACKER, R.A (1943) The use of sodium azide as an inhibition substance of gram-negative bacteria. J. Infect. Dis. 67:113

29 Blood Media

RUOFF, K.L. (1995) Streptococcus p. 299-305, in P.R. Gamma- (γ)-haemolysis means no haemolysis Murray et al. (ed.) Manual of Clinical Microbiology. 6th and there is no change in the medium ed. ASM Washington DC. Alpha-prime- (α’)-haemolysis is a small zone of complete haemolysis that is surrounded by area Blood Tryptose Agar Base of partial lysis around the colony. In the haemolysis studies must be in mind that the haemolytic reactions are affected by the environmental Ref. 01-551 conditions (aerobic, anaerobic or CO2 enriched) of incubation, the composition of the culture media (presence Speciﬁcation of sugars or growth factors) and the source of the blood Solid highly nutrient medium developed as Base for (horse rabbit, sheep, human…) Blood Agar for the isolation and cultivation of fastidious microorganisms. References ATLAS, R.M. and L.C. PARKS (1993) Handbook of Formula (in g/L) Microbiological Media. CRC Press. London. Tryptose ...... 10,00 BALLOWS, A. & W.J. HAUSLER (1981) Diagnostic Pro- Meat extract ...... 3,00 cedures for Bacterial, Mycotic and Parasitic Infections 6th Sodium chloride ...... 5,00 Ed. APHA Washington D.C. Agar ...... 15,00 CASMAN, E.P. (1947) A non-infusion blood agar base for Final pH 7,3 ± 0,2 neisseriae, pneumococci and streptococci. Am. J. Clin. Pathol. 17:281-289 Directions HARMON, S.M. et al. (1998) FDA Bacteriological Suspend 33 g of powder in 1 L of distilled water and let Analytical Manual. 8th ed. AOAC International. Gaithers- it soak. Bring to the boiling and distribute into suitable burg. containers. Sterilize autoclaving at 121°C for 15 minutes. ISENBERG, H.D. (1992) Clinical Microbiology Proce- Let it cool to 45-50ºC and then add 5-10% deﬁbrinated dures Handbook. Vol. I ASM. Washington DC. blood or the suitable enrichment. Homogenize and pour plates.

Description Casman proposed Blood Tryptose Agar Base in 1947 as alternative medium without tissue infusion components. Their original formulations include dextrose that interferes with haemolytic reactions and it is omitted in the present formulation. This medium, with the addition of blood, is very suitable for studies in haemolytic activity, but to isolate pathogens Blood Agar Base Columbia type (Ref. 1-034) is more suitable. This medium support the growth of a wide variety of fastidious microorganisms but several species of Streptococcus and Neisseria require the addition of 1 g/L of Yeast Extract for the optimal growth.

Technique The plates are inoculate by the surface striking and stabbing the agar several times to deposit inoculum beneath the agar surface, to show the hemolytic reaction of both oxygen-stable and oxygen-labile streptolysins. After an incubation of 18-24 and 48 ours in a suitable environment the hemolytic reactions are displayed: Alpha- (α)-haemolysis is the reduction of haemoglobin to met-haemoglobin that sows a greenish decolourisation of the medium surrounding the colony. Beta- (β)-haemolysis is the total lysis of erythrocytes that produce a clear zone surrounding the colony.

30 BPRM Broth Base

Ref. 02-409 Description BPRM (Bacteroides Phage Recovery Medium) was Specification formulated according the specifications by the Microbi- Base medium to cultivate Bacteroides fragilis and phage ology Department of the Barcelona University. Tartera recovery from the environmental samples according to and cols. verified its efficiency in the Bacteroides fragilis ISO Standard 10705-4. phage recovery used as human fecal pollution indicators in environmental samples. Initially Kanamycin (100 Formula (in g/L) mg/L) and Vancomycin (7,5 mg/L) were added to the medium to prevent the growth of unwanted accompany- Peptone ...... 10,00 ing bacteria, but later the Vancomycin was replaced by Tryptone ...... 10,00 Nalidixic acid (100 mg/L). Yeast extract ...... 2,00 BPRM Basal Broth can be used in double strengh and, Sodium chloride ...... 5,00 with the addition of adequate amounts of agar, in the Glucose ...... 1,80 preparation of solid or semisolid media. Magnesium sulfate ...... 0,12 L-Cysteine HCl ...... 0,50 Final pH 6,8 ± 0,2 References TARTERA, C., R. ARAUJO, T. MICHEL and JOFRE Directions (1992) Culture and decontamination methods affecting enumeration of phages infecting Bacteroides fragilis in Dissolve 29,42 g of powder in 1 L of distilled water. Add sewage. Appl. Environm. Microbiol. 58:8:2670-2673. 1 mL of a CaCl 5% solution. Distribute in flasks and 2 ISO Standard 10705-4 (2001) Water Quality-Detection sterilize in the autoclave at 121°C for 15 minutes. Cool and enumeration of bacterigophages. Part 4: Enumera- to 40°C and add 1 mL/L of 1% sterile soution of hemin tion of bacteriophages infecting Bacteroides fragilis. prepared in 0,02% NaOH. Mix well. Just before the utilization add 25mL/L of a sterile solution of 10,6% (w/v)

NaCO3. Adjust the pH to 7,0 with HCl.

31 Brain Heart Infusion Media (BHI Media)

Brain Heart Infusion Agar Description (BHI Agar) Brain Heart Infusion is used for the cultivation of fastidious bacteria (streptococci, pnemococci, meningococci, etc.) and also is recommended for the cultivation of Ref. 01-599 pathogenic fungi. Growth of the accompanying bacterial flora can be Specification almost completely suppresed by adding 20 I.U. penicillin General purpose solid medium for various fastidious and 40 µg streptomycin per mL culture medium. pathogenic microorganisms. If this medium is to be used for the selective isolation of fastidious fungi (especially of Histoplasma capsulatum Formula (in g/L) and Blastomyces) add 10% sterile defibrinated blood Brain Extract ...... 12,5 and from mixinfected samples add also 0,05 µg cy- Heart Extract ...... 5,0 cloheximide/mL and 0,5 µg choramphenicol/mL. Proteose peptone ...... 10,0 This medium is not suitable for obtaining the character- Sodium chloride ...... 5,0 istic hemolytic reactions even after addition of the blood Di-sodium phosphate ...... 2,5 because of its glucose contents. Dextrose ...... 2,0 Agar ...... 15,0 References Final pH 7,4 ± 0,2 ROSENOW, E.C. (1919) Studies on elective localization. J. Dent. Research. 1:205-209 Directions HAYDEN R.L. (1923) Elective localization in the eye of Suspend 52 g of powder in 1 L of distilled water and bacteria from infected teeth. Arch. Int. Med. 32:828-849 bring to the boil . Distribute in tubes or flasks and steri- HOWELL(1948) The efficiency of methods for the isola- lize by autoclaving at 121°C for 15 minutes. tion of Histoplasma capsulatum Public. Health. Reports 63, 173, 178. Brain Heart Infusion Broth CONANT (1950) Diagnostic Procedures and Reagents 3rd Ed., APHA, Inc., New York, 452. (BHI Broth) DIN Standard 10163. Bestimmung koagulase positiver Staphylokokken. Ref. 02-599 APHA-AWWA-AWPC (1998) Standard methods for the examination of Water and Wastewater. 20th ed. Wash- Specification ington. Liquid culture medium for general purpose according to FDA (1998) Bacterilogical Analitical Manual. 8th ed. Rev. ISO 5944. A. APHA International. Gaitherburg, VA ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- Formula (in g/L) biological Media. CRC Prss. London. Brain extract ...... 12,5 DOWNES, F.P. & K. ITO (2001) Compendium of Meth- Heart extract ...... 5,0 ods for the Microbiological examination of Foods. APHA. Proteose peptone ...... 10,0 Washington Dextrose ...... 2,0 ISO 5944 Standard (2001) Milk and milk based products: Sodium chloride ...... 5,0 Detection of coagulase-positive staphylococci - MPN Di-sodium phosphate ...... 2,5 Technique. Final pH 7,4 ± 0,2

Directions Dissolve 37 g of powder in 1 L of distilled water, heating up if necessary. Distribute into containers and sterilize in the autoclave at 121°C for 15 minutes.

32 Brilliant Green Media

Brilliant Green Agar (BGA) References (Eur. Phar. Agar Medium L) U.S.PHARMACOPOEIA (2002) 25 ed. Chap. <61> “Mi- crobial Limit Tests”. USP/NF Conv.Inc. Rockville.MD KAUFFMAN, F. (1935) Weitere Erfahrungen mit der Ref. 01-203 kombinierten Anreicherungsverfahren für Salmonellaba- zillen Z. Hyg. Infekt. Krhn, 117; 26-32 Specification EUROPEAN PHARMACOPOEIA,Supplement 4.2 Medium for Salmonella isolation, according to the Euro- (2002), 4th Ed., Council of Europe,Strasbourg, France pean Pharmacopoeia Brilliant Green Bile 2% Broth Formula (in g/L) Meat peptone ...... 5,0000 Ref. 02-041 Casein peptone ...... 5,0000 Sodium chloride ...... 5,0000 Yeast extract ...... 3,0000 Specification Lactose ...... 10,0000 Liquid medium for the detection of coliforms in water, Sucrose ...... 10,0000 as recommended by APHA and ISO 4831 and 9308-1 Phenol Red ...... 0,0800 Standards. Brilliant Green ...... 0,0125 Agar ...... 15,0000 Formula (in g/L) Final pH 7,0 ± 0,2 Bile ...... 20,000 Lactose ...... 10,000 Directions Peptone ...... 10,000 Suspend 53 g of powder in 1 L of distilled water and heat Brilliant Green ...... 0,013 to boiling with constant stirring. Dispense into containers Final pH 7,2 ± 0,2 and sterilize at 121°C for 15 minutes. Directions Description Dissolve 40 g of powder in 1 L of distilled water and BGA is a differential selective medium, able to detect bring to the boil. Distribute into containers containing the presence of enteropathogenic bacteria in different Durham tubes and sterilize by autoclaving at 121°C for samples. This medium is a modification to Kauffman’s 15 minutes. original formulation, and it complies with the ISO, HMO, Eur. Phar., USP and APHA Description specifications. Brilliant Green Bile 2% Broth has been widely used as Since it has a high brilliant green concentration, it inhibits a medium for the assay of presumptive colimetries in notably the growth of most bacteria, except Salmonella. food, milk and water, through the Most Probable Number However, S. typhi and S. paratyphi are also inhibited. Technique. This broth offers some advantages over Therefore, when their presence or Shigella is suspected, other similar broths as its balanced composition of bile it is recommended to use other media in parallel, as and Brilliant Green effectively suppresses the growth of Deoxycholate Lactose Agar (Ref. 01-057), MacConkey gram-positive bacteria, even that of the more fastidious Agar (Ref. 01-118), Salmonella Shigella Agar (Ref. 01- Clostridium perfringens. 171), Xylose Lysine Deoxycholate Agar (Ref. 01-211 or It is recommended by the APHA for colimetries in water, 1-552) or Endo Agar Base (Ref. 01-589) which are less milk and food. inhibitory. British and Australian methodology use the broth as an intermediate stage between presumptive and confirma- Presence of lactose and sucrose allows a good dif- tive colimetry, as it was an enrichment at 32°C. Other ferentiation between Salmonella, which produce pink authors suggest it as an optimal base for the Eijkman or colourless colonies with a red halo or zone, and the testing of gas production at 44°C, for the identification companion flora, which produce smaller and green yel- of E. coli. lowish colonies with yellow halo, due to acid created by This medium can be used as Presumptive broth for lactose and/or sucrose fermentation. E.coli (by fluorescent reaction) if before sterilization Osborn and Stokes suggested the addition of 0,08 g/L of MUG (Ref. 06-102CASE) is added. sulfadiacine or 1 g/L of sulfapyridin in order to make this medium more selective for Salmonella and provide suit- References able qualities to this medium to perform the examination APHA (1985) Standard Methods for the Examination of of food and eggs and their derivatives. Water and Wastewater, 16th ed. Washington. VANDERZANT, C., SPLITTSTOESSER, D.F.(1992) Compendium of Methods for the Microbiological Exami- nation of Food. 3rd. Ed. APHA. Washington.

33 Brilliant Green Media

DOWNES F.P. & K. ITO (2001) Compendium of Meth- This formulation was subsequently adopted by the ISO ods for the Microbiological Examination of Food. 4rd. Ed. and DIN official method for detecting Salmonellae in APHA. Washington. meat. FDA (1998). Bacteriological Analitical Manual.8th ed. Rev. A AOAC International, Gaithersburg MD.. Technique PASCUAL ANDERSON, MªRª (1992) Microbiologia A previous enrichment in Tetrathionate Base Broth Alimentaria. Diaz de Santos, S.A.,Madrid. (Ref.2-033) is recommended. Inoculate on the surface ISO 4831 Standard (1991) General guidance for the of this plate medium in order to get separate colonies. enumeration of coliforms. MPN technique. Incubate at 35-37°C for a 18-24 hours period. ISO 9308-1. Standard (1990) Water Quality-Detection Salmonella colonies (except S.typhi) are red, pinkish or and enumeration of coliforms, thermotolerants coliforms white, but they are always surrounded by a red halo or and presumptive E.coli. MPN Method. zone, which shows the non lactose or sucrose fermentation. Colonies of lactose and/or sucrose fermenting Brilliant Green Modified Agar bacteria produce yellow-green colonies surrounded by a yellow halo. Sometimes, Proteus or Pseudomonas may Ref. 01-309 appear, and they produce red pointed colonies. In very polluted samples, it is recommended to include 1 g/L of sodium sulfacetamide and 250 mg/L of sodium Specification mandelate. Solid culture medium for selective isolation of Salmonel- lae in food (except S. typhi) according ISO 6579, 6340, 6785 and IDF 93 Standards. References DIN. 10181 Mikrobiologische Milchuntersuchung. Nach- weis von Salmonellen. Referenzverfahren. Formula (in g/L) DIN 10160. Untersuchung von fleisch und fleischerzeng- Peptone ...... 10,000 nissen. Nachweis von Salmonellen. Referenzverfahren Meat extract ...... 5,000 ISO Standard 6579 Meat and meat Product. Detection Yeast extract ...... 3,000 of Salmonellae. Reference Method. (1993) Lactose ...... 10,000 PASCUAL ANDERSON MªRª (1992) Microbiología Ali- Sucrose ...... 10,000 mentaria. Diaz de Santos, S.A.Madrid,. Disodium phosphate ...... 1,000 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Sodium phosphate ...... 0,600 biological Media. CRC Press, Inc.,Boca Raton, Fla. Phenol red ...... 0,090 ISO 6785 Standard (2001) Milk and milk products - De- Brilliant green ...... 0,005 tection of Salmonella spp. Agar ...... 15,000 FIL-IDF Standard 93 (2001) Milk and milk based prod- Final pH 6,9 ± 0,2 ucts - Detection of Salmonella spp. ISO 6340 Standard (1995) Water Quality - Detection of Directions Salmonella spp. Suspend 54,5 g of powder in 1 L of distilled water. Let it soak and heat up to boiling with constant stirring. Distrib- ute in plates. Do not autoclave.

Description In this modiﬁcation of the classical medium for Salmo- nellae, the concentration of brilliant green has been reduced to obtain a less inhibitory medium. At the same time, the nutrient basis has been enriched to enhance the recovery of those microorganisms that are weakened during the food production process.

Ref. 01-203 Brilliant Green Agar.

Salmonella typhimurium E. coli ATCC 14028 ATCC 25922

Ref. 02-041 Brilliant Green Bile 2% Broth. Left: control; center: E.coli ATCC 25922; right: Salmonella typhimurium ATCC 14028 Control 34 Bromcresol Purple Base Broth

Ref. 02-031 Description Bromcresol Purple Base Broth is the liquid version suit- Speciﬁcation able to determine gas production (by Durham´s tubes) Liquid culture medium with indicator to carry out sugar by enterobacteria which are sensitive to phenol red. fermentation studies. In the SCHARLAU formulation, meat extract has been omitted as it was found unnecessary and it also provid- Formula (in g/L) ed low concentrations of fermentable sugars that could change or give erroneous results. Gelatin peptone ...... 10,00 The bacteria when ferment the carbohydrates, changes Sodium chloride ...... 5,00 the medium colour to yellow due to Bromcresol purple Bromocresol purple ...... 0,02 pH indicator, and if they produce gas, it is retained in the Final pH 6,8 ± 0,2 Durham’s tube. Directions Dissolve 15 g of powder in 1 litre of distilled water. Add References ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- substrate to assay in the desired concentration and biological Media. CRC Press, Inc.,Boca Raton, Fla. distribute into containers provided with Durham’s tubes. Sterilize in the autoclave at 121°C for 10 minutes. Heat up the autoclave before putting in the tubes to avoid sugar caramelization. Addition of some kind of sugars may require a pH adjustment. To study the fermentation of some sugars like Glucose (Ref. 06-048), Lactose (Ref. 06-051), Maltose (Ref. 06- 052), Mannitol (Ref. 06-050) and Sucrose (Ref. 06-049) it is advisable to add 10 g/L of each one.

Brucella Media

Brucella Agar Brucella Broth

Ref. 01-042 Ref. 02-042

Speciﬁcation Speciﬁcation Solid medium for the cultivation of Brucella and other Liquid culture medium for Brucella and other fastidious fastidious microorganisms microorganisms

Formula (in g/L) Formula (in g/L) Peptone of casein ...... 10,0 Peptone of casein ...... 10,0 Meat Peptone ...... 10,0 Meat Peptone ...... 10,0 Yeast Extract ...... 2,0 Yeast Extract ...... 2,0 D(+) Glucose ...... 1,0 D(+) Glucose ...... 1,0 Sodium chloride ...... 5,0 Sodium chloride ...... 5,0 Sodium bisulphite ...... 0,1 Sodium bisulphite ...... 0,1 Agar ...... 15,0 Final pH 7,0 ± 0,2 Final pH 7,0 ± 0,2 Directions Directions Dissolve 28 g of powder in 1 L of distilled water, heat- Suspend 43 g of powder in 1 L of distilled water and ing if necessary. Distribute in suitable containers and bring to the boil. Distribute into suitable containers and sterilize in autoclave at 121ºC for 15 minutes. If a highly sterilize in autoclave at 121ºC for 15 minutes. If a high selectivity is wanted, add aseptically after autoclave, selectivity medium is wanted, add 2 ﬂasks of Brucella Brucella Selective Supplement (Ref. 06-025CASE). Selective Supplement (Ref. 06-025CASE). When Ethyl When Ethyl Violet 1,25 mg/L is added to the culture Violet 1,25 mg/L is added to the culture medium, the medium, the growth of biotype 2 of Brucella abortus is growth of biotype 2 of Brucella abortus is inhibited. inhibited. Some microbiologists promote this medium as suitable for Campylobacter maintenance.

35 Brucella Media

Description References The Brucella Media are prepared from composition of ALTON, G.G, L.M. JONES & D.E. PIETZ (1976) Las the APHA’s Albimi Broth used for isolation of Brucella técnicas de laboratorio en la Brucelosis, 21 ed. Mono- species and the only difference between Broth and Agar graph nº.55 FAO/WHO. Geneve. is the solidifying agent. Both media are suitable for the CRUICKSHANK.(1965) Medical Microbiology. 11th ed. isolation and cultivation of a lot of fastidious microorgan- E.S. Livingstone. Edimburgo. isms including Streptococcus, Neisseria and Campylo- ISENBERG H.D. (1992) Clinical Microbiology Proce- bacter, but they became selective with the addition of dures Handbook. ASM. Washington D.C. antibiotics like polymyxin or bacitracin or chemical inhibi- MacFADDIN J.D. (1985) Media for Isolation-cultivation- tors like cycloheximide and ethyl violet. With some dyes identification-maintenance of medical bacteria. William & (fuchsin and thionin) the media became differential. See Wilkins Baltimore MD. the suitable reference for the technique in every case. VANDERZANT, C & D.F. SPLITTSTOESSER (1992) Compendium of methods for the microbiological exami- Caution nation of food 3rd Ed. APHA. Washington D.C. Brucella species are classified as Biosafety Level 3 pathogens. All manipulations with live cultures and antigens must be confined to a Class II Biological Safety Cabinet. Follow proper established laboratory procedures in handling and disposing of infectious materials.

Bryant & Burkey Media

Bryant & Burkey Lactate Broth Description (BB-Lactate Broth) BB medium is used to enumerate, by MPN technique, the spores of gasogenic clostridia that are the producers of the swelling and rancidness of cheese in the dairy Ref. 02-421 industry (late blowing or butyric swelling). In normal conditions of use,the medium allows the growth of other Speciﬁcation microorganims also which are not directly related to the Liquid medium for the enumeration of spores of lactate- cheese alteration, e.g. Cl. butyricum or Cl. sporogenes, fermenting clostridia in milk and dairy products besides the main responsible organism Cl. tyrobutyri- cum, in presence of enough acetate, Clostridium tyrobu- Formula (in g/L) tyricum ferments lactate, producing acetic and butyric

Peptone ...... 15,0000 acids, CO2 and hydrogen. Meat extract ...... 7,5000 Yeast extract ...... 5,0000 Technique L-Cysteine HCl ...... 0,5000 Recommended technique is to enumerate the spores by Resarzurine ...... 0,0025 the MPN technique with fresh made medium, covered Sodium acetate ...... 5,0000 with a vaspar layer of 2 mL that acts as a cap that as- Sodium lactate ...... 5,0000 sures a low redox potential and at the same time retains Final pH 6,2 ± 0,2 gas produced in the reaction. Sample must be previously decontaminated by heat- Directions ing up for 10 minutes at 75°C in order to destroy all the Dissolve 38 g of powder in 1 L of distilled water, heating vegetative forms and only leaving the alive spores. up only if necessary. Distribute in suitable containers for Incubation is performed for 7 days at 37°C. Tubes will every procedure and sterilize in the autoclave at 121°C be declared positive if they show a clear gas produc- for 15 minutes. tion. Despite the fact that results are often expressed as butyric spores/mL, the limitations of the method suggest that they should be expressed as lactate fermenting clostridial spores.

36 Bryant & Burkey Media

Bryant & Burkey Modiﬁed maintaining its anaerobic condition by the thioglycollate Fluid Medium added. Also the amount of lactate is reduced because the density of the medium retains easily the gas bubbles produced. Ref. 03-557 Technique Speciﬁcation Samples are processed by established procedures in Fluid medium for the enumeration of spores of lactate- every product. fermenting clostridia in dairy products References Formula (in g/L) BERGÈRE, J. L. & S. SIVELA (1989) Detection and enu- Tryptose ...... 15,0000 meration of clostridial spores related to cheese quality. Meat Extract ...... 7,5000 Classical and new method. FIL-IDF Bull. 51:18-23 Yeast Extract ...... 5,0000 BRYANT M.P. & L.A. BURKEY (1956) The characteris- Cystine HCl...... 0,6000 tics of lactate-fermenting spore-forming anaerobes from Resazurine ...... 0,0025 silage. J. Bacteriol. 71:43-46 Sodium lactate ...... 3,0000 ROSENBERGER, K.F. (1951) The development of meth- Sodium acetate ...... 5,0000 ods for the study of obligate anaerobes in silage. Proc. Sodium thioglycollate ...... 0,2000 Soc. Appl. Bacteriol. 14:161-164 Agar ...... 0,7500 Final pH 5,9 ± 0,2

Directions Suspend 37 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers for every procedure and sterilize in autoclave at 121ºC for 15 minutes.

Description This modiﬁcation of the Bryant & Burkey Lactate Broth (Ref. 02-421) add a little amount of agar that makes the medium more thick to be used in greater volumes

Buffered Peptone Water Eur Phar (Buffered Sodium Chloride-Peptone Solution pH 7.0)

Ref. 02-494 Description This solution is recommended by European Pharmaco- Speciﬁcation poeia to dilute the sample for microbiological examina- Diluent for the homogenization of samples according to tion. Depending on the amount of fat in the sample to the European Pharmacopeia and ISO 21149. examine it is the technician’s decision regarding the kind and quantity of emulsifying agent to be used. Formula (in g/L) Peptone ...... 1,00 References Sodium chloride ...... 4,30 EUROPEAN PHARMACOPOEIA (2007) 5ª ed. Suppl. Disodium phosphate ...... 7,23 5.6 § 2.6.13 Microbiological examination of non-sterile Potassium phosphate ...... 3,56 products. EDQM Council of Europe. Strasbourg. Final pH 7,0 ± 0,2 ISO 21149:2006 Cosmetics – Enumeration and detection of aerobic mesophilic bacteria. Directions Dissolve 16 g of powder in 1 L of distilled water, heating up if necessary. Add 1 to 10 mL of Polysorbate 80 (Ref. 06-088) or Polysorbate 20 depending on the type of food to be diluted. Homogenize and distribute into containers. Sterilize by autoclaving at 121ºC for 15 minutes.

37 Casein Lecithin Polysorbate Broth Base

Ref. 02-539 Description This medium is produced according the formulation of Speciﬁcation the U.S. Pharmacopoeia. In the Section <61> “Microbial Liquid medium to dilute and neutralize samples of phar- Limit Tests” is proposed as alternative system to neutral- maceutical, cosmetic, raw material or end-products for ize preservatives and didsinfectants before to proceed the purpose of enumeration with the enumeration process, specially by the membrane ﬁltration method. Formula (in g/L) Casein peptone ...... 20,00 References Soya lecithin ...... 5,00 US PHARMACOPOEIA (2002) <61> Microbial Limits Final pH 7,3 ± 0,2 Tests. 25th ed. US Pharmacopoeial Conv. Inc. Rockville. MD Directions Dissolve 25 g of powder in 960 mL of distilled water pre- warmed at 50ºC . Add 40 mL of polysorbate 20, homogenize and distribute in suitable containers. Sterilize in autoclave at 121ºC for 15 minutes.

Caseinate Agar

Ref. 01-569 Technique Pour the caseinate agar into plates so that the medium Speciﬁcation is 2 mm thick (e.g. 12 mL for a 9-cm plate). After the me- Solid medium, acc. Standard Methods, for the detection dium is hardened, plate 0,1 mL quantities of the sample and enumeration of proteolytic organisms in dairy prod- or diluted sample on the agar surface and spread evenly ucts with a sterile Drigalski rod. To ensure absorption of the sample allow inoculated plates to dry for 15 minutes, Formula (in g/L) and incubate them for 48 to 72 hours at 32±1ºC or for 72 hours at 21±1ºC. Colonies surrounded by a white or off- Tryptone ...... 5,00 white zone of casein precipitate are proteolytic. Highly Yeast extract ...... 2,50 proteolytic bacteria will also produce a clear inner zone. Dextrose ...... 1,00 Report results as “proteolytic count per gram or mL”. tri-Sodium citrate ...... 4,41 Designate the medium used and the incubation condi- Sodium caseinate ...... 10,00 tions. Calcium chloride ...... 4,40 Agar ...... 15,00 Final pH 7,0 ± 0,2 References MARTLEY, F.G., S.R. JAYASHANKAR & R.C. LAW- Directions RENCE. (1070) An improved agar medium for the detection of proteolytic organisms in total bacterial counts. J. Suspend 42,3 g of powder in 1 L of distilled water and Appl. Bacteriol. 33:363-370 bring to the boil. Distribute in suitable containers and FRANK, J.F. G.L CHRISTEN & L.B. BULLERMAN sterilize in autoclave at 121ºC for 15 minutes. (1992) Test for groups of Microorganisms. In R. T. Marshall (ed) Standard Methods for the examination of Description Dairy Products. APHA. Washington D.C. Caseinate Agar is preferred to other media like Skimmed Milk Agar for the detection of proteolytic organisms because its better recovery of stressed cells and the absence of false positives.

38 Cetrimide Agar (Pseudomonas Selective Agar) (Eur. Phar. Agar Medium N)

Ref. 01-160 N References LOWBURY,E.J.L. & A.G. COLLINS (1955) The use of a R-52/53 Speciﬁcation S-61 new cetrimide product in a selective medium for Pseu- Solid culture medium for selective isolation of Pseu- domonas aeruginosa J. Clin. Path. 8.47 domonas aeruginosa acc. to ISO 22717. BROWN, V.I. & J.L. LOWBURY (1965) Use of an improved Cetrimide Agar Medium and of culture methods Formula (in g/L) for Pseudomonas aeruginosa. J. Clin. Path. 18.752 FDA (1998) Bacteriological Analytical Manual. 8th ed. Gelatin peptone ...... 20,0 Rev. A. AOAC International. Gaitherburg. VA. Magnesium chloride ...... 1,4 ATLAS, R.M. and L.C. PARKS (1993) Handbook of Potassium sulfate ...... 10,0 Microbiological Media. CRC Press Inc.,Boca Raton,Fla. Cetyltrimethyl-Ammonium Bromide ...... 0,3 EUROPEAN PHARMACOPOEIA,(2002) Supplement4.2 Agar ...... 15,0 4th ed., 2.6.13 Microbiological examination of non-sterile Final pH 7,2 ± 0,2 products. Council of Europe,Strasbourg,. U.S. PHARMACOPOEIA (2002) 25 ed. <61> Microbial Directions limit tests. Us Pharmacopoeial Conv. Inc. Rockville, MD Suspend 46,7 g of powder in 1 L of distilled water and ISO 22717:2006 Cosmetics – Detection of Pseu- add 10 mL of Glycerol. Bring to the boil and distribute domonas aeruginosa. into suitable containers. Sterilize at 121°C for 15 minutes.

Description control The Cetrimide Agar is based on the enormous resistance of Ps. aeruginosa strains to the Quaternary Ammonium Compounds (QAC’s). With regard to the Cetyltrimethyl- Ammonium Bromide there has been growth at 1 g/L concentrations, but in such cases it has been very poor and slow. An inhibitor concentration of 0,3-0,5 g/L does not seem to affect the viability of the pyogenic species. Neverthe- less, it does inhibit the rest of the fastidious accompanying bacteria, both gram-positive and gram-negative, as well as other species of Pseudomonas which may Pseudomonas aeruginosa ATCC develop at lower inhibitory concentrations. 27853

Although Ps. aeruginosa prevails over any other fastidious bacteria after a 48 hour incubation at 35°C, it is recommended to ﬁrst isolate at 42°C with an incubation of 48 hours. By this method, almost complete inhibition of other microorganisms is obtained.

39 Chapman-Stone Agar

Ref. 01-052 ment mannitol (biqger size) and the colonies that do not ferment mannitol (smaller size) greater, since the latter Speciﬁcation must grow solely depending on peptone as the nutrient Solid and differential medium with a high selective ability, source. for the isolation of staphylococci from food. Technique Formula (in g/L) Material under test is inoculated on the surface to pro- Peptone ...... 10,0 duce separated colonies, and is incubated at 30°C for Yeast extract ...... 2,0 a 48 hours period. After this time, examine and select Gelatin ...... 30,0 colonies on the basis of these criteria: D-Mannitol ...... 10,0 White or non pigmented colonies are discarded, even if Sodium chloride ...... 55,0 they show a gelatin liquefaction halo (coagulase nega- Ammonium sulfate ...... 75,0 tive). Dipotassium phosphate ...... 5,0 Golden yellow pigmented colonies, surrounded by a Agar ...... 15,0 clear zone of gelatin hydrolysis (positive Stone’s reac- Final pH 7,0 ± 0,2 tion) are selected to verify mannitol fermentation and further, coagulase and hemolysis.

Directions Mannitol fermentation is checked by adding a few drops Suspend 202 g of powder in 1 L of distilled water and of Bromcresol purple indicator (aq. sol. 0,04%) over the heat up in boiling water bath until the total dissolution of colonies. Any colour change indicates positive mannitol gelatin. Bring to the boiling. Distribute in tubes or flasks fermentation. and sterilize at 121°C for 15 minutes. Pour into plates Coagulase production and hemolysis tests are per- immediately. Avoid overheating. formed in later subcultures on suitable media. Chapman-Stone medium composition is very selective, Description and so, medium can be employed without real steriliza- Chapman-Stone medium is according to a modification tion. However, it is always advisable to sterilize it and of the classical 110 medium for Staphylococcus. The keep one control before use. main modification consists the inclusion of ammonium sulfate, that allows the direct reading or observation of References gelatin hidrolysis, instead of adding reagents to the plate CHAPMAN (1948).An improved Stone Medium for the medium. Another modification is the reduction in the isolation and testing of food-poisoning staphylococci amount of sodium chloride. The saline content is selec- Food Research 13:100-105 tive itself, but it is reinforced by the ammonium sulfate, ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- which is also selective. Finally, lactose has been omitted biological Media. CRC Press, Inc.London , making the difference between the colonies that fer-

Ref. 01-053 Chapman TTC Agar.

control E. coli ATCC 25922 Salmonella typhimurium ATCC 14028

Salmonella typhimurium E. coli ATCC 25922 ATCC 14028 (MF technique) (MF technique)

40 Chapman TTC Agar (Tergitol® 7 Agar)

Ref. 01-053 For every water sample two volumes or double quantity must be filtered over different membranes and incubated Specification on Tergitol 7 Agar at 35°C and 44°C respectively. Medium for the detection of coliforms by membrane fil- After 48 hours the typical colonies have the appearance tration technique in water analysis acc. ISO 9308-1:2000 shown in the table. standard. Most coliform can not grow on this medium when incubated at 44°C, except E. coli which forms a characteristic appearance. Formula (in g/L) Results are always expressed per 100 mL sample in- Meat peptone ...... 10,00 cluding any applied dilutions. Estimation is done by tak- Meat extract ...... 5,00 ing typical colonies which have grown at 35°C as fecal Lactose ...... 20,00 coliform, together with those grown at 44°C as E.coli. Yeast extract ...... 6,00 Nevertheless, according to the legislation and despite Bromothymol blue ...... 0,05 the medium’s selectivity, results can only be considered Sodium heptadecyl sulfate ...... 0,10 as presumptive and all coliform colonies have to be con- Agar ...... 15,00 firmed by following the criteria stated below: Final pH 7,2 ± 0,2 Typical appearance in EMB Agar (Ref.1-068) or Endo Agar Base (Ref.1-589); characteristic reactions in Kligler Directions Iron Agar (Ref.1-103). For the confirmation of faecal E. Suspend 56,2 g in 1 L of distilled water and bring to the coli, verification of it is: a motile,gram-negative bacillus boil. Distribute into suitable containers and sterilize by and lactose fermentator with acid and gas production, autoclaving at 121°C for 15 minutes. Cool to 45-50°C. which gives negative results on the citrate test and Add 2-3 mL/L of filter sterile 1% aqueous 2,3,5-triphe- indole production positive. nyltetrazolium chloride (TTC) (Ref. 06-023) and pour plates. Do not reheat. References ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Description biological Media. CRC Press, Inc. London. This medium is formulated for the presumptive iden- CHAPMAN G.H. (1951). A culture medium for detect- tification of coliforms in drinking water, by membrane ing and confirming E. coli in ten hours. Am. J. Publ. Hlth filtration technique. 41:1381-1386. Due to the instability the triphenyltetrazolium is provided DOWNES, F.P. & K. ITO (2001) Compendium of Meth- in a separate container, sterilized and ready to use. ods for the Microbiological Examination of Foods. 3rd Poured plates can be stored refrigerated for up to 8 days ed. APHA.Washington. without losing their effectiveness. They should not be GUINEA, SANCHO,PARES (1979). Análisis Microbiológ- used if any dehydration or drying signs appear. ico de Aguas. Ed. Omega. Barcelona. ISO 9309-1:2000 Standard. Water Quality. Detection and Technique enumeration of Escherichia coli and coliform bacteria.- While using the membrane filter technique for the pre- Part 1: Membrane filtration method. sumptive identification of coliforms in water, it should be SPECK, M (Ed.) (1982) Compendium of Methods for the kept in mind that to every type of water corresponds a Microbiological Examination of Foods. 2nd. Ed. APHA. minimum volume to be filtered and depends on the type Washington. of water. Dilute with sterile phosphate buffer if necessary to obtain the number of colonies on the membrane which are easy to count.

41 Chloramphenicol Glucose Media

Chloramphenicol Glucose Agar Chloramphenicol Glucose Broth (CGA) (Yeast Extract Dextrose (CGB) (Yeast Extract Dextrose Chloramphenicol Agar Chloramphenicol Broth acc. FIL-IDF) acc. FIL-IDF)

T T Ref. 01-366 Ref. 02-366 R-45 R-45 S-53-45 S-53-45 Specification Specification Solid and selective medium for the isolation and enu- Liquid and selective medium for the enumeration of fungi meration of fungi in milk and dairy products according in milk and dairy products using the MPN technique. ISO 7954. Formula (in g/L) Formula (in g/L) Dextrose ...... 20,0 Dextrose ...... 20,0 Yeast extract ...... 5,0 Yeast extract ...... 5,0 Chloramphenicol ...... 0,1 Chloramphenicol ...... 0,1 Final pH 6,6 ± 0,2 Agar ...... 15,0 Final pH 6,6 ± 0,2 Directions Dissolve 25 g of powder in 1 L of distilled water. Distrib- Directions ute in tubes and sterilize in the autoclave at 121°C for 15 Suspend 40 g of powder in 1 L of distilled water and let minutes. it soak. Bring to the boil and distribute into containers. Sterilize in the autoclave at 121°C for 15 minutes. Description This is the liquid version of the medium with the same Description name (Ref. 01-366) recommended by the Federation This is the medium recommended by the Federation In- International Laitrere - International Dairy Federation ternational Laitrere - International Dairy Federation (FIL- (FIL-IDF) for the enumeration of fungi (moulds and IDF) for the isolation and enumeration of fungi (moulds yeasts in liquid products). and yeast) in milk and dairy products. This medium has Medium is specially adapted to enumerate yeast and, also been adopted by the DIN and ISO standard. eventually, moulds by the Most Probable Number technique (MPN). Medium relies its selectivity on the bactericidal action of Chloramphenicol which, due to its thermostabity, may References be sterilized with the complete medium in the autoclave. FIL-IDF 94B Standard (1991). Enumeration of yeast and Moreover, pH may be adjusted near to neutrality, and moulds. Colony Count Technique at 25 °C. this fact allows the medium to be remelted several times ISO 7954 Standard (1987) General guidance for enu- without affecting its stability, selectivity and efficacy. meration of yeast and moulds - Colony count at 25ºC. Remeltings and overheatings may make the medium DIN Standard 10186. Mikrobiologische Milch Untersu- darker. chung. Bestimmung der Anzahl von Hefen und Schim- melpilzen. Referenzverfahren. Technique Generally the mass seed method or poured plate method is used to inoculate the medium, and an incubation at 22-25°C for 4 to 5 days is carried out.

42 Citrate Azide Agar

Ref. 01-558 T Technique

R-45-22-32-52/53 Pour the melted medium cooled to 45-50ºC over the Speciﬁcation S-53-45-7-61 sample in a petri dish. Let it solidify and dose another Selective differential solid medium for the detection and 5-7 sterile volume of medium on the surface to facilitate enumeration of streptococci in dairy products. the micro-aeroﬁlic environment. The plates are incubated at 37ºC for 48-72 hours and then the blue colonies are Formula in g/L counted. Express the results as “streptococci per g or mL of sample” Tryptone ...... 10,00 Yeast extract ...... 10,00 Sodium citrate ...... 20,00 References Sodium azide ...... 0,40 MARSHALL, R.T. (1992) Standard Methods for the Tetrazolium blue ...... 0,10 Examination of Dairy Products. 16th Ed. APHA. Washing- Agar ...... 15,00 ton. DC Final pH 7,0 ± 0,2 HARTMANN P.A, R.H. DEIBEL, & L.M. SIEVERDING (1992) Enterococci. en Vanderzant & Splittstoesser: Directions Compendium of methods for the microbiological examination of foods. 3rd Ed. APHA. Washington DC. Suspend 55,5 g of powder in 1 L of distilled water and EFTHYMIOU C.J., P. BACCASH, V.J. LABOMBARDI, & bring to the boil. Distribute in suitable containers and D.S. EPSTEIN (1974) Improved isolation and differentia- sterilize in autoclave at 121ºC for 15 minutes. tion of enterococci in cheese. Appl. Microbiol. 28:417- 422 Description SARASWAT, D.S. G.W. REINBOLD & W.S. CLARK Citrate Azide Agar is the streptococci detection medium (1963) Selection of a medium for the isolation and enu- preferred in dairy microbiology over the other media like meration of enterococci in dairy products. J. Milk Food KF-Streptococcal Agar (Ref. 01-294) used in food micro- Technol. 26:114-118 biology because its high selectivity and security of use.

43 CLED Agar (Brolacin Agar)

Ref. 01-047 Staphylococci coagulase(-ve): White or light yellow colonies, with halo and the same size as of Specification Enterococci. Cystine,lactose,electrolyte deficient medium, recom- Proteus sp.: Blue, translucent and smaller than E.coli. mended for the isolation and identification of urinary Pseudomonas aeruginosa: Plain, matt and wrinkled pathogenic bacteria. colonies. Green colour and irregular boundary. Corynebacteria: Pointed and grey colonies Lactobacilli: Matt colonies, like Corynebacteria. Formula (in g/L) Peptone ...... 4,000 Trypsic Peptone ...... 4,000 References Meat extract ...... 3,000 SANDYS, G.H. (1960) A new method of preventive Lactose ...... 10,000 swarming of Proteus sp. J.Med.Lab.Tech. 17:224 L-Cystine ...... 0,128 MACKEY J.P. and G.H.SANDYS (1966) Diagnosis of Brom Thymol Blue ...... 0,020 urinary tract infections. Brit. Med. J. 1.173 Agar ...... 15,000 Final pH 7,4 ± 0,2

Directions Add 36 g of powder to 1 L of distilled water and heat to boiling. Sterilize by autoclaving at 121°C for 15 minutes.

Description This general purpose medium has been recommended for bacteriological urine analysis. Current formulation is a modiﬁcation of the original one reported by Sandys, that achieves an excellent colony differentiation without inhibitors. This fact, and also the careful selection of nutritive components, makes this medium a substrate able to support growth of most urinary pathogenic bacteria. Presence of lactose as a fermentable sugar allows classic differentiation and, at the same time, lack of electro- lytes suppresses swarming waves on the members of the Proteus species and sometimes growth of Shigella sp. also. The characteristics of colonies that grow on C.L.E.D. Agar (after 18 hours of incubation): Escherichia coli: Yellowish colonies, opaque, with core, 1,25 mm diameter. Non fermentative strains give control blue colonies. Klebsiella sp.: Very mucuous colonies of variable colour, from yellow to blue-white. Salmonella sp.: Plain and blue colonies. Enterococcus faecalis: Yellow colonies. 0,5 mm diameter. Staphylococcus aureus : Convex yellow colonies. 0,75 mm diameter.

Proteus mirabilis ATCC 10975

44 CN Selective Agar Base for Pseudomonas

Ref. 01-609 N Technique A volume of the sample is filtered thorough a filtering R-52/53 Specifications S-61 membrane of 0,45 µm pore and the membrane is depos- Selective solid medium for the detection of Pseu- ited on the surface of the CN medium. The plates are domonas aeruginosa according the EN 12780-2002 and incubated at 36±2ºC for a period of 44±4 h with a partial ISO 16266 Standard. examination at 22±2 h. All the colonies producing a green or blue (pyocianin) Formula (in g/L) pigmentation in this period must be considered as Pseu- domonas aeruginosa without any other confirmation. Gelatine Peptone ...... 16,00 All the colonies that produces fluorescence under the Casein Peptone ...... 10,00 Wood’s light (without pyocianin production) era consid- Potassium sulphate ...... 10,00 ered presumptive Ps. aeruginosa and must be confir- Magnesium chloride ...... 1,40 mate on Acetamide Medium (Ref. 03-428). Cetrimide® ...... 0,20 All the colonies producing a brown-reddish pigment and Agar ...... 15,00 no produces fluorescence nor pyocianine are considered Final pH 7,1 ± 0,2 presumptive Ps. aeruginosa and must be confirmed by the oxidase test and typical growth on Acetamide Me- Directions dium (Ref. 03-428) and King B Agar (Ref. 01-029) Add 52,6 g of powder to 1 L of distilled water with 10 mL of glycerol. Heat to complete solution and sterilize in autoclave at 121ºC for 15 min. Cool to 45-50ºC and add References KING, E.O., M.K. WARD & E.E. RANEY (1954) Two to 500 mL of medium a flask of the Nalidixic Acid Selec- simple media for the demonstration of pyocianin and tive Supplement (Ref. 06-124CASE). Homogenise and fluorescein. J. Lab. Clin. Med. 44:301 pour plates. BROWN, V.L. & E.J.L. LOWBURY (1965) Use of an im- Do not maintain the complete medium melted for more proved Cetrimide Agar Medium and of culture methods than 4 hours. Do not re-melt. The finished plates can be for Ps. aeruginosa. J., Clin. Pathol. 18:752 used without lose its efficiency for one month if they are GOTO S. & S. ENOMOTO (1970) Nalidixic acid cetrim- refrigerated and in a dark place. ide agar. A new selective plating medium for the selective isolation of Ps. aeruginosa. Jpn. J. Microbiol. 14:65 Description ROBIN, T. & J.M. JANDA (1984) Enhanced recovery of The CN Selective Medium for Pseudomonas was pro- Ps. aruginosa from diverse clinical specimens on a new gressively developed from the basic media of King, Ward selective agar. Diag. Microbiol. Infect Dis. 2:207 and Raney for the production of pigments. Browne and EN STANDARD 12780 (2002) Water Quality. Detection Lowbury add the cetrimide as selective agent and Goto and enumeration of Ps. aeruginosa by membrane filtra- and Enomoto improves efficiency by adding nalidixic tion. Brussels. acid. The presence of both inhibitors eliminates the con- ISO 16266:2006 Standard. Water Quality – Detection taminant microbiota from heavily polluted specimens and and enumeration of Pseudomonas aeruginosa. Method was adopted by the CEN (Centre Europeen de Normali- by membrane filtration. sation) in its EN Standard 12780 for the detection of Ps. aeruginosa by filtering membrane in water.

control Pseudomonas aeruginosa ATCC 27853

45 Cosmetic Diluent of Beerens

Ref. 02-257 Description Cosmetic diluent of Beerens has all the necessary Speciﬁcation compounds to neutralize most of the chemical agents Diluent to neutralize preservative systems in common included in cosmetic products to maintain and preserve it examination of cosmetic products. free of microorganisms. It complies with the EU recommendation that states that Formula (in g/L) before any microbiological examination, a treatment to remove all the growth inhibitor systems in the cosmetics Lecithine ...... 3,00 must be performed. Sodium thiosulfate ...... 5,00 However, this standard also declares that later dilutions L-Histidine HCl ...... 1,00 have to be performed in less aggressive media, that Peptone ...... 1,00 may be considered as an enrichment and revitalization Sodium chloride ...... 8,50 system, and it suggests Letheen Broth (Ref. 02-236) or Dipotassium phosphate ...... 1,00 Letheen Modiﬁed Broth (Ref. 02-237). Final pH 7,0 ± 0,2

Directions References BEERENS, H., RAMONS, C., LEMAIRE, D. (1976). Rev. Dissolve 19,5 g of powder in 1 L of distilled water con- Inst. Past. Lyon. 9:127. taining 30 mL of polysorbate 80.(Ref. 06-080). Distribute BRIGIDI, P., MATTEUZZI, D. (1982) II Farmaco Ed. Pr. into suitable containers and sterilize by autoclaving at 37:8:260. Commission del Communautes Europeennes, 121°C for 15 minutes. Groupe Special. Methodes de Controle Microbiologique Let it cool to 50°C and shake gently to redissolve polys- des Produits Cosmetiques: Limites Numeriques Appli- orbate. cables au controle Ofﬁciel de la Qualité Microbiologique des Produits Cosmetiques. XI/405A, ISPRA, 1976.

m-CP Agar Base

Ref. 01-513 Homogenize avoiding bubble formation and pour in plates. Specification Solid medium for the enumeration and isolation of Description Clostridium perfringens in water acc. the European The m-CP Agar Base is a solid medium for counting Directive 12767/97 and isolating vegetative cells and spores of Clostridium perfringens by the membrane filtration method. Its use is Formula (in g/L) compulsory in determining the quality of water for human Tryptose ...... 30,00 consumption in European Union by Directive 12767 (12- Yeast extract ...... 20,00 07-1997) of the European Council. Sucrose ...... 5,00 L-Cisteine HCl ...... 1,00 Technique A suitable volume of water is filtered through a mem- Magnesium sulfate.7H2O ...... 0,10 Bromocresol purple ...... 0,04 brane filter of 47 mm diameter and 0,45 mm pore. Put Agar ...... 15,00 the membrane on the surface of a plate of m-CP medium Final pH 7,6 ± 0,2 freshly prepared and incubate in an anaerobic atmosphere at 44±1ºC for 21±3 hours. Expose the growth Directions obtained to amonium hydroxide vapours for 20-30 sec- Suspend 71 g of powder in 1 L of distilled water and onds. Count as Clostridium perfringens all the opaque bring to the boil. Distributein suitable containers and yellow colonies that turn to pink or red after the ammo- sterilize in autoclave at 121ºC for 15 minutes. Cool to nium hydroxide exposure. Express the results as cfu/mL. 45-50ºC and aseptically add 2 flasks of Selective Sup- plement m-CP (Ref. 06-125CASE) with the following References composition: European Council (1998) Directive 98/83/CE on the D-Cicloserine ...... 400 mg/L quality of the water destined to the human consuption. Polymyxin B sulphate ...... 25 mg/L EC Bull. 11-03-1998. Indoxil-b-D-Glucoside ...... 60 mg/L Fenolftalein diphosphate ...... 100 mg/L Iron (III) Chloride ...... 90 mg/L

46 m-CP Agar Base

control Clostridium perfringens Clostridium perfringens before the exposure after the exposure to Ammonia vapours (AM0257) to Ammonia vapours (AM0257)

Cystine-Tryptone Fluid Medium (CTA Medium)

Ref. 03-045 Technique On this medium, we can maintain by deep stab a lot of Specification fastidious microorganisms like Neisseria, Enterococcus, Basic medium for strain maintenance or sugar fermenta- Pasteurella, Shigella and in most cases without needing tion assays. Its fluidity makes it suitable to observe the any additives or CO2. Even some light-sensitive anaero- motility. bic microorganisms can grow without special conditions though in reduced atmopheres they give ideal growth on Formula (in g/L) this medium. In the few cases as per the nutritive requirements of the L-Cystine HCl ...... 0,500 microorganisms like serum or ascitic liquid, which can Casein peptone ...... 20,000 be added in an aseptic way to the molten medium, when Sodium chloride ...... 5,000 cooled to 45-50°C. Sodium sulfite ...... 0,500 Microorganism motility can be easily detected by growth Phenol red ...... 0,017 in the stab, and helped by the medium fluidity. Agar ...... 2,500 Medium, with added sugars, may be used to study sugar Final pH 7,3 ± 0,2 fermentation with microorganisms that do not grow on phenol red classical media because of the additional Directions nutritional requirements. Acidification can be easily ob- Suspend 28,5 g of powder in 1 L of distilled water and served with the change in colour of phenol red indicator. heat to boiling. Dispense and sterilize at 121°C for 15 minutes. Addition of certain sugars may require a pH readjustment. References VERA, H.D. (1948) A simple medium for identification and maintenance of the gonococcus and other bacteria. Description J.Bact. 55:531 Cystine and Tryptone fluid medium is appropiate for the ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- propagation and maintenance of bacterial strains, even biological Media. CRC Press, Inc. the fastidious ones, without additives. The lack of fermentative sugars allows the long survival of microorganims, and sometimes it has been used for studies on severe microaerials fermentation, due to the fact that these microorganisms do not grow on current basal media. Medium has its maximum efficacy when freshly prepared, but it can be stored for long periods of time, taking care to avoid its dehydration. To achieve this, screw caps or hermetic sealing are strongly recommended. In any case, if medium has been stored for a long time, it is better to put it in a boiling water bath for a few minutes before using it.

47 Czapek-Dox Media

Czapek-Dox Agar lactic acid 10% solution, producing a pH drop to 3,5. In this medium, Warcup adds 5 g/L of Yeast extract (Ref. Ref. 01-051 07-079) and an antibiotic mixture (Streptomycin 30 mg/L and Tetracycline 2 mg/L) to achieve total efficacy and selectivity. In any case, bacterial growth is very poor. Specification Semisynthetic solid medium for the cultivation of fungi, Czapek-Dox medium has been adopted for the morpho- with sodium nitrate as the only nitrogen source. logical studies of soil fungi, and it aids chlamydospora formation by Candida albicans in shorter periods (Daw- Formula (in g/L) son, 1962). Temperatures and times of incubation al- Sucrose ...... 30,00 ways varies, and they can go from 1 to 5 weeks at room Sodium nitrate ...... 2,00 temperature. Usually, it is 8-15 days at 15°C. In case of Magnesium glycerophosphate ...... 0,50 Candida, 28°C for 48 hours seems to be optimum ; for Potassium sulfate ...... 0,35 Penicillium, 22°C and nonetheless, Aspergillus grows Potassium chloride ...... 0,50 better at 30-32°C. Ferrous sulfate ...... 0,01 Agar ...... 15,00 References Final pH 6,8 ± 0,2 CZAPEK, F. (1903) Untersuchung uber die sticstoff- gewinnungund einweissbildung der Pflanze.Beitr. Chem. Directions Physiol. Pathol. 1:540 Suspend 48,5 g of powder in 1 L of distilled water and DOX, A.W. (1910) The intracellular enzymes of Penicil- heat to boiling. Dispense and sterilize by autoclaving at liumand Aspergillus with special references to those of P. 121°C for 15 minutes. Should a lower pH be desired, camemberti. US Dept Agr. Bur. Animal Ind. Bull. 120:70 acidify the medium with sterile lactic acid after cooling to APHA-AWWA-WPCF (1992) Standard Methods for the 45°C. examination of Water and Wastewater. 18th ed. APHA. Washington. Czapek-Dox Broth RAPER, K.B. & D.J.FENELL (1965) The genus Aspergil- lus. William & Wilkins Co. Baltimore. Ref. 02-051 THOM,C & M.B. CHURCH (1926 The aspergilli. William & Wilkins Co. Baltimore. WARCUP, J.H. (1950) The soil-plate method for isolation Specification of fungi from soil. Natur 166:117-118 Semisynthetic liquid culture medium with sodium nitrate as the only nitrogen source.

Formula (in g/L) Sucrose ...... 30,00 Sodium nitrate ...... 2,00 Potassium chloride ...... 0,50 Magnesium glycerophosphate ...... 0,50 Ferrous Sulfate ...... 0,01 Potassium sulfate ...... 0,35 Final pH 6,8 ± 0,2

Directions Dissolve 33,5 g of powder in 1 L of distilled water. Dis- pense in suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Do not overheat

Description Czapek-Dox medium, both liquid and solid version, is a general cultivation medium of deﬁned chemical composition, where the sole nitrogen source is sodium nitrate, and the carbon source is sucrose. It has been employed successfully in the isolation and cultivation of soil microorganisms especially fungi.

Its original version had a pH near neutrality, but it can be Fusarium sp. rendered selective medium for the fungi by adding, (after sterilization and before solidiﬁcation), 10 mL of sterile

48 D/E Neutralizing Agar

Ref. 01-610 Xi Technique R-43 When the RODAC (Replicate Organisms Detection and Specification S-24-37 Counting) plates are filled in the laboratory be careful Solid culture medium for the neutralization and testing of with the meniscus of the agar: It should rise above the antiseptics and disinfectants acc. ISO 22717 and 22718 rim of the plate to give a slightly convex surface to make standards. a proper contact with the surface to be sampled. For sampling remove the cover of the RODAC plate and Formula (in g/L) carefully press the agar surface to the surface being sampled. Make certain that the entire agar meniscus Tryptone ...... 5,00 contacts the surface. Replace the cover and incubate Yeast extract ...... 2,50 in an inverted position under the time and temperature Dextrose ...... 10,00 conditions for the microorganisms in question. Express Lecithin ...... 7,00 the results as “colonies per RODAC plate” or “Colonies Sodium thiosulphate ...... 6,00 per cm2” Sodium sulphite ...... 2,50 Sodium thioglycollate ...... 1,00 Polysorbate 80 ...... 5,00 References Bromcresol purple ...... 0,02 DEY, B.P. & F.B. ENGLEY (1983) Methodology for Agar ...... 15,00 recovery of chemically treated Staphilococcus aureus Final pH 7,6 ± 0,2 with neutralizing medium. Appl. Environm. Microbiol. 453:1533-1537 Directions HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW (1992) Microbiological tests for equipment, containers, Suspend 54 g of powder in 1 L of distilled water and water and air. In R.T. Marshall (Ed.) Standard Methods bring to the boil. Distribute in suitable containers and for the examination of Dairy Products 16th ed. APHA sterilize in autoclave at 121ºC for 15 minutes. The ap- Washington. pearance of precipitates is normal and do not interferes EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F. the results. FRANK (2001) Microbiological Monitoring of the Food Processing Environment. In DOWNES & ITO (Eds) Description Compendium of Methods for the Microbiological Exami- Dey & Engley developed this medium in 1983 to recov- nation of Foods. 4th ed. APHA. Washington DC. ery chemically damaged staphylococci, At the present ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- its use is generalized for testing by the contact method biological Culture Media. CRC Press. Boca Ratón, Fla. (RODAC Plates) the efficiency of antiseptics and disin- ISO 22717:2006 Standard. Cosmetics – Detection of fectants on impervious surfaces. The present formulation Pseudomonas aeruginosa. incorporate neutralizing substances for almost all the ISO 22718:2006 Standard. Cosmetics – Detection of active products used as antiseptics and disinfectants. Staphylococcus aureus. Lecithin neutralizes quaternary ammonium compounds (QAC’s); Polysorbate acts on phenolics and formalin; thioglycollate neutralizes the organic-mercurial compounds; thiosulfate-sulfite inactive halogen-compounds and lecithin + polysorbate neutralizes ethanol and other alcoholic compounds.

49 Decarboxylase Lysine Broth acc. to Taylor

Ref. 02-336 Technique It is advisable to use a vaseline seal to avoid spontane- Specification ous oxidation. The use of glucose in anaerobic condi- Liquid medium to differentiate enteric bacteria in the tions produces an acidification of the medium, the indica- L-Lysine decarboxylation assays according to ISO 6785, tor will then turn to yellow if there is growth. The use of 21567 and IDF 93 Standards the aminoacid will acidify the medium again, and then it turns to grey and finally to violet, thereby showing the Formula (in g/L) positive reaction. The observations of these biochemi- Yeast extract ...... 3,000 cal tests are performed after an incubation period of 24 D(+)Glucose ...... 1,000 hours at 37°C. Bromcresol purple ...... 0,016 L-Lysine ...... 5,000 References Final pH 6,8 ± 0,2 DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. APHA. Directions Washington. DC. TAYLOR, W.I. (1961) Isolation of Salmonellae from Food Dissolve 9 g of powder in 1 L of distilled water. Distribute Supplies. V Determination of the Method Choice for in thin tubes in an amount of 2 or 5 mL per tube. Steri- Enumeration of Salmonella. App. Microbiol. 9, 487-490. lize in the autoclave at 121°C for 10 minutes. ISO 6785 Standard (2001) Milk and milk products - De- tection of Salmonella spp. Description FIL-IDF 93 Standard (2001) Detection of Salmonella The capacity to decarboxylate some aminoacids has spp. been widely employed to classify Enterobacteriaceae. ISO 21567:2004 Food and feeding stuffs – Horizontal Taylor’s formulation, including lysine, has been recently method for the detection of Shigella ssp. included in several standards for the identification of Sal- monella. This modification shows an improved performance, in comparison to Falkow’s formulation.

Ref. 01-057 Deoxycholate Lactose Agar

control

Salmonella typhimurium ATCC 14028 Escherichia coli ATCC 25922

50 Deoxycholate Media

Deoxycholate Citrate Agar References (Eur. Phar. Medium J) LEIFSON, E. (1935) New culture media based on sodium deoxycholate for the isolation of intestinal patògens and for the enumeration of colon bacilli in milk and water. Ref. 01-056 J. Path.. Bact. 40:581-599. HYNES, M. (1942) The isolation of intestinal pathogens Specification by selective media. J. Path. Bact. 54:193-207 Differential and moderately selective plating medium for EUROPEAN PHARMACOPOEIA (2002) 4th ed. Suppl. enteric pathogenic bacteria, according the European 4.2 . § 2.6.13 Test for specified micro-organisms. Council Pharmacopoeia. of Europe. Strasbourg. MAC FADDIN, J.F. (1985) Media for isolation-cultivation- Formula (in g/L) identification-maintenance of medical bacteria. William & Peptone ...... 10,00 Wilkins, Baltimore, MD. Meat extract ...... 10,00 ATLAS, R.M. & L.C. PARKS (1991) Handbook of Micro- Lactose ...... 10,00 biological Media. CRC Press London. Ferric Citrate ...... 1,00 Sodium Citrate ...... 20,00 Deoxycholate Lactose Agar Sodium deoxycholate ...... 5,00 Neutral red ...... 0,02 Ref. 01-057 Agar ...... 15,00 Final pH 7,3 ± 0,2 Specificacion Directions Differential solid medium for the isolation of enterobacteria according to APHA. Suspend 71 g of powder in 1 L of distilled water and bring to the boil. Immediatelly pour into plates. Plates may be used at once or refrigerated for a few days. Do Formula (in g/L) not autoclave or overheat. Peptone ...... 10,00 Lactose ...... 10,00 Description Sodium chloride ...... 5,00 Sodium citrate ...... 2,00 The European Pharmacopoeia formulation is one of Sodium deoxycholate ...... 0,50 these modifications to rhr original medium developed by Neutral red ...... 0,03 Leifson in 1935. The inhibition of gram-positive microor- Agar ...... 15,00 ganisms is due primarily to its content of sodium deoxy- Final pH 7,1 ± 0,2 cholate, although the two citrate compounds also are active inhibitors. The lactose achieves differentiation of enteric bacilli. Organisms that ferment lactose produce Directions acid that, in presence of neutral red indicator, results in Suspend 42,5 g of powder in 1 L of distilled water and the formation of red colonies. Lactose non-fermenting heat to the boil. Do not autoclave and pour into sterile produces colourless colonies. The black centres due to petri plates. The medium loses its efficiency if overheat- the ferric sulphide depot detect the production of SH2. ed and so avoid autoclaving and remelting.

Technique Description Inoculate the specimen as soon as possible directly onto The Deoxycholate-Lactose Agar is very close to the surface of medium. Incubate the plates at 35 ± 2ºC for Deoxycholate Agar, differing only in the deoxycholate 18-24 hours. Plates can be incubated for an additional amount and in its reduced inhibitory power. The present 24 hours if no lactose-fermenting are observed. formulation is made according to the recommendation of Typical colonial morphology on Deoxycholate Citrate APHA and AOAC. Agar is as follows: Escherichia coli: Large, ﬂat, rose-red References Enterobacter / Klesiella: Large, mucoid, pale with pink GREENBERG, A.E., L.S. CLESCERI & A.D. EATON centre. (1995) Standard Methods for the examination of Water Proteus: Large, colourless to tan. and Wastwater. 19th ed. APHA-AWWA-WEF. Washing- Salmonella: Large, colourless to tan. ton D.C. Shigella: Colourless to pink SPECK, M.L (1984) Compemdium of methods for the Pseudomonas: Irregular, colourless to brown microbiological examination of food.2nd ed. APHA. Gram-positive bacteria: No growth to slight growth Washington D.C. ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press, Boca Raton, Fla.

51 Dextrose Media

Dextrose Agar Dextrose Broth

Ref. 01-089 Ref. 02-089

Speciﬁcation Speciﬁcation General purpose solid culture medium. Liquid culture medium for general purposes.

Formula (in g/L) Formula (in g/L) Meat peptone ...... 5,0 Casein peptone ...... 10,0 Casein peptone ...... 5,0 D(+)Glucose ...... 5,0 Meat extract ...... 3,0 Sodium chloride ...... 5,0 D(+)Glucose ...... 10,0 Final pH 7,2 ± 0,2 Sodium chloride ...... 5,0 Agar ...... 15,0 Directions Final pH 6,9 ± 0,2 Dissolve 20 g of powder in 1 L of distilled water, heating up if necessary. Dispense into containers and sterilize by Directions autoclaving at 121°C for 10 minutes. Suspend 43 g of powder in 1 L of distilled water and heat to boiling . Sterilize by autoclaving at 121°C for Description 15 minutes. Should the acid pH is required, add sterile This version of Dextrose Broth is formulated without tartaric acid solution when the medium is at 45°C. Do meat extract in order to make dextrose the single carbo- not reheat. hydrate in the medium. It is a liquid culture medium for the faster growth, since most microorganisms can use Description glucose as their energy source, but it has one draw- This solid culture medium is suitable for many objectives back that it is not buffered, and the strong acidification and it supports growth of most non fastidious microor- produced by fermentation may damage its maintenance. ganisms. Adding 5% sterile defibrinated blood to this me- Hence, though it is used many times in blood cultures, it dium, makes it an excellent culture medium which satisfy has been replaced by other, more buffered media. nearly all kinds of nutritive needs, even for meningococci Due to the relatively high proportion of glucose, it is and pneumococci, however due to its high glucose con- advisable to use it freshly prepared, and with short tent it is not suitable for hemolytic reaction studies. sterilization period, as otherwise toxic furfurales may be This formulation is also in accordance with the one developed. suggested for the study of frozen food, and also for On the other hand, its simple formulation makes it the fruit juices. In this latter case, it is suggested to use it in best medium for checking gas production from glucose duplicate, and one of the samples must be acidified in if Durham’s tubes are used, as it has no indicator that order to facilitate the growth of moulds and yeast in such could interfere with it. a selective way. Acidification can be easily achievevd by adding 7-8 mL References of 10% sterile tartaric acid to the sterile, and cooled me- APHA (1958) Recommended Methods for the Microbio- dium at 45°C,producing a pH drop to 3,5 ± 0,2. In these logical Examination of Food. APHA. Inc, New York. conditions, do not remelt the medium, as then agar DOWNES, F.P. & K. ITO (2001) Compendium of Meth- tends to get hydrolysed and do not solidify again. ods for the Microbiological Examination of Food.4th ed. APHA. Washington. References MacFADDIN, J.D. (1985) Media for isolation-cultivation- APHA (1958) Recommended Methods for the Microbio- identification-maintenance of medical bacteria. William & logical Examination of Food. APHA. Inc, New York. Wilkins Co. Baltimore SPECK, M. L. (1984) Compendium of Methods for the Microbiological Examination of Food. 2nd ed. APHA. Dextrose Purple Broth Washington. VANDERZANT & SPLITTSTOESSER (1992) Compen- dium of Methods for the Microbiological Examination of Ref. 02-416 Food. 3rd ed. APHA. Washington. Specification Liquid medium for E.coli detection in water, according to German legislation (DEV).

52 Dextrose Media

Formula (in g/L) of foods, like Bacillus coagulans (causing the typical Peptone ...... 10,00 “ﬂat-sour”) other Bacillus and Sporolactobacillus and the Meat extract ...... 3,00 thermophilic Bacillus stearothermophilus. Sodium chloride ...... 5,00 Dextrose ...... 10,00 Technique Bromocresol purple ...... 0,02 The samples or its dilutions are inoculated in the me- Final pH 7,1 ± 0,2 dium, melted and cooled to 50ºC. Then are poured in petri dishes and incubated for 72 h at 32ºC (mesophiles) Directions or for 48 hours at 55ºC (thermophiles). After incubation Dissolve 28 g of powder in 1 L of distilled water. Dis- the acid-producing colonies can be easily enumerated tribute into tubes with Durham’s tubes and sterilize by because they show a yellow zone that contrast with the autoclaving at 121°C for 15 minutes. purple medium.

Description References Medium can be used for any assay of degradation of NATIONAL CANNERS ASSOCIATION (1968) Labora- sucrose. It has been adopted by the German Federal tory Manual for food canners and Processors. Vol. 1. Government for detecting E.coli in water, according to NCA. Washington Eijkman’s test, which is based on the production of acid DOWNES, F.P. & K. ITO (2001) Compendium of meth- and gas (yellow change of indicator) from sucrose after ods for the Microbiological Examination of Foods. 4th ed. an incubation of 20 hours at 44°C (±0,5). APHA. Washington. HORWITZ, W. (2000) Official Methods of Analysis. References AOAC International, Gaithersburg. MD. DIN Normative (Standards) 38411 (1991) Teil 6 (Juni 1991): Mikrobiologische Verfahren (Gruppe K): Nach- Dextrose Tryptone PB Broth weis von Escherichia coli und coliformen keimen (K6). DEUTSCHE EINHEITSVERFAHREN zur Wasser-, Ref. 02-556 Abwasser- Und Schlammuntersuchung. VCH Verlags- gesellschaft D-6940 Weinheim. Specification VERORDNUNG über Trinkwasser und über wasser fur Liquid medium for the microbiological examination of Lebensmittelbetreibe vom 12/12/1990. Bundesgesetzbl. canned foods Teil I 2613-2629 (1990) Formula (in g/L) Dextrose Tryptone PB Agar Tryptone ...... 10,00 Dextrose ...... 5,00 Ref. 01-556 Bromocresol Purple ...... 0,04 Final pH 6,9 ± 0,2 Specification Solid medium for the cultivating the “flat-sour” food spoil- Directions ing microorganisms Dissolve 15 g of powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and steri- Formula (in g/L) lize in autoclave at 121ºC for 15 minutes. Tryptone ...... 10,00 Dextrose ...... 5,00 Description Bromocresol Purple ...... 0,04 Baumgartner and Hersom proposed this medium, in Agar ...... 15,00 1956 for the microbial examination of canned foods and Final pH 6,9 ± 0,2 at the present it is recommended for all the medium and low acidity (pH 4,5) canned or heat-processed foods. Directions The microbial growth is supported by the peptone and Suspend 30 g of powder in 1 L of distilled water and the pH indicator that turns from purple to yellow detects bring to the boil. Distribute in suitable containers and the acid-producers from glucose. sterilize in autoclave at 121ºC for 15 minutes. Technique Description Volumes of 10-20 mL of medium in duplicate are inocu- This medium was adopted in 1930 by the National Can- lated with 1-2 g or mL of sample to detect the aerobic ners Association for the detection of the microorganisms microorganisms. For the anaerobic ones it is convenient causing the “flat-sour” spoilage in the canned foods. inoculate other series of tubes with a more reducer me- Latter it was used for the detection and enumeration dium like Liver Broth (Ref. 02-098). The incubation must of all the micro-organisms related with acid spoilage be at 35 and 55ºC for both series.

53 Dextrose Media

References BAUMGARTNER, J.G. & A.C. HERSOM (1956) Canned Foods. 4th ed. Churchill Ltd. London DOWNES F.P. & K. Ito (2001) Compendium of methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington. D.C.

Dichloran Glycerin Selective Agar (DG18 Agar)

Ref. 01-485 T The inclusion of 18% (w/v) of Glycerine makes the medium with a water activity (aw) of 0,995 without caus- R-45 Specification S-53-45 ing any problems that generally appear when this water Solid differential and low water activity medium for the activity is reached with sodium chloride or sugar. determination of xerophilic fungi from low moisture food. Technique It is advisable to inoculate in mass and thus a surface Formula (in g/L) inoculation is recommended. This seed may be by Peptone ...... 5,000 surface streaking as well as swab spreading or by Dri- Dextrose ...... 10,000 gaslky loop. Never use inoculum more than 0,1 mL. Monopotassium phosphate ...... 1,000 According to the standardized technique, plates must be Magnesium sulfate ...... 0,500 incubated at 22-25ºC, with partial readings after 3 and Dichloran ...... 0,002 5 days, and a definitive readings after 7-8 days. Results Chloramphenicol ...... 0,100 are expressed in cfu xerophiles/g or mL of food sample. Agar ...... 15,000 Final pH 5,6 ± 0,2 References HOCKING, A.D., J.I. PITT (1980) Dichloran-glycerol me- Directions dium for enumeration of xerophilic funghi from low-mois- Suspend 31,7 g of powder in 820 mL of distilled water ture food. Appl. Environm. Microbiol. 39:488-492 and heat to the boiling. Add 180 mL of glycerin. Distrib- PITT, J.I., A.D. HOCKING, D.R. GLENN (1983) An ute into suitable containers and sterilize by autoclaving improved medium for the detection of Aspergillus flavus at 121ºC for 15 minutes. and A. parasiticus. J. Appl. Bacteriol. 54:109-114. PITT, J.I., A.D. HOCKING (1985) Fungi and Food Spoil- age. Academic Press: Sydney Description SAMSON, R.A., E.S. HOEKSTRA (2001) Introduction Among the culture media for xerophilic fungi, those that to the Food Borne fungi. 6th. Ed. Centralbureau voor have played a more successful role are the ones which Schimmelcultures: Baarn. include any agent that restrains the continuous growth of zygomicete fungal colonies. Dichloran (Dichloreben- zalkonium cloride) and Rose bengal are two of those inhibitors. DG18 Agar medium is according to the formulation proposed by Hocking & Pitt in 1980, and it includes Dichlo- ran which limits the size of fungal colonies more ef- ficiently than Rose bengal. Chloramphenicol inhibits the bacterial growth very well and its thermostability allows it to be included in the medium before sterilization.

54 Differential Reinforced Clostridial Medium (DRCM)

Ref. 02-410 of anaerobiosis in the medium in the same assay. L- Cysteine acts as reducing agent in this medium. Specification Liquid medium for the enumeration of clostridia in food Technique samples and other products by MPN technique. Sample to be examined is distributed in tubes as per the MPN technique, and is covered with paraffin or vaseline oil to help the anaerobiosis. The bank of tubes is kept in Formula (in g/L) a boiling water bath at 75°C for 30 minutes to remove Peptone ...... 10,000 all the dissolved oxygen and vegetative cells. Then, Meat extract ...... 8,000 incubate at 30°C up to 7 days before concluding any Yeast extract ...... 1,000 negative results. Starch ...... 1,000 Generally, the spores of sulfate reducing clostridia Glucose ...... 1,000 germinate between the second and fourth day, and the L-Cysteine HCl ...... 0,500 medium turns black, in which case the test is positive. Sodium acetate ...... 5,000 The medium can be rendered selective by the addition of Sodium bisulfite ...... 0,500 70 IU/mL of Polymyxin sulftate. Ferric-ammonium citrate ...... 0,500 Prepared tubes without the inoculation may be stored Resazurine ...... 0,002 up to 2 weeks provided the resazurine band does not Final pH 7,0 ± 0,2 show an excessive oxidation (more than a 1/3 part of the column). Directions Dissolve 27,5 g of powder in 1 L of distilled water. Bring References to the boil, distribute in tubes and sterilize in the auto- GIBBS, B.M., FREAME, B. (1965) Methods for the re- clave at 121°C for 15 minutes. covery Clostridia from food. J.Appl. Bact. 36:23-33 FREAME, B., FITZPATRICK, B.W.F. (1972) The use of DRCM for the isolation and enumeration of Clostridia Description from Food. In Isolation of Anaerobics. Ed. Shapton AND This medium is a modification by Freame and Fitzpatrick Board. Academic Press. London. of the Gibb’s classic medium, to easily detect the pres- DIN Standard 38411 (1991) Teil 6 (Juni 1991): Mikro- ence of sulfite reducing clostridia. The modification is, biologische Verfahren (Gruppe K): Nachweis von Es- mainly, an addition of sodium bisulfite and ferric citrate, cherichia coli und coliformen keimen (K6). that make colonies black and thus more conspicu- ously visible. The current version of this medium has no agar in order to facilitate the blackness of the medium. Resazurine,the redox indicator allows the verification

55 DNAse Agar

Ref. 01-346 Mannitol fermentation may be simultaneously determined if 10 g of mannitol and 0,025 g of phenol red are Specification added to 1 L of DNAse Agar, before sterilization. Positive Solid culture medium for the determination of the deox- results in both tests will determine with more certainty yribonuclease activity of microorganisms, especially of that the microorganism is a pathogenic Staphylococcus staphylococci and Serratia sp. aureus. This medium is also useful to identify Serratia marcescens in clinical specimens, since it is a DNAse producer. Formula (in g/L) Davis (1967) modified the medium by adding toluidine Tryptose ...... 20,00 blue and crystal violet, and stated that gramnegative DNA ...... 2,00 DNAse producing bacilli that grew on this medium may Sodium chloride ...... 5,00 be accepted as Serratia species. Agar ...... 15,00 Final pH 7,3 ± 0,2 Tehnique DNAse Agar plates are inoculated with the microorgan- Directions ism to be studied by thick streak or inoculating at the Suspend 42 g of powder in 1 L of distilled water and heat bottom and are incubated at 35-37°C for a 18-24 hours to the boiling with constant stirring. Distribute into suit- period. able flasks and sterilize in the autoclave at 121°C for 15 To read, flood the plates with 1N chlorhydric acid and minutes. Cool it to 50°C and pour it into the plates. observe if there are any clear or transparent zones surrounding the streak. If the plate becomes totally turbid Description without any clear zone then the test is Negative however Jeffries, Holtman and Guse (1957) incorporated DNA if any clear zone developes around the growth, then the into a general medium with agar to study bacterial and test is accepted as Positive. fungal DNAse production. Microorganisms that are able to produce DNAse, break DNA, reducing it to nucleotide References fragments. JEFFRIES, C.D., D.F. HOLTMAN y D.G. GUSE (1957). This reaction is observed by the appearance of a clear Rapid Method for Determining the Activity of Microorgan- zone surrounding the growth, the rest of the plate re- isms on NucleicAcids. J. Bacteriol. 73:590-591 maining turbid. The process is as follows: Hydrochloric DISALVO, J.W. (1958). Desoxyrribonuclease and acid reacts with DNA producing white precipitates that Coagulase Activity of Micrococci. Med. Tech. Bull. U.S. make the medium turbid, although it does not react with Armed Forces. Med. J. 9:191 nucleotide fragments. SMITH, P.B., G.A. HANCOCK & D.L. RHODEN (1969) The same authors also observed that there is a correla- Improved médium for detecting deoxyrribonuclease-pro- tion between coagulase production and DNase activity, ducing bacteria. Appl. Microbiol. 18:991-993 thus DNAse Medium may be used as a laboratory test to diagnose pathogenic staphylococci.

control

Serratia marcescens ATCC 13880 Staphylococcus aureus ATCC 25923

56 EC Broth

Ref. 02-060 dence of coliform bacteria. Later confirmation will have to be done using any of the classical methods. Specification Should the incubation take place at 44,5°C, gas forma- Selective medium for the detection of enterobacteria, in tion could be interpreted as a confirmation of the pres- water and foodstuff according to ISO 9308-2 and 7251 ence of Escherichia coli. Nevertheless, it must be taken standards. into account that the validity of this test is highly limited by technical variations. A maximum incubation time of 24 hours in a water bath with a very precise temperature Formula (in g/L) regulation, is therefore recommended. Peptone ...... 20,0 When using the samples more than 10 mL, the medium Bile Salt n.3 ...... 1,5 must be reconstituted at a concentration equivalent Lactose ...... 5,0 to that specified on the directions, once the sample is Dipotassium phosphate ...... 4,0 added. Potassium dihydrogen phosphate ...... 1,5 Sodium chloride ...... 5,0 Final pH 6,9 ± 0,2 References APHA-AWWA-WEF (1998). Standard Methods for the Examination of Water and Wastewater, 20th ed. APHA, Directions Inc. Washington D.C., U.S.A. Dissolve 37 g of powder in 1 L of distilled water. Distrib- PERRY and HAJNA, (1943). Am. J. Pub. Hlth. 33:550. ute into tubes or containers with inverted Durham tubes DOWNES, F.P. & K. ITO (2001) Compendium of meth- (for gas production). Sterilize at 121°C for 15 minutes. ods for the microbiological examination of foods. 4th ed. APHA. Washigton DC Description ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- EC Broth is a buffered medium containing lactose. It is in biological Media. CRC Press, Inc. London. the range of selective broths for Enterobacteriaceae. Its ISO Standard 9308-2 (1990) Water Quality Detection efficiency or selectivity is based on bile salts’ inhibitory and enumeration of coliform organisms, thermotoler- effect on other microorganisms. ant coliform organisms and pressumptive E. coli - MPN This broth may be used for routine testing of water and method. food, either alone or by using the Most Probable Number ISO Standard 7251 (1993). Microbiology General method of enumeration. Guidance for enumeration of presumptive E. coli. M.P.N. The type of sample will determine how precise the Technique. results are. If the incubation is at 35-37°C for 48 hours, gas formation may be interpreted as presumptive evi-

E. coli Direct Agar (ECD Agar)

Ref. 01-484 Description Specification This medium is formulated according to the Swiss Stand- Solid culture medium for the detection of coliforms and ards for the detection of coliforms in the food. Although E.coli in water and food. the medium is complete by itself, it is recommended to add 5 g/L of glucose or lactose in order to make the Formula (in g/L) colonial growth more evident. Tryptone ...... 20,00 Direct detection is achieved by adding to the preparation Yeast extract ...... 5,00 the fluorescent agent (MUG, Ref. 06-102). After incuba- Bile salts ...... 1,50 tion of the sample dilution or filter membrane, E.coli Disodium phosphate ...... 5,00 colonies show a light blue fluorescence when examined Monopotassium phosphate ...... 1,50 under UV light. Sodium chloride ...... 5,00 Agar ...... 15,00 References Final pH 7,2 ± 0,2 ANONYMOUS. Schweizerisches Lebensmittelbuch. 5th. Ed. Chap. 56A. Directions ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Suspend 53 g of powder in 1 L of distilled water and add biological Media. CRC Press. Boca Raton. Fla. 5 g/L of carbohydrate. Heat to the boiling. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Cool to 50ºC and add 2 flasks/L of MUG Supplement (Ref. 06-102CASE)

57 EE Broth (Eur. Phar. Enrichment Broth Medium E)

Ref. 02-064 Usual recommended technique is as follows: sample to be studied is added to sterile broth at a proportion of Specification 10%. After strong homogenization, the mixture is incu- Liquid culture medium for the enrichment of enterobacte- bated for a period of18-20 hours at 35-37°C. Afterwards, ria from food samples according to ISO 8523 standard. subcultures are performed on a solid media appropiate for the selective enterobacteria isolation. For this step, Violet Red Bile Agar (Ref. 01-164) is specially recom- Formula (in g/L) mended, though there are also the MacConkey (Ref. Gelatin peptone ...... 10,000 01-118), Deoxycholate or Brilliant green based media. Dextrose ...... 5,000 From the suspected colonies on this media, identification Ox bile ...... 20,000 can be performed following the common methodology. Di-sodium phosphate ...... 6,450 Monopotassium phosphate ...... 2,000 Brilliant green ...... 0,015 References Final pH 7,2 ± 0,2 MOSSEL, VISSER and CORNELISSEN (1963) The examination of foods for Enterobacteriaceae using a test of the type generally adopted for the detection of salmonel- Directions lae J. Appl.Bact. 26:444-452 Suspend 43,5 g of powder in 1 L of distilled water and PASCUAL ANDERSON, MªRª. (1992) Microbiología heat at 100°C for 30 min. and cool immediately. Alimentaria. Díaz de Santos, S.A. Madrid,. EUROPEAN PHARMACOPOEIA,Supplement 4.2 Description (2002),2.6.13 Test for specified micro-organisms 4th As the name suggests, this medium is for the enrichment ed.,Council of Europe, Strasbourg. of enterobacteria, and is a modification by Mossel(1963) ISO 8523 Standard (1991) General guidance for the of the classic Brilliant Green Bile 2% Broth (Ref. 02-041). detection of Enterobacteriaceae with pre-enrichment. Substitution of lactose by glucose makes it more suitable for enteric bacteria detection, whether gas or non-gas- producer, in food and different samples.

Elliker Broth

Ref. 02-288 Description Medium was formulated by Elliker, Anderson and Han- Speciﬁcation nesson to cultivate lactobacilli and streptococci from milk Liquid culture medium for the enrichment of lactobacilli and other dairy products. Sodium acetate, in this me- and streptococci in the dairy industry. dium at this concentration, gives two advantages: on one hand it restrains gramnegative bacteria growth, and on Formula (in g/L) the other hand, it enhances lactic bacteria growth. The latter produce massive growth due to the high amount of Casein peptone ...... 20,0 sugars. Yeast extract ...... 5,0 Gelatin ...... 2,5 Dextrose ...... 5,0 Technique Lactose ...... 5,0 Readings are performed after 3-5 days of incubation at Sucrose ...... 5,0 37°C. Should a solid medium be desired, add 15 g/L of Sodium chloride ...... 4,0 Agar Bacteriological (Ref. 07-004). Sodium acetate ...... 1,5 Ascorbic acid ...... 0,5 References Final pH 6,8 ± 0,2 ELLIKER, P.R., ANDERSON, A.W., HANNESSON, G. (1956) An Agar Culture Medium for Lactic Acid Strepto- Directions cocci and Lactobacilli. J. Dairy Sci. 39:1611. Dissolve 48,5 g of powder in 1 L of distilled water, heat- HAUSLER, W.J. (1976) Standard Methods for the ing up to 50°C to totally dissolve gelatin. Distribute into Examination of Dairy Products, 14 Ed. (APHA). Wash- suitable containers, and sterilize by autoclaving at 121°C ington, D.C. for 15 minutes. MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products, 16 Ed. (APHA). Wash- ington, D.C

58 Endo Media

Endo Agar Base Endo Dev Agar Base

Ref. 01-589 Ref. 01-606

Specification Specification Solid selective medium for the detection of coliform and Selective agar for the isolation and differentiation of other enteric organisms, in milk and water, according to E.coli in water, according to the german legislation. the APHA specifications. Formula (in g/L) Formula (in g/L) Meat extract ...... 10,00 Peptone ...... 10,0 Peptone ...... 10,00 Lactose ...... 10,0 Lactose ...... 10,00 Sodium sulfite ...... 2,5 Sodium chloride ...... 5,00 Di-potassium hydrogen phosphate ...... 3,5 Sodium sulfite ...... 2,50 Agar ...... 15,0 Agar ...... 20,00 Final pH 7,2 ± 0,2 Final pH 7,3 ± 0,2

Directions Directions Suspend 41 g of powder in 1 L of distilled water. Bring Suspend 57,5 g of powder in 1 L of distilled water and to the boil and add 2 vials of Basic Fuchsin 250 Supple- bring to the boil. Add 2 vials of Basic Fuchsin 250 Sup- ment (Ref. 06-607CASE). Homogenize and distribute plement (Ref. 06-607CASE). Homogenize and distribute into suitable containers. Sterilize at 121°C for 15 min- into suitable containers. Sterilize in the autoclave at utes.Cool to 45-50°C, homogenize and pour into plates. 121°C for 15 minutes. Cool it to 45-50°C, homogenize Medium must appear slightly pinkish. If colour is very in- and pour into plates. tense red, it can be decolourised by adding a few drops In these conditions, medium must appear slightly pink- of a sterile solution of sodium sulfite 10% before pouring ish. If colour is very intense red, it can be decolourised it into the plates. Medium must be freshly prepared for by adding a few drops of a sterile solution of sodium the use, and must not be used when it is red. sulfite 10% before pouring it into the plates. Medium must be freshly prepared for the use, and must not be Description used when it is red. Endo Agar is used to confirm the detection of and to count coliform bacteria following presumptive test of Description drinking water, as well as for the detection and isolation Medium is a modification over the classical ENDO (Ref. of coliforms and fecal coliforms from milk, dairy products 01-589), according to the German legislation, to obtain a and other food. better detection of damaged coliforms. Since the buffer Inoculate the plates by the streak-plate method and system is removed in this medium, this formulation incubate for 24 hours at 37°C. includes a more rich nutrient base and sodium chloride Colonies of coliform bacilli, which ferment lactose, are to restore the osmotic balance. Agar concentration has pink to rose red, with or without green metallic sheen: been increased to keep the strengh of gel after the water marked reddening of the medium may occur. Colonies sample is incorporated. of other enteric bacilli, including Salmonella and of non On this medium, E.coli colonies appear red, with metallic lactose fermentors are about the same colour as the sheen, meanwhile Klebsiella and Enterobacter only take medium, being almost colourless to faint pink. on the red colour. Colonies of other enteric bacteria are On exposure to oxygen, the plated medium gradually colourless. becomes red due to the oxidation of sulfite and can thus no longer be used. It can only be kept for a few days Technique even if it is stored in the dark and at refrigerator tem- DEV standards recommends this medium to incubate perature. membrane filters used in the coliform detection and in the re-seed on the surface of suspicious colonies References for their confirmation and isolation. However, the agar APHA /AWWA/WEF, (1985). Standard Methods for the strength allows the incorporation of the sample to be as- Examination of Water and Wastewater, 15th ed., Inc. sayed in the medium mass, without any loss of consist- Washington D.C., U.S.A. ency. APHA (1967). Standard Methods for the Examination of For times and temperatures of incubation, it is recom- Dairy Products, 12th ed. , APHA Inc. Washington D.C., mended to follow the standard for every purpose, or fol- U.S.A. low the technician’s criteria. In any case, an incubation at ENDO, S (1904), Über ein Verfahren Zum Nachweis von 25-30°C for 24-48 hours usually provides good results. typhusbazillen. Zbl BaKt. Hyg. Abt. I, Orig, 35:109 MARSHALL, R.T. (1992) Standard Methods for the Examination of Dairy Products, 16th ed. , APHA Inc. Washington D.C., U.S.A. 59 Endo Media

References References DEUTSCHE EINHEITSVERFAHREN zur Wasser-, APHA-AWWA-WEF (1985) Standard Methods for the Abwasser- Und Schlammuntersuchung. VCH Verlags- Examination of Water and Wastewater. 16th. Ed. Wash- gesellschaft D-6940 Weinheim. ington DC. BUNDESGESUNDHEITSAMT: Amtliche Sammlung von ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Untersuchungverfahren nach. 35 LMBG Beuth Verlag biological Media. CRC Press Inc.,London Berlin Köln. DIN 38411: Teil 6 (Juni 1991): Mikrobiologische Ver- Endo MF Broth Base fahren (Gruppe K): Nachweis von Escherichia coli und coliformen keimen (K6). Ref. 02-605 Endo LES Agar Base Speciﬁcation Liquid medium especially formulated for the incubation Ref. 01-604 of membrane ﬁlters over the absorbent pads.

Specification Formula (in g/L) Medium for detecting and enumerating coliforms in water Casein peptone ...... 10,00 and food, by the membrane filtration method. Meat peptone ...... 10,00 Yeast extract ...... 1,50 Formula (in g/L) Lactose ...... 12,50 Peptone ...... 15,00 Sodium chloride ...... 5,00 Yeast extract ...... 1,20 Potassium phosphate ...... 5,75 Lactose ...... 9,40 Sodium lauryl-sulfate ...... 0,05 Sodium chloride ...... 3,70 Sodium deoxycholate ...... 0,10 Dipotassium phosphate ...... 3,30 Sodium sulfite ...... 2,10 Potassium phosphate ...... 1,00 Final pH 7,2 ± 0,2 Sodium deoxycholate ...... 0,10 Sodium laurylsulfate ...... 0,05 Directions Sodium sulfite ...... 1,60 Suspend 47g of powder in 1 L of distilled water. Heat Agar ...... 15,00 to boiling. Add 4 vials of Basic Fuchsin 250 Supplement Final pH 7,2 ± 0,2 (Ref. 06-607CASE) and homogenize. Optimum results are obtained using the media on the same day it is pre- Directions pared. Do not sterilize in the autoclave. Suspend 50,3 g of powder in 1 L of distilled water and bring to the boil. Add 4 vials of Basic Fuchsin 200 Sup- Description plement (Ref. 06-617CASE). Homogenize and distribute Endo Broth for membrane filtration has the suggested in sterile plates. Do not autoclave. Final medium is composition according to “Standard Methods” for detec- intense red in colour. tion and enumeration of coliforms by the membrane filtration method. Description Basically, it is the classical Endo medium, but selectivity Medium is a modification of the classical Endo’s me- has been improved by adding lauryl-sulfate and sodium dium, reformulated to use it with the membrane filtration deoxycholate and also the sodium sulfite and basic technique and a previous enrichment in Tryptose Lauryl fuchsin. The mixture of peptones and yeast extract pro- Sulfate Broth (Ref. 02-108) is always helpful and recom- vide the medium with a nutritive substrate by which most mended for achieving in more growths and more brilliant of the enteric bacteria can have the better recovery and colonies. the colonies of coliforms take on a characteristic metallic sheen. So,the members of Enterobacter, Citrobacter Technique and Klebsiella types, after 20-22 hours of incubation, Membrane(s) that have filtered the sample are incu- take a metallic green-blue sheen, but not so intense as bated at 35°C for 2-3 hours over a pad with Tryptose Escherichia coli. Lauryl Sulfate Broth (Ref. 02-108), and after this they are transferred to a plate with Endo LES Agar. Incubate at Technique 35°C for 24 hours. Coliform colonies appear red with a Endo Broth for membrane filtration may be used directly characteristic metallic sheen. or soaked in a pad as just by solidifying it with agar. In some cases, it seems that a better results are obtained if double-step method is used. That is, a short incubation of 2-3 hours on Tryptose Lauryl Sulfate Broth (Ref.

60 Endo Media

02-108) and a later incubation of 20-22 hours over Endo References Broth at 35°C (see Endo LES Agar Base, Ref. 01-604). APHA-AWWA-WEF (1995) Standard Methods for the The procedure suggested by APHA is ﬁltration of mini- Examination of Water and Wastewater, 19th ed. APHA mal amounts of water (50 mL), but in any case, mem- Washington. D.C. branes with more than 400 colonies must be rejected for FIFIELD, C.W. & C.P. SCHAUFUS (1958) Improved enumeration. In the direct method, as in the double-step membrane ﬁlter medium for the detection of coliform method, lactose fermenting colonies show a characteris- organisms.J. Amer.Water Works Assoc. 50:193 tic sheen, and they have to be considered as presumptive coliforms, meanwhile the lactose non fermenting colonies are colouress and transparent.

Salmonella typhimurium ATCC 14028 Escherichia coli ATCC 25922

control

Eosin Methylene Blue Agar (EMB Agar)

Ref. 01-068 It has been repeatedly recommended for the detection, enumeration and differentiation of members of the Specification coliform bacteria. Selective differential medium for the isolation of coliforms from water acc. ISO 21150 Standard. Technique The Weld method for the identification of Candida albi- Formula (in g/L) cans uses this medium with Chlortetracycline (100 mg/l) in a 10% CO environment. The method’s effectivity has Peptone ...... 10,000 2 Lactose ...... 10,000 been tested with a variety of samples, such as sputum, Dipotassium Hydrogen phosphate ...... 2,000 oral secretions, faeces, nails and vaginal secretions, all Yellowish Eosin ...... 0,400 of which provide definite results within 24-48 hours. sta- Methylene Blue ...... 0,065 phylococci are also easily identified, particularly coagu- Agar ...... 15,000 lase-positive strains. These have a very characteristic Final pH 7,1 ± 0,2 appearance: small colourless colonies with a central red nucleus. Nevertheless, the medium’s prevailing application is in Directions the differentiation of E. coli and E. aerogenes. Add 37,5 g to 1 L of distilled water. Bring to the boil and The medium should be sterilized once distributed into distribute in suitable containers. Sterilize in the autoclave tubes containing 20 mL of product each, and then be at 121°C for 15 minutes. refrigerated. Melt in a boiling water bath before use and stir until it acquires a dark purple colour. Pour a tube into Description each sterile plate and allow to solidify. It is advisable to A very versatile medium originally developed for the dry the medium’s surface before use, leaving the plate differentiation of E. coli and Enterobacter aerogenes. It open but inverted on a heater. has also proved very effective in the rapid identification For each doubtful lactose broth tube,inoculate one plate of Candida albicans and presents a high correlation with by streaking , and incubate for 24 to 48 hours at 37°C. the coagulase test for staphylococci. Examine afterwards.

61 Eosin Methylene Blue Agar (EMB Agar)

Escherichia coli and Citrobacter form ﬂat colonies of 2-3 References mm in diameter and are dark violet in colour with CLESCERI, L.S., A.E. GREENBERG & A.D. EATON. a black centre which produces a distinctive green (1998) Standard Methods for the Examination of Water metallic glow when light is reﬂected on it. and Wastewater. 20th edition. APHA-AWWA-WEF. Enterobacter and Klebsiella form convex colonies Washington D. C. which are twice as big as the very smooth E. coli HOLT-HARRIS, J. E. y TEAGUE O.A. (1916) A New Cul- , have no metallic glow and are pink in colour with ture Medium for the Isolation of Bacillus typhosus from a dark blue centre. Non-lactose fermenting organ- Stools J. Infect. Dis. 18:596-600. isms produce colourless colonies. ISO 21150:2006 Cosmetics – Detection of Escherichia

Candida albicans colonies incubated in a CO2 atmos- coli. phere have a very peculiar cotton-like appear- LEVINE, M (1918) Diferentation of E. coli and A. aero- ance which distinguishes them from other genes on simplified Eosin-ethylene Blue Agar. J. Infect. Candida species that produce classical yeast like Dis. 23:43-47. colonies. MENOLASINO, N.I., GRIEVES B. Y PAYNE P. (1960) Isolation and Identification of Coagulase Positive Sta- phylococci on Levine’s Eosin-Methylene Blue Agar. J. Lab. Clin. Med. 56(6) 908-910. WELD, J. (1953) Candida albicans: Rapid Identification in Cultures made directly from Human materials Arch. Dermat. Syph. 67(5):473-478. WINDLE TAYLOR, E. (1958)The Examination of Water and Water Supplies. Churchill Ltd. 7th ed. Londres. USP 29 – NF 24 (2006) 2nd Suppl. <61> Microbial Tests. USP Con. Inc. Rockville, MD, USA

Ethyl Violet Azide Broth (EVA Broth)

Ref. 02-028 Xn

R-22-32-52/53 Technique Specification S-7-46-61 Each of the EVA Broth tubes is inoculated with one or Medium for the confirmation of enterococci in water. two loops from a presumed positive Rothe Azide Broth (Ref. 02-027) flask, and are incubated for a 24-48 hours Formula (in g/L) period at 37°C. Enterococcus presence is noted by the Meat Peptone ...... 10,0000 turbidity in the medium. Casein Peptone ...... 10,0000 Occasionally a slight turbidity may appear accompanied Dextrose ...... 5,0000 by ample violet sediment at the bottom of the tube. Sodium chloride ...... 5,0000 Commonly, growth confirmation in this medium is Monopotasium phosphate ...... 2,7000 considered enough to state Enterococcus presence. Dipotassium phosphate ...... 2,7000 However, confirmative identification must be carried out Sodium Azide ...... 0,3000 by isolation in solid media and classification in one of the Ethyl Violet ...... 0,0005 four faecal enterococci species: Enterococcus faecalis, Final pH 6,8 ± 0,2 Enterococcus faecium, Enterococcus bovis and Entero- coccus equinum. Directions Dissolve 35,6 g of powder in 1L of distilled water, heating References up slightly if necessary. Distribute in tubes or flasks and LITSKY, MALLMAN and FIFIELD (1953) Amer.J.Publ. sterilize in the autoclave at 121°C for 15 minutes. Hlth 43:873 APHA-AWWA-WPCF (1995) Standard Methods for the Examination of Water and Wastewater. 19th. ed. APHA Description Washington. EVA Broth is a highly selective medium for some kinds SPECK, APHA/Intersociety (1976). Compendium of of enterococci, and it has been adopted by many Official methods for the microbiological examination of food. Organisations, National and International. Washington. Medium´s high selectivity is due to the presence of GUINEA, SANCHO and PARES (1979). Análisis micro- sodium azide and ethyl violet, as they inhibit other ac- biologicos de aguas: aspectos aplicados. Ed. Omega, companying bacteria, blocking their respiratory chains, Barcelona. leaving enterococci unaffected. In general, this medium is always used as a confirmation medium in the second stage, recommending an inoculum from a suitable medium such as Rothe Azide Broth (Ref. 02-027) to be inoculated in this medium.

62 FDA Broth (AATCC Bacteriostasis Broth)

Ref. 02-532 and disinfectants for antibacterial activities. It has the formula required by the AOAC for “Nutrient Broth” used Specification in Phenol Coefficient and others determinations. The Liquid medium used in routine antibacterial testing of AATCC uses this medium in the procedures for the de- antiseptics and disinfectants. tection of antibacterial activity of fabrics and in the production of the solid medium for this type of evaluations. Formula (in g/L) Meat peptone ...... 10,00 References Meat Extract ...... 5,00 AATCC (1985) 1986 Technical Manual of the AATCC Sodium chloride ...... 5,00 Vol. 61. Research Triangle Park. N.C. Final pH 6,9 ± 0,2 MACFADDIN J.F. (1985) Media for Isolation Cultivation- Identification-Maintenance of Medical Bacteria. William & Wilkins. Baltimore Directions ATLAS R.M, & LC PARKS (1993) Handbook of Microbio- Dissolve 20 g of powder in 1 L distilled water, heating if logical Media. CRC Press. Boca Raton Fla. necessary. Distribute in suitable containers and sterilize HORWITZ, W. (2000) Official Methods of Analysis. 17th. in autoclave at 121ºC for 15 minutes. Ed. AOAC International. Gaithersbourg, M.D. Description This medium is produced according the formulation specified in U.S. Food ad Drug Administration (FDA), Association of Official Analytical Chemists (AOAC) and American Association of Textile Chemists and Colour- ists (AATCC) procedures for the testing of antiseptics

Fecal Coliforms Media (FC Media)

Fecal Coliforms Agar (FC Agar) Fecal Coliforms Broth (FC Broth) Ref. 01-287 Ref. 02-287 Specification Solid, selective and differential medium for coliform enu- Specification meration by membrane filter technique. Liquid, selective and differential medium for coliform enumeration by membrane filter technique. Formula (in g/L) Tryptose ...... 10,0 Formula (in g/L) Yeast extract ...... 3,0 Tryptose ...... 10,0 Proteose peptone ...... 5,0 Yeast extract ...... 3,0 Bile salts # 3 ...... 1,5 Proteose peptone ...... 5,0 Sodium chloride ...... 5,0 Bile salts # 3 ...... 1,5 Lactose ...... 12,5 Sodium chloride ...... 5,0 Aniline blue ...... 0,1 Lactose ...... 12,5 Agar ...... 15,0 Aniline blue ...... 0,1 Final pH 7,4 ± 0,2 Final pH 7,4 ± 0,2

Directions Directions Suspend 52,1 g of powder in 1 L of distilled water and Suspend 37,1 g of powder in 1 L of distilled water and heat to boiling. Add two vials of Rosolic Acid Selective heat to boiling. Add two vials of Rosolic Acid Selective Supplement (Ref. 06-085CASE). Mix and pour into ster- Supplement (Ref. 06-085CASE). Do not autoclave nor ile Petri plates. Do not autoclave nor overheat. Use overheat. Use freshly prepared medium. freshly prepared medium.

63 Fecal Coliforms Media (FC Media)

Description of coliforms. Should a total E.coli selectivity be desired, FC Agar and Broth are formulated according to Geldre- incubate at 44,5°C. ich et al., to detect the faecal coliforms in polluted water. The bile salts included in these media make these References media selective for enterobacteria, and also selective GELDREICH, E.E, H.F.CLARK, C.B. HUFF and for coliforms due to the high temperature of incubation: L.C.BEST (1965) Fecal-Coliform-organism medium for 44,5°C±0,5°C. the membrane ﬁlter technique. J. Am. Water Works As- Freshly prepared medium has a red-garnet colour. Fae- soc, 57:208-214 cal coliform colonies are greenish-blue, and the medium APHA-AWWA-WPCF (1995) Standard Method for the also turns to this colour. In case of other bacteria, when examination of water and wastewater. 19th ed. APHA they grow, show pinkish colonies, and then the medium Washington DC. turns to ﬂuorescent red.

Technique Without Essentially, the technique consists of filtering the test Rosolic Acid sample to be examined through a membrane filter of suitable porocity (0,22-0,45 µm), assisting the filtration by pressure or suction, so that the microorganisms are retained on the membrane. Remove the membrane carefully and aseptically and take it to the culture medium. With Put the membrane over the agar,if using the solid me- Rosolic Acid dium, or over the impregnated pad if using the liquid version. Cover the Petri plates and incubate them at 37°C for 24 hours. After incubation, proceed with the counting Escherichia coli ATCC Salmonella typhimurium 25922 ATCC 14028 GE Motility Medium

Ref. 03-593 plates for motility studies, improving the original formulation by Jordan et al. Specification Semi-solid culture media for the motility test perform- Technique ance. After sterilization, cool the tubes by placing in cool water bath up to the depth of the medium (in a 16x160 Formula (in g/L) mm tube 8 cm depth or 15 mL of medium). For plates, Gelatin ...... 52,00 cool flasks of medium to 50ºC and pour into sterile petri Tryptose ...... 10,00 dishes to a dept of 5 mm or more and allow solidifying. Heart extract ...... 10,00 From an overnight culture spot the inoculum on the Sodium chloride ...... 5,00 surface or stab just below the medium surface. If tubes Agar ...... 5,00 are used, inoculate by depth stab inoculation. Incubation Final pH 7,2 ± 0,2 must be suitable in time and temperature to the suspected organism being tested. Periodically examine tubes or plates for growth and signs of motility at last for 7 days. Directions Suspend 82 g of powder in 1 L distilled water, heating until total solution. Distribute in suitable containers and References sterilize in autoclave at 121ºC for 15 minutes. ATLAS, R.M. & L.C. PARKS. (1993) Handbook of Microbiological Media. CRC Press. London MACFADDIN, J.F. (1985) Media for isolation-cultivation- Description identification-maintenance of medical bacteria. William & In this medium, the presence of diffuse growth away Wilkins, Baltimore MD from the line or spot of inoculation evidences motil- D’AMATO, R.F. & K.M. TOMFOHRDE (1981) Influence ity. Non-motile organisms growth only along the line of of media on temperature-dependant motility test for inoculation. In the medium all the components supplies Yersinia enterocolitica. J. Clin Microbiol. 14:347-348 nutrients and the little amount of agar introduces enough JORDAN, E.O. M.E CALDWELL & D. REITER (1934) strength to maintain solidity even at incubation tempera- Bacterial motility. J. Bacteriol. 27:165-173. tures, therefore, it is adaptable to use in both tubes and

64 Giolitti-Cantoni Broth

Ref. 02-230 first dilution (other products) may be inoculated in double strength medium. MPN procedures need at least three Specification tubes for at least three dilution steps. If no anaerobic jar Liquid medium for the recovery and enumeration of low is available, overlay with a layer of sterilized vaseline numbers of coagulase-positive staphylococci in foods (Ref. 6-077) or vaspar. Incubate anaerobically for 24-48 acc. to the ISO 5944:2001, IDF 60:2001 and EN-ISO h at 37°C. 6888-3:2003 Standards After 24 hours, subculture any tubes showing blackening Formula (in g/L) or black precipitate by streaking onto Baird-Parker Agar Tryptone ...... 10,0 (Ref. 01-030). Incubate the remainder of the tubes for Meat extract ...... 5,0 a further 24 h and subculture all tubes showing growth Yeast extract ...... 5,0 (irrespective of blackening) to Baird-Parker Agar. Lithium chloride ...... 5,0 D-Mannitol ...... 20,0 When determining the bacterial count by the MPN Sodium chloride ...... 5,0 method, all tubes showing growth are considered as Glycine ...... 1,2 presumptive positive for staphylococci and they are Sodium pyruvate ...... 3,0 confirmed only if they produce a positive result in the Polysorbate 80 ...... 1,0 coagulase test. Final pH 6,9 ± 0,2 Directions References Dissolve 55,2 g of powder in 1 L of distilled water. The CHOPIN, A. et altri (1985) ICMSF Methods Studies XV. medium can be prepared at single strength or double Comparison of four media and methods for enumerat- strength using double quantity of powder Distribute into ing Staphylococcus aureus in powdered milk. J. Food tubes dispensing 10 mL/tube (Single strength) or 20 Protect. 48:21-27 mL/tube (double strength). Sterilize by autoclaving at EN-ISO 6888-3 Standard (2003) Microbiology of food 121°C for 15 minutes. Cool and add 1% Potassium Tel- and animal feeding stuffs. Horizontal method for the enu- lurite Sterile Solution (Ref. 06-089) using 0,1 mL/tube for meration of coagulase positive staphylococci (Staphylo- single strength and 0,2 mL/tube for double strength. coccus aureus and other species). Part 3: Detection and MPN technique for low numbers. Description FIL-IDF (2001) Milk and milk based Products. Detection This medium for the selective enrichment of staphyloco- of coagulase-positive staphylococci. MPN technique. cci was formulated by Giolitti and Cantoni in 1966. Standard 60:2001. Brussels. GIOLITTI, G. A. CANTONI, C (1966) A medium for the The growth of staphylococci is promoted by pyruvate, isolation of staphylococci from foodtuffs. J.Appl. Bact. glycine and above all by a high concentration of man- 29, 395-398. nitol. Addition of Polysorbate 80 is necessary for the suc- HARRIGAN, WF. a. McCANCE, M.E. (1976) Labora- cessful recovery of Staphylococcus aureus (Chopin et tory Methods in Food and Dairy Microbiology. Academic al., 1985). Competitive microbiota is inhibited by lithium Press. London. chloride and potassium tellurite. Anaerobic growth condi- ISO 5944 Standard (2001) Milk and milk based Prod- tions increase the selectivity of the medium. Generally, ucts. Detection of coagulase-positive staphylococci. growth of staphylococci can be recognized by a blacken- MPN technique. ing or black precipitates in the culture medium due to reduction of tellurite to metallic tellurium.

The prepared basal culture medium can be stored for about 1-2 weeks in the refrigerator. The ready-to-use medium must be used the same day of preparation. It is advisable that the stored basal medium be degasiﬁed at the moment of use by heating for 15 minutes at 100°C, cool rapidly and add sterile potassium tellurite solution. Technique Refer to the standard protocol for speciﬁc products (Food and animal feeding stuffs EN-ISO 6888-3:2003; Milk and milk based products ISO 5944:2001 and FIL- IDF 60:2001). As a general technique the following is suggested:

Use food macerates or decimal dilutions and inoculate 1 mL to single strength medium. To lower the detection limit 10 mL of the test sample (liquid products) or of the

Left: control; right: Staphylococcus aureus ATCC 25923

65 Glucose Bromcresol Purple Agar

Ref. 01-502 Description This medium is used to confirm enterobacteria by their Specification ability to ferment glucose. Glucose fermentation can be Solid medium for the confirmation of enterobacteria detected by the production of acid that change the colour in diverse samples according to ISO 4702 and 8523 of the medium to yellow. standards. References Formula (in g/L) ISO 4702 Standard (1993) Microbiology General Guid- Tryptone ...... 10,000 ance for the enumeration of entrobacteriaceae without Yeast extract ...... 1,500 ressucitation. MPN technique and colony count tech- Dextrose ...... 10,000 nique. Sodium chloride ...... 5,000 ISO 8523 Standard (1991) Microbiology General Guid- Bromcresol purple ...... 0,015 ance for the detection of Enterobacteriaceae with pre- Agar ...... 15,000 enrichment. Final pH 7,0 ± 0,2 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press. Boca Raton. Fla. Directions Suspend 41,5 g of powder in 1 L of distilled water and bring to the boil. Distribute into containers and sterilize by autoclaving at 121ºC for 15 minutes.

Glutamate Starch Pseudomonas Agar (GSP Agar)

Ref. 01-092 tion of starch, which is degraded by Aeromonas with acid production which causes change in colour of phenol Specification red to yellow. Pseudomonas also grows on this medium Solid, semiselective and differential solid medium for the but do not degrade starch and there is no acid produc- isolation of Pseudomonas and Aeromonas from very tion and thus their colonies remain blue-violet. contaminated samples. Technique Formula (in g/L) Antibiotics are added to this medium after sterilization Sodium L(+) glutamate ...... 10,00 and cooling to 50°C, in such a way that final concentra- Soluble starch ...... 20,00 tions in the medium are 100 u/mL of penicillin and 10 Monopotassium phosphate ...... 2,00 mcg/mL of piramicine (it may be replaced by amphoter- Magnesium sulfate ...... 0,50 icin or nystatin). Phenol red ...... 0,36 After solidification in plates, medium may be used by Agar ...... 15,00 surface inoculation or by leaving for the membrane Final pH 7,2 ± 0,2 filters. Aeromonas colonies turn to yellow, and Pseu- domonas ones do not. The incubation is performed at room temperature (20- Directions 25°C) for 3 days. Sometimes, enterobacteria may also Suspend 48 g of powder in 1 L of distilled water and grow but very slowly with pinpoint colonies. bring to the boil. Dispense in tubes or flasks and sterilize by autoclaving at 121°C for 15 minutes. Cool to 50°C and add 100.000 I.U. of sodium G penicillin and 0,01g of References pimaricine per litre. Pour into sterile plates. KORTH, H. (1963) Ein Nährboden zur Züchtung von Pseudomonaden. Zbl.Backt.Parasit. Hyg. Abt. 190:225 STANIER, R., N. PALLERONI, M. DOUDOROFF. (1966) Description The aerobic pseudomonads: A taxonomic study. J. Gen. This formulation is according to Kielwein’s modification Microbiol. 42:159-271 to Korth’s medium, improving the latter because it sup- KIELWEIN, G. (1971) Die Isolierung und Differenzierung ports growth of almost all types of Pseudomonas and von Pseudomonaden aus Lebensmitteln. Arch G. Leb- Aeromonas. ensmillelhyg. 22:29-37. Selectivity is achieved due to the antibiotics (penicillin and pimaricine) and glutamate, which is hard to metabo- lize by gram-negative bacteria. Differentiation between Pseudomonas and Aeromonas is based on the utiliza-

66 Gram Negative Broth (GN Broth)

Ref. 02-093 content of citrate and deoxycholate. On the other hand, mannitol restrains the growth of Proteus and facilitates Specification the proliferation of Salmonella and Shigella. Liquid culture medium for enteric bacteria according The medium is strongly recommended for primary Hajna’s formulation. enrichment, 14-16 first hours, before going to selective media such as EMB (Ref. 01-068) or MacConkey (Ref. 01-118). Its author, Hajna, declares an extraordinary Formula (in g/L) selectivity of the medium, whatever may the origin of the Peptone ...... 20,0 sample, if everything is kept in a transport medium upto Dextrose ...... 1,0 the inoculation. D-Mannitol ...... 2,0 Sodium citrate ...... 5,0 Sodium deoxycholate ...... 0,5 References Di-potassium phosphate ...... 4,0 HAJNA, A.A. (1955) A new enrichment medium for gram- Monopotassium phosphate ...... 1,5 negative organisms of the intestinal group Pub.Hlth.Lab Sodium chloride ...... 5,0 13:83 Final pH 7,0 ± 0,2 EDWARDS and EWING (1973). Identification of Entero- bactericeae. Burgess Pub.Co. Minneapolis. VANDERZANT & SPLITTSTOESSER (1992). Compen- Directions dium of Methods for the Microbiological Examination of Dissolve 39 g of powder in 1 L of distilled water. Dis- Food. 3rd. Ed. APHA. Washington. pense in tubes or flasks and sterilize in the autoclave at DOWNES, F.P. & K.ITO (2001) Compendium of Meth- 121°C for 15 minutes. ods for the Microbiological Examination of Food. 4th ed. APHA. Washington. Description GN Broth (Gram Negative Broth) is an enrichment and selective medium for enterobacteria, with a strong inhibitory action against gram-positive because of its high

HC Agar Base

T Ref. 01-298 Description R-45 The HC Agar was developed by Mead & O’Neill in 1986 S-53-45 Speciﬁcation to attain reliable enumeration of moulds in cosmetic Selective solid medium for the enumeration of molds in products in short time. The nutrient basis of the medium cosmetic products. s the dextrose with the peptones and yeast extract that supplies the energy, nitrogen and vitamins and growth Formula (in g/L) factors. The inorganic ions are given by ammonium chloride and magnesium sulphate and both phosphates acts Tryptone ...... 2,50 buffering the medium. Sodium carbonate and polysorb- Proteose peptone ...... 2,50 ate are detoxiﬁers and neutralising preservatives and Yeast Extract ...... 5,00 others toxic substances. The selectivity against bacteria Dextrose ...... 20,00 is due to the chloramphenicol. Disodium phosphate ...... 3,50 Monopotasium phosphate ...... 3,40 Ammonium chloride ...... 1,40 Technique Magnesium sulphate ...... 0,06 Suitable sample is inoculated into surface of me- Sodium carbonate ...... 1,00 dium plates per duplicate and incubate aerobically at Chloramphenicol ...... 0,10 27,5±0,5ºC for 72 hours. Count colonies of moulds from Agar ...... 15,00 duplicate plates and record average count of mould Final pH 7,0 ± 0,2 count per g or mL of sample.

Directions References Suspend 54,6 g of powder in 1 L of distilled water and MEAD, C. & J. O’NEILL (1986) A three-day mould as- bring to boil. Add 20 mL of polysorbate 80 and homog- say for cosmetics and toiletries. J. Soc. Cosmet. Chem. enize. Distribute in suitable containers and sterilize in 37:49-57 autoclave at 121ºC for 15 minutes.

67 Heart Extract Broth

Ref. 02-564 Description The Heart Extract Broth is a very old and classical gen- Speciﬁcation eral purpose medium that can be used for the cultivation Liquid medium for fastidious microorganisms cultivation of fastidious microorganisms. Its components supply a very rich nutritive base which supplemented with suitable Formula (in g/L) additives can be used in all laboratory purposes, from Blood Agar Base until Base for carbohydrate fermenta- Heart Extract ...... 10,00 tion studies. Tryptose ...... 10,00 Sodium Chloride ...... 5,00 Final pH 7,4 ± 0,2 References HUNTOON, F.M. (1918) “Hormone” Medium. A simple Directions medium employable as a substitute for serum medium. J. of infect. Dis. 23:169-72 Dissolve 25 g of powder in 1 L of distilled water, heating ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- if necessary. Distribute in suitable containers and steri- biological Media. CRC Press Boca Ratón Fl. lize in autoclave at 121ºC for 15 minutes. MAC FADDIN, J.F. (1985) Media for Isolation-Cultiva- tion-Identiﬁcation-Maintenance of Medical Bacteria. William & Wilkins. Baltimore. FDA (1998) Bacteriological Analytical Manual. 8th ed. Rev.A. AOAC International. Gaithersburg. MD.

Hektoen Enteric Agar

Ref. 01-216 Technique In order to avoid the spreading of Proteus, it is neces- Specification sary that the agar surface be perfectly dry at the moment Solid, selective and differential culture medium for isola- of inoculation. Inoculation must be carried out by surface tion of pathogenic enterobacteria from very contami- streaking, directly from rectal swabs or faeces dilutions. nated samples acc. ISO 21567. If colonies are well separated after 18 hours of incubation, first characteristic appearances or colony characters may be observed, as then those colonies become Formula (in g/L) more prominent after a longer period: Peptone ...... 12,00 Shigella spp., Proteus inconstans: Raised colonies, Yeast extract ...... 3,00 humid, green colour. Bile salts ...... 9,00 Salmonella sp.: Green-blue colonies, with or without Lactose ...... 12,00 black core. Sucrose ...... 12,00 Pseudomonas spp.: Irregular colonies, plain, green or Salicin ...... 2,00 brown. Sodium chloride ...... 5,00 Companion and non pathogenic bacteria : Salmon colour Sodium thiosulfate ...... 5,00 colonies. Ammonium ferric citrate...... 1,50 Acid fuchsin ...... 0,10 References ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- Bromothymol blue ...... 0,06 biological Media CRC Press. BocaRaton. Fla. USA Agar ...... 15,00 HORWITZ, W. (2000). Official Methods of Analysis of the Final pH 7,7 ± 0,2 AOAC Internacional 17th ed. Gaithersburg Md. USA DOWNES, F.P. & K. ITO (2001) Compendium of Directions Methods for the Microbiological Examination of Suspend 77 g of powder in 1 litre of distilled water and Foods.4th ed. APHA. Washington DC. USA. let it soak. Heat up by constant stirring until boiling. FORBES, B.A., D.F SAHM & A.S. WEISSFELD (Eds) Cool to 55-60°C and pour into sterile plates. Do not (1998) Bailey & Scott’s Diagnostic Microbiology 10th ed. autoclave. This medium is very thermolabile and thus Mosby. St Louis, Mo. USA MURRAY, P.R., E.J. BARON, J.H. JORGENSEN, M.A. overheating should be avoided. PFALLER & R.H. YOLKEN (Eds) (2003) Manual of Clini- cal Mcrobiology 8th ed. ASM Press. Washington DC, USA Description KING S.y METZGER W. Y. (1968). A new plating method This culture medium, originally developed by King and for the isolation of the enteric pathogens. Appl. Microbiol. Metzger, has a high nutrient content like peptones, 16:577. fermentable sugars and combination of indicators which US FDA (1998) Bacteriological Analytical Manual 8th makes this medium less toxic. All these characteristics ed. AOAC Internacional. Gaithersburg, Md. USA. and the bile salts makes it a very selective and effective ISO 21567. Standard (2004) . Horizontal method for the medium. detection of Shigella ssp. 68 m-HPC Agar

Ref. 01-593 Technique Sampling Specification Refer to the Section 9060 of the Standard Methods or Solid medium for the enumeration of heterotrophic mi- the ISO 5667 Standard. In any case the time between cro-organisms from water by filter membrane technique the sampling and analysis must exceed 8 hours (6 for transfer and 2 for analysis). The samples can be refriger- Formula (in g/L) ated but never chilled. After 24 hours refrigeration the samples must be rejected. Peptone ...... 20,00 Procedure Gelatine ...... 25,00 A suitable volume to obtain 20-200 colonies on the 47 Agar ...... 15,00 mm filter must be filtered. Grilled filter of 0,45 µm pore Final pH 7,1 ± 0,2 are preferred. The funnel and the filter are washed three times with 20-30 mL of sterile water. The filter is placed Directions on the surface of a m-HPC Agar plate and incubated Suspend 60 g of powder in 1 L of distilled water and for 48 h. at 35±2ºC. Express the results as “Cfu/unity of bring to the boil. Add 10 mL of glycerol and homogenize. volume filtered” Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. References TAYLOR, R.H & E.E. GELDREICH (1979) A new mem- Description brane filter procedure for bacterial counts in potable wa- The membrane-Heterotrophic Plate Count Agar was ter and swimming pool samples. J. Amer. Water Works developed in 1979 by Taylor and Geldreich as an adap- Assoc. 71:402-405 tation to the membrane filtration method of the Standard EATON, A.D., .S. CLESCERI & A.E. GREENBERG (Eds) Plate Count Medium and it is also know as m-SPC. (1995) Standard Methods for the Examination of Water and In this medium peptone supplies all he nutrients and Wastewater. 19th Ed. APHA Washington DC. gelatine overcomes the trouble of liquefaction and overgrowth of the colonies. The m-HCP is recommended by Standard Methods for Examination of Water and Waste- water in the filtration technique applied to the greats volumes of water with a low microbial population. Indole Nitrite Fluid Medium

Ref. 03-101 such as Lactobacillus or Clostridium grow well on this medium, and indole and nitrite tests can be carried out. Speciﬁcation Adding 2 g/L of Bacteriological Agar to this medium can General purpose medium for indole production tests and make it suitable for motility test. Indole production can nitrite detection. be observed with Kovacs’ Reagent (Ref. 06-018) with or without previous extraction with chloroform, and in another tube, nitrate assay can be carried out with Nitrate Formula (in g/L) reagents (Ref. 06-003 and Ref. 06-004). Casein peptone ...... 20,0 This medium is not the most suitable one for indole Di-sodium phosphate ...... 2,0 production detection in enterobacteria, because some Dextrose ...... 1,0 times there are some interferences. For that assay, SIM Potassium nitrate ...... 1,0 Medium (Ref.3-176) or Nitrate Broth (Ref. 02-138) are Agar ...... 1,0 more suitable. Final pH 7,2 ± 0,2 Nonetheless, Indole Nitrite Broth is the medium of choice for nitrate reduction assays or denitriﬁcation assays with Directions any other bacterial biotype especially the grampositive Suspend 25 g of powder in 1 L of distilled water. Heat to ones . boiling and dispense in tubes. Sterilize at 121°C for 15 minutes. If tubes are kept in refrigerator, heat them up in References boiling water bath for 2 minutes before using them. VANDERZANT & SPLITTSTOESSER (1992). Compen- dium of Methods for the Microbiological Examination of Description Food.3rd. Ed. APHA. Washington. The extraordinary richness of this medium, due to high quality and quantity of peptone, allows its usage as a general medium. Even the fastidious microorganisms

69 Inhibitory Substances Test Media

Speciﬁcation Assay Agar at pH 7,2 Solid media for the veriﬁcation of antimicrobial inhibitor substances in food and packaging material. Ref. 01-439

Assay Agar at pH 6,0 Formula (in g/L) Casein peptone ...... 3,60 Ref. 01-437 Meat peptone ...... 3,60 Sodium chloride ...... 5,00 Formula (in g/L) Trisodium phosphate ...... 0,80 Casein peptone ...... 3,45 Agar ...... 15,00 Meat peptone ...... 3,45 Final pH 7,2 ± 0,1 Sodium chloride ...... 5,10 Agar ...... 15,00 Directions Final pH 6,0 ± 0,1 Suspend 27 g of powder in 1 L of distilled water and heat to the boiling. Distribute into suitable containers and Directions sterilize in the autoclave at 121°C for 15 minutes. Check Suspend 27 g of powder in 1 L of distilled water and heat the pH and adjust it if necessary. Cool to 50°C and add to the boil. Distribute in the suitable containers and steri- 50 mcg/L of Trimetoprim. Inoculate with the spore sus- lize in the autoclave at 121°C for 15 minutes. Check the pension of Bacillus subtilis to obtain a confluent culture. pH and adjust it if necessary. Cool to 50°C and inoculate Homogenize well and pour the medium into sterile with the spore suspension of Bacillus subtilis to obtain a plates, 15 mL per plate. Once the agar is solidified, put confluent culture. the plates into the refrigerator until their use. Homogenize well and pour the medium into sterile All the plates must be sealed with an adhesive and plates, (15 mL per plate). Once the agar is solidified, put water-proof tape, named, and packed in plastic bags the plates into the refrigerator until their use. before storing them into the refrigerator until their use. If they are stored at 3-6°C, plates may be thus stored up Assay Agar at pH 8,0 to 3 weeks. Do not freeze the plates, and never put them back in the refrigerator once they have reached 10°C.

Ref. 01-438 Technique Assay may be performed with 2 or 4 plates. If two plates Formula (in g/L) are used, one must be at pH 6.0 and the other one at pH Casein peptone ...... 3,45 8.0, and both must be inoculated with Bacillus subtilis. Meat peptone ...... 3,45 If four plates are used, include another plate at pH 7,2, Sodium chloride ...... 5,10 inoculated with Bacillus subtilis too, and another plate at Trisodium phosphate ...... 2,40 pH 8,0 inoculated by Micrococcus luteus. Agar ...... 15,00 If the sample is solid, use a punch to extract 8 mm x 2 Final pH 8,0 ± 0,1 mm diameter cylinders. If the sample is liquid, use antibiogram assay discs. Sample pieces or discs are placed Directions over the plates surface, 2 per plate, on all the plates. Suspend 29,4 g of powder in 1 L of distilled water and As a control, put discs alternatively with the samples heat to the boiling. Distribute into suitable containers and in each plate, impregnated with the following standard sterilize in the autoclave at 121°C for 15 minutes. Check substances: the pH and adjust it if necessary. Cool to 50°C. Inoculate Assay Medium at pH 6,0: Penicillin 0,01 IU/disc a part of medium with the spore suspension of Bacillus Assay Medium at pH 8,0: Streptomycin 0,5 mcg/disc subtilis to obtain a conﬂuent culture. Inoculate the other Assay Medium at pH 7,2: Sulfamidin 0,5 mcg/disc part of medium with a cell suspension of Micrococcus luteus ATCC 9341. Plates are incubated at 30°C for 18-24 hours, except Homogenize well and pour the medium into sterile those inoculated with M. luteus, which are incubated at plates, (15 mL per plate). Once the agar is solidiﬁed, put 37°C. all the plates into the refrigerator until their use. After the incubation period, read the inhibition halos (zones of inhibition), but do not consider the diameter of the discs or cylinders. Assay is considered reliable when control discs provide ø12 mm halos, and then samples with average diameter greater than ø4 mm are considered positive (with inhibitor presence), and samples with ø1-4 mm halos must be considered doubtful.

70 Inhibitory Substances Test Media

References BAUR, E. (1975) Untersuchungen von Fleisch und Wurstwaren mit dem Hemmstoff im Rahmen das Tierärt- zlichen Lebensmittelüberwachung. Fleischwirtschaft 55:843-845. BOGAERTS, R., F. WOLF (1980) Eine standardisierte Methode sum Nachweis von Rückständen antibakterial wirksauer substanzen in ﬁschen Fleich. Fleischwirtschaft 60: 667-675 DEUTSCHES FLEICHBESCHANGESETZ: Aus- führungsbestimmungen A über die Untersuchung und Gesundheitspolizeiliche Behandlung der Schlachtiere und des Fleisches bei Schlachtungen im Inland. Anlage 1 zu § 20 Abs. 4: Vorschriften über die Bakteriologische Fleischuntersuchung.

Iron Sulﬁte Agar

Ref. 01-328 suitable for clostridia. The actual formulation is according to the one widely experimented by several authors, and Specification provides a relatively low false negative results. Medium for detection and identification of sulfite reduc- Most of clostridia have sulfite reductases in their cito- ing clostridia. plasm, but they are unable to expel them to the exterior. So, when H2S is produced from sulfite, the colony becomes dark due to the formation of precipitates of iron Formula (in g/L) sulfide from citrate, which is less toxica than the alum Tryptone ...... 10,0 described by Wilson and Blair. Sodium sulfite ...... 0,5 Note that there are many gram negative bacteria able to Ferric citrate ...... 0,5 reduce sulfite with iron sulfide production in this medium, Agar ...... 15,0 but in these cases the enzymes are extracellular and all Final pH 7,1 ± 0,2 the medium becomes dark, rendering their enumeration impossible. Directions Dissolve 26 g of powder in 1 L of distilled water, heating Technique up to boiling with constantl stirring. Distribute in suitable To enumerate sulfite reducing clostridia, vegetative containers and sterilize in the autoclave at 121°C for 15 cells have to be eliminated by heating up the sample to minutes. 80°C for 10 minutes. Sample is cooled under water and mixed aseptically with an equal volume of sterile, double Description concentrated, melted and cooled to 60°C medium. Let it Although the medium was originally described by Wilson solidify and incubate at 37°C for 48 hours, with visual ex- Blair, it remained unused because it was not safe. There amination after 24 hours. Enumerate the black colonies, have been many modifications, and the one by Tanner which clearly contrast with the medium, and express in 1944 for the National Canners Association of America the result as spores of sulfite reducing clostridium per was the more lasting. However, it was also modified volume of sample. because it was demonstrated that clostridia were highly Tubes with blackish medium, without discrete colonies, inhibited at concentrations of sulfite over 0,1%, this be- must be rejected and the assay has to be restarted heating the reason why the Wilson Blair formulation was un-

71 Iron Sulfite Agar ing up the sample at the same temperature but for additional 5 minutes more than the previous heating time. Should thermophilic species are desired to be enumerated, incubate at 55°C. If the assay is performed by membrane filtration, it is advisable to put the membrane over a layer of medium solidified in the plate, and then cover it with another layer of medium (20 mL) melted and cooled to 60°C.

References TANNER, F.W. (1944) The Microbiology of Food. 2 Ed. Garrad Press U.S.A. BUTTON, A.W.J. (1959) A note on the enumeration of thermophilic sulfate-reducing bacteria. J. Appl. Bact., 22(2) 278-280. SCARR, M.P. (1959) Selective Media Used in the Micro- biological Examination of Sugar Products. J. Sci. Food Agri., 678-681.

Left and center: Clostridium perfringens ATCC 13124; right: control.

Kanamycin Esculin Azide Media (KAA Media)

Kanamycin Esculin Azide Agar Kanamycin Esculin Azide Broth (KAA Agar) (KAA Broth)

Xn Xn Ref. 01-263 Ref. 02-263 R-22-32-52/53 R-22-32-52/53 S-7-46-61 S-7-46-61 Specification Specification Solid medium for confirmative detection and isolation of Liquid medium for the presumptive detection of Lance- Lancefield’s group D streptococci in food samples, ac- field’s group D streptococci in food samples, according cording to Mossel et al. to Mossel et al.

Formula (in g/L) Formula (in g/L) Tryptone ...... 20,00 Tryptone ...... 20,00 Yeast extract ...... 5,00 Yeast extract ...... 5,00 Sodium chloride ...... 5,00 Sodium chloride ...... 5,00 Disodium citrate ...... 1,00 Disodium citrate ...... 1,00 Esculin ...... 1,00 Esculin ...... 1,00 Ferric-Ammonium citrate ...... 0,50 Ferric-Ammonium citrate ...... 0,50 Sodium azide ...... 0,15 Sodium azide ...... 0,15 Kanamycin sulfate ...... 0,02 Kanamycin sulfate ...... 0,02 Agar ...... 15,00 Final pH 7,0 ± 0,2 Final pH 7,0 ± 0,2 Directions Directions Dissolve 33 g of powder in 1 L of distilled water. Distrib- Suspend 48 g of powder in 1 L of distilled water and let it ute in suitable containers and sterilize in the autoclave at soak. Heat to boiling and distribute into suitable contain- 121°C for 15 minutes. ers. Sterilize in the autoclave at 121°C for 15 minutes.

72 Kanamycin Esculin Azide Media (KAA Media)

Description in the hypersaline broth, and therefore, definitive identifi- KAA Presumptive Broth and Confirmative Agar are the cation has to be performed by serological methods. two media that the Spanish National Center for Food and Nutrition (CeNAN) recommend to detect, enumerate This methodology does not allow the enumeration of and isolate Lancefield’s group D streptococci in samples cells from the original sample, and as this is a neces- of food and beverages like: bottled water, fresh, refriger- sary data, it is recommended to use the Most Probable ated, frozen or mashed meat, fish and molluscs, soft Number (MPN) technique with the presumptive broth, drinks, pastries, spices and semiconserves. Kanamycin doing it at double strength if necessary. For bottled and sodium azide are the selective inhibitory com- water, soft drinks and molluscs, CeNAN suggest the fol- pounds. lowing technique: Prepare broth tubes at normal concentration and at Technique double strength. Using a sterile pipet, inoculate five broth Prepare tubes with 9 mL of broth, and Petri plates with tubes of double strength with 10 mL of sample. Inoculate the agar. Make a decimal dilution bank from the sample five tubes of normal concentration with 1 mL of sample in duplicate, and inoculate 1 mL fractions in the tubes. and five tubes of normal concentration with 0,1 mL of Incubate at 37°C for 24 hours. sample. Homogenize them well and incubate at 37°C for 48 hours. Tubes that show a blackish-brown colour Presumptive presence of streptococci is indicated by the after the incubation period, are considered positive. Note development of a blackish-brown colour and the loss of down the results and carry out the counting using the fluorescence behind Wood’s light. These tubes are con- MPN tables. sidered as positive, and then inoculate 0,1 mL aliquotes from them over the surface of plates with Confirmative References Agar, spreading with a Drigalsky loop (Ref. 5-010). GUINEA, J., SANCHO, J., PARES, R. (1979). Análisis Incubate those plates, in inverted position, at 37°C for 24 Microbiológico de Aguas. Ed. Omega, Barcelona,. hours. Colonies that appear surrounded by a black halo MOSSEL, D.A.A., P.G.M. BUKER, J. ELDERING (1978) are considered as group D streptoccoci, and are isolated Streptokokken der Lancefield Gruppe D in Lebensmitteln to confirm them biochemically and morphologically with und Trinkwasser. Arch F. Lebensmittelhyg. 29:121-127 the following tests: microscopical examination, catalase PASCUAL ANDERSON, MªRª (1992) Microbiología assay (that should be negative) in an azideless medium, Alimentaria. Diaz de Santos, S.A. Madrid. growth at 45°C and resistance to a high saline concen- VANDERZANT & SPLITTSTOESSER (1992). Compen- tration (6,5% of NaCl in BHI Broth (Ref. 02-102). Finally, dium of Methods for the Microbiological Examination of they have to grow in Bile Esculin Agar (Ref. 01-265) with Food.3rd. Ed. APHA. Washington. a similar appearance of the colonies in the Confirmative Agar. Nonetheless, there are some exceptions to this rule, i.e. Streptococcus equinus and S.bovis do not grow

73 Kenner Fecal Media (KF Media)

Kenner Fecal Agar (KF Agar) Directions

Xn Suspend 56,4 g of powder in 1 L of distilled water. If it Ref. 01-294 has to be used immediately, it may not to be sterilized, R-22-32-52/53 just heat to boiling for one or two minutes, with constant S-7-46-61 Specification stirring. Otherwise, sterilize in the autoclave in small volumes, at 121°C and 10 minutes maximum. In both Solid and selective medium for enterococci enumeration cases, let it cool to 50°C and add 10 mL/L of TTC Sterile and detection. Solution 1% (Ref. 06-023). Homogenize well and distribute in sterile tubes. Formula (in g/L) Note: Non homogeneous appearance of medium is nor- Proteose peptone ...... 10,000 mal, and it does not affect the medium´s quality Yeast extract ...... 10,000 and efficacy. Sodium chloride ...... 5,000 Sodium glycerophosphate ...... 10,000 Description Maltose ...... 20,000 Kenner, Clark and Kabler (1960,1961) discovered that Lactose ...... 1,000 KF media were excellent for detecting enterococci in pol- Sodium azide ...... 0,400 luted water. Carbohydrates in this medium viz. lactose Bromocresol purple ...... 0,015 and maltose, are utilised by most of enterococci, produc- Agar ...... 20,000 ing a big amount of acid and making the indicator turn Final pH 7,2 ± 0,2 from violet to yellow. streptococci that do not belong to D group may also grow in the medium, but they do not Directions produce enough acid to change the indicator colour. Suspend 76,4 g of powder in 1 L of distilled water and Other microorganisms, are strongly inhibited by sodium heat to boiling by constant stirring. If it has to be used azide. enterococci reduce TTC to formazan and so their immediately, it may not to be sterilized. Otherwise, colonies are red coloured. sterilize in the autoclave in small volumes, at 121°C and for 10 minutes maximum. In both the cases, let it cool to Technique 50°C and add 10 mL/L of TTC Sterile Solution 1% (Ref. Media can be used by following several techniques 06-023). Homogenize well and distribute in sterile plates. depending on the sample. When a marginal contami- Note: Non homogeneous appearance is normal, and it nation is thought, then often it is performed using the does not affect the medium´s quality and efficacy. membrane filter or MPN method. On the contrary, if a high population of enterococci is suspected, it is more advisable to make a plate count.

To use the membrane filter technique, incubate at 37°C Kenner Fecal Broth (KF Broth) for 48 hours on the surface of KF Agar or on an absorb- Xn ent pad impregnated in KF Broth. Ref. 02-294 To use MPN technique, directly inoculate samples, of up R-22-32-52/53 S-7-46-61 to 1 mL to tubes with 10 mL of KF Broth in normal con- Specification centration, and for larger samples, to tubes with 10 mL of Liquid and selective medium for enterococci enumera- KF Broth in double strength. tion by the MPN or membrane filter methods. If the sample is suspected of being highly contaminated, prepare a decimal dilution bank and inoculate on the Formula (in g/L) surface 0,1 mL with a Drigalsky Loop (Ref. 5-010) or, if desired, inoculate by stabbing 1 mL. Anyway, incubation Proteose peptone ...... 10,000 should be carried out at 37°C and for a 48 hours period. Yeast extract ...... 10,000 After the incubation, readings are performed by observ- Sodium chloride ...... 5,000 ing the indicator colour turning from violet to yellow, and Sodium glycerophosphate ...... 10,000 on the solid medium, colonies will be also pink or red Maltose ...... 20,000 coloured. Lactose ...... 1,000 Sodium azide ...... 0,400 It is very important to maintain the pH of the medium Bromocresol purple ...... 0,015 over 7,0, or otherwise, false results may appear. Steri- Final pH 7,2 ± 0,2 lization longer than the specified period could result in caramelization and thereby a decrease in the pH.

74 Kenner Fecal Media (KF Media)

References KENNER, B.A., CLARK, H.F., KABLER, P.W. (1961) Fecal streptococcci I. Cultivation and Enumeration of streptococci in Surface Waters. Appl. Microbiol. 9:15. KENNER, B.A., CLARK, H.F., KABLER, P.W. (1960) Fecal streptococci. II. Quantiﬁcation of streptococci in faeces. Am. I. Publ. Health, 50:1553. VANDERZANT & SPLITTSTOESSER (1992). Compen- dium of Methods for the Microbiological Examination of Food.3rd. Ed. APHA. Washington. APHA-AWWA-WEF (1998) Standard Methods for the Examination of Water an WasteWater. 20th. Ed. Wash- ington.

King Media

King A Agar Technique Slanted tubes or Petri dishes are inoculated by surface Ref. 01-001 inoculation by streaking and are then incubated at 30- 32°C for 4-5 days. Petri plates usage has the disadvan- Specification tage of the dehydration of the medium during incubation. Therefore, it is better to use slanted tubes being careful Solid Medium to enhance the pyocyanine production for the aeration by loosening the screw caps or replacing by Pseudomonas aeruginosa acc. ISO 16266, 22717 them with cotton or aluminium caps. standards. In the recently isolated pathogenic strains from the Formula (in g/L) pathological material, pigment production is often shown Peptone ...... 20,0 early i.e. after 24-48 hours of incubation, however if Magnesium Chloride ...... 1,4 the material is non pathogenic or if they come from Potassium Sulfate ...... 10,0 water, food or soil,then the pigmentation can be later or Agar ...... 15,0 delayed. Final pH 7,2 ± 0,2 When pigment has not got the usual blue colour, it is due Directions to the production of two or more coloured substances. Suspend 46,4 g of powder in 1 L of distilled water with At this time, and if it is not confirmed on other culture glycerol 10 mL and let it soak . Heat with constant stirring medium, it is recommended to confirm by extraction: until it boils. Distribute into suitable containers and steri- on the culture slant, 0,5-1 mL chloroform is added, lize by autoclaving at 121°C for 15 minutes. If tubes are and it is shaken for a few minutes until the pyocyanine used, let them solidify with short slant and good butt. is diffused, which makes the solvent blue. After that, chloroform is acidified with a few drops of HCl, obtaining Description a rapid change in colour from blue to red,the fact that This A medium was formulated by King, Ward and confirms the presence of pyocyanine. Raney in 1954 to enhance the pyocyanine production by Pseudomonas aeruginosa. The blue pigment Pyocyanine References diffuses into the culture medium and its production varies KING E.O., M. WARD y D.E. RANEY (1954) Two simple depending on the strains of Pseudomonas aeruginosa media for the demonstration of pyocyanin and fluores- and on the growth conditions. cein. J.Lab.Clin.Med. 44:301-307 US Pharmacopeia (2002) 25th ed. <61> Microbial Limit Sometimes, although this medium enhances especially Test. US Pharmacopoeial Conv. Inc. Rockville MD. blue pigment production, it is possible that green (piover- ISO 16266:2006 Standard. Water Quality.– Detection dine) or brown (piomelanine) pigments also appear and and enumeration of Pseudomonas aeruginosa. Method mask the pyocyanine. Anyway, fluorescence and other by membrane filtration pseudomonas pigments can be noticed on other more ISO 22717:2006 Standard. Cosmetics – Detection of suitable media, like King B Agar (1-029). Pseudomonas aeruginosa.

75 King Media

King B Agar (F Agar) Technique Slanted tubes are inoculated with Pseudomonas strains Ref. 01-029 and incubated at 30-31°C for a 2-4 days period. If after this time a green-yellowish colour does not appear on Specification the medium, the tubes should be kept under observation at room temperature for a period of 6-20 days more Culture media for enhancing the fluorescein production before the culture can be rejected as negative. It can by Pseudomonas species.acc. EN 12780:2002 and ISO be observed that Pseudomonas aeruginosa and Pseu- 16266, 22717 standards. domonas putida strains coming from water, soil or food, produce pigments in a very slow way. Formula (in g/L) Pioverdine is not soluble in chloroform, so the confirma- Meat peptone ...... 10,0 tion of presence is usually done by characteristic fluo- Casein peptone ...... 10,0 rescence verification under Wood’s light, comparing the Dipotassium phosphate ...... 1,5 dubious tube to another uninoculated F medium , which Magnesium sulfate ...... 1,5 is considered as the control. Agar ...... 15,0 Final pH 7,2 ± 0,2 References KING, E.O., M.WARD and D.E. RANEY (1954) Two Directions simple media for the demonstration of pyocyanin and Suspend 38 g of powder in 1L of distilled water with fluorescein J.Lab.Clin.Med. 44:30-307 10 mL of glycerol and let it soak . Heat to boiling and LENNETTE, E.H., E.H. SPAULDING and J.P. TROUANT distribute in suitable containers. Sterilize in the autoclave (1974) Manual of Clinical Microbiology. 2nd. Ed. ASM. at 121°C for 15 minutes. Cool by solidifying in slanted Washington. position with a long slant. US PHARMACOPOEIA (2002) <61> Microbial Limit Tests. 25th ed. US Phamacopoeial Conv. Inc. Rockville. Description MD. F medium was formulated by King, Ward and Raney DIN Standard 38411Teil 6 (Juni 1991): Mikrobiologische in 1954 to enhance green fluorescent pigment (pio- Verfahren (Gruppe K): Nachweis von Escherichia coli verdine) production by Pseudomonas fluorescens und coliformen keimen (K6). and Ps.aeruginosa, in which pyocyanine production is PALLERONI, N. (1984) The genus Pseudomonas, in restricted. Bergey’s Manual of Systematic Bacteriology. Green-yellowish pigments, soluble and from fluorescent EUROPEAN STANDARD EN 12780:2002. Water quality. type, define the Pseudomonas group I according to the Detection and enumeration of Pseudomonas aeruginosa 9th. edition of Bergey’s Manual of Systematic Bacteriol- by membrane filtration. CEN. Brussels. ogy, and therefore, detection of its productive capacity is ISO 16266:2006 Standard. Water Quality.– Detection critical. and enumeration of Pseudomonas aeruginosa. Method by membrane filtration ISO 22717:2006 Standard. Cosmetics – Detection of Pseudomonas aeruginosa.

76 Kligler Iron Agar (KIA)

Ref. 01-103 from tiosulfate) with the Fe ions from ammonium iron citrate. Specification Solid and differential medium for primary identification of Technique enterobacteria based on the fermentation of two sugars Kligler Iron agar is used in slanted tubes with short slant and the hydrogen sulphide production according ISO and plenty of butt, that are inoculated on the surface as Standard 6340. much as in stab. Inoculum must be copious, it has to come from a solid medium, because otherwise, readings Formula (in g/L) may be delayed (up to 2-3 days more). Normal incubation Meat extract ...... 3,00 is 18 hours at 37°C. Yeast extract ...... 3,00 It is recommended to use tubes with caps that allow Peptone ...... 20,00 ventilation, like cotton caps, cellullose caps or cap-o- Lactose ...... 10,00 test. Should screw caps be used, do not tighten them Sodium chloride ...... 5,00 because otherwise they can hinder the reoxidation of the Dextrose ...... 1,00 indicator. Ammonium iron citrate ...... 0,50 Kligler medium provides excellent results if it is used Sodium tiosulfate ...... 0,50 freshly prepared, however if it has been prepared before Phenol red ...... 0,03 a few days then, it is advisable to remelt it and solidify it Agar ...... 15,00 again to obtain more precision. When H S production is more, it may make the read- Final pH 7,4 ± 0,2 2 ings difficult, and hence the early readings are strongly recommended. Anyway, one can obtain more precise Directions readings if Three Sugar Iron Agar (Ref. 01-192) is used, Add 58 g of powder to 1 L of distilled water and heat to since this one has the sucrose that allows a greater dif- the boiling. Distribute in tubes and sterilize in the auto- ferentiation between members of Proteus, Salmonella clave at 121°C for 15 minutes. Let it solidify with short and Shigella types. slant and plenty of butt. References Description KLIGLER (1918) Modification of Culture Media used in Kligler Agar is a differential medium that has all the the Isolation and Differentiation of Typhoid, Dyesentery characteristics of the 2-Sugar Russell Agar and the and allied Bacilli. J. Exper. Med. 28:319-332 Lead Acetate Medium for H S detection. In this medium, 2 KLIGLER (1917) A simple medium for the differentiation lactose fermentation and hydrogen sulphide production of members of typhoid-paratyphoid groups. Am. J. Pub. can be detected, so it allows a presumptive diagnostic Hlth 7:1042-1044 of most enterobacteria. Glucose fermentation is shown VANDERZANT & SPLITTSTOESSER (1992) Compen- by acid production, which makes the indicator turn from dium of Methods for the Microbiological Examination of red to yellow, but since there is little sugar (dextrose), Food. 3rd. Ed. APHA. Washington. acid production is very limited and then a reoxidation of DOWNES, F.P. & K.ITO (2001) Compendium of Methods the indicator is produced on the surface of the medium, for the Microbiological Examination of Food. 4th. ed. and the indicator remains red. Otherwise, when lactose APHA. Washington. is fermented, the large amount of acid produced avoids reoxidation and then all the medium turns to yellow. Hydrogen sulphide production is indicated by the medium turning black, due to the reaction of H2S (liberated

Left: control; center: Escherichia coli ATCC 25922; right: Salmonella typhimurium ATCC 14028.

77 Kligler Iron Agar (KIA)

Typical reactions of enterobacteria on Kligler Iron Agar

78 Lactose Media

Lactose Broth US PHARMACOPOEIA (2005) <61> Microbial limit test. (Eur. Phar. Broth Medium D) US Pharmacopoeial Conv. Inc. Rockville. Md USA APHA-AWWA-WPCF (1998) Standard methods for the examination of water and wastewater. 20th ed. APHA Ref. 02-105 Washington

Speciﬁcation Lactose Peptone Broth Medium for the pre-enrichment and the detection of enterobacteria and coliforms in milk and water according ISO 9308-2 and 21150 standards. Ref. 02-414

Formula (in g/L) Specification Peptone ...... 5,0 Liquid medium for the enrichment and enumeration of Meat extract ...... 3,0 coliforms in water. Lactose ...... 5,0 Final pH 6.9 ± 0,2 Formula (in g/L) Casein peptone ...... 17,00 Directions Soy peptone ...... 3,00 Add 13 g of powder to 1 L of distilled water, or in the Lactose ...... 10,00 quantity required for the desired concentration. Dissolve Sodium chloride ...... 5,00 it and distribute into containers fitted with Durham tubes. Bromocresol purple ...... 0,02 Sterilize by autoclaving at 121°C for 15 minutes. Avoid Final pH 7,2 ± 0,2 any further reheating. Directions Description Dissolve 35 g of powder (or 70 g if double concentrated Lactose Broth is a classical medium for use in the medium is desired) in 1 L of distilled water. Distribute in presumptive testing for coliforms and for the enrichment tubes provided with Durham’s tubes and sterilize in the of Salmonella. This formulation is as per the standards autoclave at 121°C for 15 minutes. recommended by APHA, AWWA, USP-NF and Euro- pean Pharmacopoeia. Description It is commonly used with Durham fermentation tubes for This medium is according to the German standards for the examination of gas formation. If the volume of sam- quality control of water. ple to inoculate is important, reconstitute the medium at a concentration such, as to remain normal once the Technique sample has been added to it. German standards suggests the use of MPN technique Although it is not the original formulation, this broth pro- with 0,1, 1 and 10 mL of sample and an incubation at vides excellent results in Eijkman assays of gas produc- 36±1°C for 44±4 hours. Tubes that change to yellow tion at 45°C, which is a characteristic of Escherichia coli. and eventual gas production/accumulation in Durham´s While preparing this medium it is important to avoid tubes are considered positive. overheating and to distribute it into tubes before sterilization. References DEUTSCHE EINHEITSVERFAHREN zur Wasser-, References Abwasser- Und Schlammuntersuchung. Mikrobiologie FDA (1998) Bacteriological Analitical Manual 8th ed. Verfahren (Gruppe K) Nachweiss vom E. coli und colifor- Rev A. AOAC International. Gaithersburg. Va. USA.. men keimen. VANDERZANT & SPLITTSTOESSER (1992) Compen- VERORDNÜNG über Trinkwasser und Wasser für Leb- dium of Methods for the Microbiological Examination of ensmittelbetriebe vom 12- Dezember-1990- Bundesges- Foods. 3rd. Ed. APHA. Washington. etzblatt; Teil I, 2613-2629 (1990) DOWNES, F.P. & K. ITO (2001) Compendium of Meth- MURRAY, PR, EJ BARON, MA PFALLER, FC TENO- ods for the Microbiological Examination of Foods. 4th ed. VER & RH YOLKEN (eds) (1995) Manual of Clinical APHA. Washington. Microbiology, 6th ed. ASM Washington. EUROPEAN PHARMACOPOEIA (2005) 5th ed. §2.6.13 MaCFADDIN J.A. (1985) Media for Isolation-Cultivation- Test for specified micro-organisms. EDQM. Council of Identification-Maintenance of Medical Bacteria. William& Europe. Strasbourg. Wilkins.Baltimore. ISO 9308-2 Standard. (1990) Water Quality – Detection and enumeration of coliform organisms, thermotolerant coliform and presumptive E. coli – MPN technique. ISO 21150:2006 Standard. Cosmetics – Detection of Escherichia coli

79 Lactose Media

Lactose Purple Modified Broth Directions Dissolve completely 10,1 g of powder in 500 mL of Ref. 02-417 distilled water and sterilize in the autoclave at 121°C for 15 minutes. Cool and aseptically add a flask sodium Specification meta-bisulfite sterile solution (Ref. 06-114CASE) and a flask of sterile solution of ammonium ferric citrate (Ref. Liquid medium for coliforms and E. coli identification. 06-113CASE). Mix well and distribute into sterile tubes provided with Durham´s tubes. Formula (in g/L) Meat peptone ...... 10,00 Description Meat extract ...... 3,00 This is a simple medium that selects Cl. perfringens Sodium chloride ...... 5,00 among other sulfite reducing clostridia by their ability to Lactose ...... 10,00 produce gas from lactose, at 46°C. It has interferences Bromocresol purple ...... 0,02 only with Cl. paraperfringens, however this microorgan- Final pH 7,2 ± 0,2 ism is very rare in food samples.

Directions Technique Dissolve 28 g of powder in 1 L of distilled water. Distrib- All of the freshly prepared media tubes (or regenereated) ute in tubes provided with Durham’s tubes. Sterilize in are inoculated in duplicate with 1 mL of sample dilution. the autoclave at 121°C for 15 minutes. Sample dilution must be previously kept in boiling water bath, boiling it for 10 minutes. Tubes are incubated in Description anaerobic conditions at 46°C for a period of 18-24 hours. This medium is according to the German standards for Cl. perfringens presence is observed by an iron sulfide quality control of water. precipitate appearing in the tubes. It indicates the sulfite reducing activity. Accumulation of gas in the Durham’s Technique tubes is a sign of lactose fermentation. German standards suggests the use of MPN technique with 0,1, 1 and 10 mL of sample and an incubation at References 36±1°C for 44±4 hours. Tubes that change to yellow PASCUAL ANDERSON, MªRª (1992) Microbiología and eventual gas production/accumulation in Durham´s Alimentaria. Diaz de Santos, S.A.,Madrid,. tubes are considered EUROPEAN PHARMACOPOEIA (2002) Suppl. 4.2 positive. (2001). Chap. 2.6.13 Tests for specified microo-organisms 4th Ed., Council of Europe, Strasbourg. References ISO 7937 Standard (2004) Microbiology of food and DEUTSCHE EINHEITSVERFAHREN zur Wasser-, animal feeding stuff. Horizontal method for enumeration Abwasser- Und Schlammuntersuchung. VCM Verlags- of Cl. perfringens. Colony-count technique. gesellschaft, D-6940. Weinhem. VERORDNÜNG über Trinkwasser und Wasser für Leb- ensmittelbetriebe vom 12- Dezember-1990- Bundesges- etzblatt; Teil I, 2613-2629 (1990)

Lactose Sulﬁte Broth Base (Eur. Phar. Medium R)

Ref. 02-519

Speciﬁcation

Liquid medium for the determination of H2S production by Clostridium perfringens according to ISO 7937 standard.

Formula (in g/L) Peptone ...... 5,000 Yeast extract ...... 2,500 Sodium chloride ...... 2,500 Lactose ...... 10,000 L-Cysteine HCl ...... 0,300 Final pH 7,1 ± 0,2

80 LB Media

Under this generic name there are several formulations Description included which are derived from the base medium that Formulation of both the media, solid and liquid, are ac- was originally described by Luria and lately modified by cording to the Luria and Bartani base, in which sodium different authors. chloride has been omitted to help in the variation of the This nutrient base has become very popular among saline concentration with other additives. culture media for the maintenance and propagation of Escherichia coli in the assays about molecular genetics. LB Agar acc. to Lennox The nutrient richness and simplicity in their composition allow easy and quick modifications, where the most common are the addition of different antibiotics and Ref. 01-406 inhibitors as well as the variations in the sodium chloride concentration. Specification Solid medium for general purposes recommended for The current SCHARLAU formulation program covers the the molecular genetics studies with E. coli. following formulations: Formula (in g/L) LB Agar Casein peptone ...... 10,0 Yeast extract ...... 5,0 Ref. 01-384 Sodium chloride ...... 5,0 Agar ...... 15,0 Final pH 7,0 ± 0,2 Specification Solid medium for general purposes recommended for the molecular genetics studies with E. coli. Directions Suspend 35 g of powder in 1 L of distilled water and heat to the boil with constant stirring. Distribute into suitable Formula (in g/L) containers and sterilize by autoclaving at 121°C for 15 Casein peptone ...... 10,0 minutes. Yeast extract ...... 5,0 Agar ...... 15,0 Final pH 7,2 ± 0,2 LB Broth acc. to Lennox

Directions Ref. 02-406 Suspend 30 g of powder in 1 L of distilled water and heat to the boil with constant stirring. Distribute into suitable Speciﬁcation containers and sterilize by autoclaving at 121°C for 15 Liquid medium for general purposes, recommended for minutes. molecular genetics studies with E. coli.

LB Broth Formula (in g/L) Casein peptone ...... 10,0 Yeast extract ...... 5,0 Ref. 02-384 Sodium chloride ...... 5,0 Final pH 7,0 ± 0,2 Speciﬁcation Liquid medium for general purposes, recommended for Directions molecular genetics studies with E. coli. Dissolve 20 g of powder in 1 L of distilled water. Distrib- ute into suitable containers and sterilize by autoclaving Formula (in g/L) at 121°C for 15 minutes. Casein peptone ...... 10,0 Yeast extract ...... 5,0 Description Final pH 7,2 ± 0,2 Formulation of both the media, solid and liquid, are according to the Luria and Bartani base modiﬁed by Directions Lennox. Dissolve 15 g of powder in 1 L of distilled water. Distrib- ute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

81 LB Media

LB Agar acc. to Miller Directions Dissolve 25 g of powder in 1 L of distilled water. Distrib- Ref. 01-385 ute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. Specification Solid medium for general purposes recommended for Description the molecular genetics studies with E. coli. Formulation of both the media, solid and liquid, are according to the Luria and Bartani base modified by Miller, Formula (in g/L) who has increased the sodium chloride concentration. Casein peptone ...... 10,0 Yeast extract ...... 5,0 References Sodium chloride ...... 10,0 AUSUBEL, F.M., R. BRENT, R.E. KINGSTON, D.D. Agar ...... 15,0 MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL Final pH 7,2 ± 0,2 (1994) Current protocols in molecular biology. Greene Pub. Assoc. Inc. Brooklyn, N.Y. Directions ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press, Inc. London. Suspend 40 g of powder in 1 L of distilled water and heat GHERNA, R., P. PIENTA, R. COTE (Eds.) 1992. ATCC to the boil with constant stirring. Distribute into suitable Catalogue of Bacteria and Bacteriophages. Media containers and sterilize by autoclaving at 121°C for 15 #1065, #1226, #1226, #1235, #1236, #1315, #1364. minutes. American Type Culture Collection. Rockville MD. USA. LURIA, S.E. & J.W. BURROUS (1955) Hybridization LB Broth acc. to Miller between Escherichia coli and Shigella. J. Bacteriol. 74:461-476 Ref. 02-385 LENNOX, E.S. (1955) Transduction of linked genetic character of the host bacteriophage P1. Virology 1:190- Specification 206. Liquid medium for general purposes, recommended for MILLER, J.H. (1972) Experiments in Molecular Genetics. molecular genetics studies with E. coli. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y. SAMBROOK, J., E.F. FITSCH & T. MANIATIS (1989) Formula (in g/L) Molecular cloning: A laboratory manual. 2nd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y. Casein peptone ...... 10,0 Yeast extract ...... 5,0 Sodium chloride ...... 10,0 Final pH 7,2 ± 0,2

82 Letheen Media

Letheen Agar Description This is the liquid version of Letheen Agar (Ref. 01-236), Ref. 01-236 recommended by AOAC to verify the germicidal activity coefficients in cationic soaps, although its formulation is Specification not the same. Solid medium for assays of antimicrobial action of quaternary ammonium compounds (QAC’s). Letheen Modified Agar

Formula (in g/L) Ref. 01-237 Tryptone ...... 5,00 Meat extract ...... 3,00 Specification Dextrose ...... 1,00 Solid medium for the primary screening of microorgan- Lecithin ...... 1,00 isms in cosmetics according to the FDA Agar ...... 15,00 Final pH 7,0 ± 0,2 Formula (in g/L) Casein peptone ...... 10,0 Directions Meat peptone ...... 10,0 Suspend 25 g of powder in 1 L of distilled water with 7 Meat extract ...... 3,0 mL of Polysorbate 80 (Ref. 06-088). Let it soak and heat Yeast extract ...... 2,0 to boiling with constant stirring. Distribute in suitable Dextrose ...... 1,0 containers and sterilize in the autoclave at 121°C for 15 Lecithin ...... 1,0 minutes. Sodium chloride ...... 5,0 Sodium bisulphite ...... 0,1 Description Agar ...... 15,0 Letheen Agar is formulated according to AOAC direc- Final pH 7.0 ± 0,2 tions, that were taken from the research work of Weber and Black for the assay of bactericidal action of quater- Directions nary ammonium compounds (QAC’s). Suspend 47 g of powder in 1 L of distilled water and add In fact, the medium is the classical one for the standard- 7 mL of Polysorbate 80 (Ref. 06-088). Allow to soak and ized counting with the addition of lecithin and polysorb- heat to the boil with constant agitation. ate, which act as the neutralizers of the QAC’s. Distribute into final containers and sterilize by autoclaving at 121°C for 15 minutes. Letheen Broth Letheen Modified Broth Ref. 02-236 Ref. 02-237 Specification Liquid culture medium for the determination of germicidal Specification activity coefficients of cationic detergents. FDA recommended liquid medium for the primary recovery of stressed microorganisms in the microbial exami- Formula (in g/L) nation of cosmetics. Peptone ...... 10,0 Meat extract ...... 5,0 Formula (in g/L) Lecithin ...... 0,7 Casein Peptone ...... 15,0 Sodium chloride ...... 5,0 Meat Peptone ...... 10,0 Final pH 7,0 ± 0,2 Meat extract ...... 5,0 Yeast extract ...... 2,0 Directions Sodium chloride ...... 10,0 Dissolve 20,7g of powder in 1 L of distilled water with 5 Lecithin ...... 0,7 mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable Sodium bisulfite ...... 0,1 containers and sterilize by autoclaving at 121°C for 15 Final pH 7,0 ± 0,2 minutes. Directions Dissolve 43 g of the dry powder in 1 L of distilled water with 5 mL of Polysorbate 80 (Ref. 06-088). Distribute in suitable containers and autoclave at 121°C for 15 minutes.

83 Letheen Media

Description References In the early 40’s, Weber and Black recommended the ASTM Standard E 640-78 (1991) Test method for pre- use of lecithin and polysorbates to neutralize the anti- servatives in water-containing cosmetics. Philadelphia. microbial action of the quaternary amonium compounds PA (QAC’s). ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- In 1965 the methodology was accepted by AOAC for the biological Media. CRC Press, Inc. London. antimicrobial assays and extendes their use to all the FDA (1998) Bacteriogical Analytical Manual. 8th ed. cationic tensoactives (detergents). Revision A. AOAC International. Gaithersburg. MD The TAT (Tryptone Azolactin Tween) medium in the HORWITZ, W. (2000) Official Methods of Analysis. 17th Newburger Cosmetic Analysis Manual (2nd. Ed., 1977) ed. AOAC International. Gaithersburg. MD is closely similar in formulation and uses to the AOAC LUCAS, J.P. (1977) Microbiological Examination of recipe. In 1978 the FDA (Bacteriological Analytical Cosmetics. Newburguers’ Manual of Cosmetic Analysis. Manual, 1978) incorporated it as previous presumptive AOAC and enrichment medium for every microbial examination WEBER, G.R. & L.A. BLACK (1948) Relative efficiency in cosmetics. of quaternary inhibitors. Soap and Sanit Chem. 24:134- 139 The SCHARLAU formulation of the Letheen Media ISO 21149:2006 Cosmetics – Enumeration and detec- covers the old recipe of Lucas (1977) in the Newburger tion of aerobic mesophilic bacteria. Manual (Ref.1-236 Letheen Agar and Ref. 02-236 ISO 22717:2006 Standard. Cosmetics – Detection of Letheen Broth) and the new recipe (Ref. 01-237 Letheen Pseudomonas aeruginosa. Modified Agar and Ref. 02-237 Letheen Modified Broth) ISO 22718:2006 Standard. Cosmetics – Detection of of the FDA (B.A.M., 1979) Staphylococcus aureus.

84 Liebermeister & Braveny Agar

Ref. 01-446 in the yeast extract. Moreover, they also included lysine, which has an effect on the hemolysis similar to that of Speciﬁcation the nucleic acids. Solid medium for the selective isolation of ß-hemolytic The result is that microorganims fail to grow or form only streptococci from throat samples. small colonies that have hemolysis zones(ß-hemolysis) of normal or greater than normal size. Viridans streptococci (alpha-hemolytic) virtually do not grow, and if Formula (in g/L) hemolysis zones form at all, they are minimal. Meat peptone ...... 1,00 Meat extract ...... 0,60 Yeast extract ...... 0,50 Technique L(+)Lysine ...... 0,02 Plates are inoculated by surface seeding and incubated Sodium chloride ...... 6,00 at 37°C for 24-48 hours. After the incubation small colo- Disodium phosphate ...... 2,00 nies form which are surrounded by a large, well deﬁned Agar ...... 15,00 hemolytic zone. Staphylococci and enterococci are Final pH 7,2 ± 0,2 almost viridans streptococci.

Directions References Suspend 25 g of powder in 930 mL of distilled water and OKAMOTO, H., S. KYODA, R. ITO (1939) Jap. J. Med. heat to the boil. Distribute into suitable containers and Sci. VI Pharmacol. 12:167. sterilize in an autoclave at 121°C for 15 minutes. Cool BERNHEIMER, A.W., M. RODBART (1948) The effect to 45°C, then add 70 mL/L of sterile defibrinated lamb’s of nucleic acids and carbohydrates on the formation of blood. Homogenize well and pour into sterile plates. streptolysin. J. Exp. Med. 88:149. LIEBERMEISTER, K., J. BRAVENY (1971) Ein Nährsub- strat zur Isolierung von haemolytischen Streptokokken. Description Z. Med. Mikrobiol. Inmunol. 156:149-153. Despite its simplicity, this medium has a better yield in MILATOVIC, D. (1981) Comparison of five selective the recovery of ß-hemolytic streptococci than the com- media for beta-haemolytic streptococci. J. Clin. Pathol. monly used Blood Agar. 34:556-558 Okamoto et al. and, later Bernheimer and Rodbart demonstrated the strong stimulatory effect of the nucleic acids on the hemolytic properties of streptococci. Lieber- meister and Braveny formulated a medium with insufficient nutrients for the development of normal microorganisms but with an increased amount of nucleic acids

85 Lingby Iron Agar Base

Ref. 01-584 Lingby Iron Agar Base has been formulated according to the Food Analysis Nordic Committee standard. Specification This standard states that cysteine addition must be Solid medium for the isolation and enumeration of done separately in order to achieve better results in the recovery of the H2S producing bacteria from fish and fish heterotrophic H2S producing bacteria from fish and fish products. products that have not been under heat treatment or with any addition of preservatives. Formula (in g/L) Tryptone ...... 20,0 Technique For the total count of H S producing bacteria, the Com- Meat extract ...... 3,0 2 Yeast extract ...... 3,0 mittee prescribes the following technique: Ferric citrate ...... 0,3 Transfer 1 mL of sample dilution to sterile Petri plates. Sodium thiosulfate ...... 0,3 Add 10-12 mL of molten medium and cool to 45ºC. Ho- Sodium chloride ...... 5,0 mogenize by shaking it gently. When cooled, add a new Agar ...... 15,0 layer of medium and incubate at 20±1ºC for 3 days. Final pH 7,4 ± 0,2 Select the adequate plates and proceed with total count. H2S producing bacteria form black colonies. If incubation temperature is too high or the covering Directions layer is too thin, black colonies loses the colour rapidly. Suspend 46,6 g of powder in 1L of distilled water and heat to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Cool to References 45-50ºC and aseptically add 8 mL/L of a filter sterilized Nordisk Metodikkommité för Livsmedil. (1994) UDC. solution of 5% L-Cysteine hydrochloride. Homogenize 637.56:576.8.08-No96. 2ntg- Bacteriological examina- well and pour into sterile plates. tion of fresh and frozen seafood.

Description

Listeria Enrichment Media

Listeriosis Detection and confirmation of Listeriosis is the generic name for the whole group of Listeria disorders caused by Listeria monocytogenes, which is Food control requires to culture the samples that use the only species of the Listeria genus that is important to need 5-7 days before provide definitive results, and as a human pathogen, howewer occasionally L.seeligeri, although the commercial production of DNA probes to L.welshimeri and L.ivanovii have been related with help the diagnosis, they are not available yet. human diseases. In anycase, all the species are patho- The traditional technique of low temperature (4ºC) en- genic between the ovin and bovine cattle. richment has been substituted for selective enrichments Positive diagnosis of listeriosis can be obtained only by at temperatures near to the optimum growth tempera- the isolation and cultivation of the responsible bacte- ture. Isolation can be also performed over selective ria from blood or cerebrospinnal fluid samples of the and differential media, that most of the cases allow to affected organism. Faecal samples have a very debat- disregard the special Henry lighting technique, but final able value, since actually it is estimated that between identification must be carried out by biochemical and 1-10% of human population may be an intestinal carrier serological methods. of L.monocytogenes. This species of bacteria have been The type of sample restrains the technique of detection isolated from 37 species of mammals, domestic or wild, because the companion flora is very important. If a pop- as well as from 17 species of birds and some species of ulation more than 100 Listeria cells/g is suspected, the fishes and shellfishes. sample can be inoculated directly over the solid selective Listeria monocytogenes bears a notorious tolerance to media, but in other cases, an enrichment technique, in heat, cold and desiccation, and it acts as a real psi- one or two stages, must be adopted. chrotrophic. These facts and the high morbidity when it infects annimals and humans increase its interest as a food-borne pathogen. Its presence has been related with food as raw milk or supposedly pasteurized milk, soft-ripped cheese, ice creams, vegetables, raw meat, cured fermented meat, raw or smoked fish, but it has been isolated also from soil, silages and other environmental samples.

86 Listeria Enrichment Media

Specific identification Directions All the Listeria species are grampositive, oxidase- and Dissolve 54,5 g of powder into 1 L of distilled water. Dis- urease-negative. They produce catalase, do not reduce tribute 500 mL in each container and autoclave at 121ºC nitrate and hydrolyze esculin. Provide +/+ result in the for 15 minutes. Cool to 50ºC and then aseptically add O/F test for glucose and they also acidify the bottom the suitable selective supplement toeach 500 mL: UVM and surface in TSI Agar. Provide a positive result in the I for primary enrichment (Ref. 06-106CASE) and UVM Voges-Proskauer and Phenol Red tests. Differential II/Fraser for secondary enrichment (Ref. 06-111CASE). characteristics are shown in the table above. Note: Prepared medium (broth+supplement) must be kept away from light, since it helps the produc- References tion of acriflavine-oxidised photocomplexes that DONNELLY, C.W., R.E. BRACHETT, S. DOORES, W.H. repress Listeria growth. LEE, J. LOVETT (1992) Listeria, in Compendium of method for the microbiologi- Description cal examination of foods, Vanderzant & Splittstoesser This basal broth for the enrichment of listeria is made ac- (eds) APHA. Washington. cording the AOAC modifications over the medium of the ADAMS, M.R., M. O. MOSS (1995) Food Microbiology . Vermont University (UVM), since it was demonstrated The Royal Society of Chemistry. Cambridge. U.K. that a slight increase of acriflavine concentration in the FDA/ Center for Food Safety & Applied Nutrition. (March secondary enrichment and a strong reduction in the 1999) Foodborne Pathogenic Microorganisms and amount of nalidixic acid in all the stages allowed more Natural Toxins Handbook: Listeria monocytogenes http:// positive isolations. vm.cfsan.fda.gov/mow Technique Following you will find the Scharlau Microbiology produc- Primary enrichment tion program in selective and differential media for the Add 25 g or 25 mL of sample to 225 mL of primary enrichment and isolation of Listeria. enrichment broth (Base Broth, Ref. 02-472, and UVM I, Ref. 06-106CASE) and homogenize it all in a stomacher Listeria Enrichment Broth Base for 2 minutes. Incubate the mixture at 30ºC for 24 hours, (UVM) but after the first 4 hours take aliquots of 0,2 mL to plates with Oxford Selective Agar (Ref. 01-471) in order to make the isolation. Ref. 02-472 Secondary enrichment After 24 hours of primary enrichment, inoculate the sec- Specification ondary enrichment broth (Base Broth, Ref. 02-472, and Liquid culture medium for the enrichment of Listeria sp. UVM II/Fraser, (Ref. 06-111CASE) at the rate of 1:100. Incubate at 30ºC. After 4 and 24 hours take aliquots of Formula (in g/L) 0,2 mL to plates with Oxford Selective Agar (Ref. 01- Proteose Peptone ...... 5,00 471) in order to make the isolation. Tryptone ...... 5,00 Isolation Meat extract ...... 5,00 Isolation is carried out on the Oxford Selective Agar (Ref. Yeast extract ...... 5,00 01-471) plates prepared during the primary and second- Sodium chloride ...... 20,00 ary enrichment, after 24-48 of incubation at 30-37ºC. Esculin ...... 1,00 Sometimes it is advisable to alkalinize the inoculum Disodium phosphate ...... 12,00 before the seeding, by mixing 1 mL of enrichment broth Dipotassium phosphate ...... 1,35 with 5 mL of 0,5% sterile KOH solution. Final pH 7,4 ± 0,2

87 Listeria Enrichment Media

Listeria Enrichment Broth Base Listeria Enrichment Broth Base acc. to Lovett acc. to Fraser

Ref. 02-498 Ref. 02-496

Specification Specification Liquid culture medium for the enrichment of Listeria, ac- Liquid culture medium for the enrichment and detection cording Lovett et al. of Listeria spp. according ISO standards 11290-1 and 11290-2. Formula (in g/L) Tryptone ...... 17,00 Formula (in g/L) Yeast extract ...... 6,00 Proteose peptone ...... 5,00 Soy peptone ...... 3,00 Tryptone ...... 5,00 Sodium chloride ...... 5,00 Meat extract ...... 5,00 Dextrose ...... 2,50 Yeast extract ...... 5,00 Dipotassium phosphate ...... 2,50 Sodium chloride ...... 20,00 Final pH 7,3 ± 0,2 Esculin ...... 1,00 Dissodium phosphate ...... 12,00 Directions Monopotassium phosphate ...... 1,35 Dissolve 36 g of powder into 1 L of distilled water and Lithium chloride ...... 3,00 distribute 500 mL per flask. Sterilize by autoclaving at Final pH 7,2 ± 0,2 121ºC for 15 minutes. Cool to 50ºC and aseptically add to each flask the content of one vial of Listeria Supple- Directions ment for Selective Enrichment acc. to FDA/IDF (Ref. Dissolve 57,4 g of powder into 1 L of distilled water. 06-107CASE). Homogenize and distribute into suitable Distribute 500 mL per flask and sterilize in the autoclave containers. at 121ºC for 15 minutes. Cool to 50ºC. Aseptically add Note: Prepared medium (broth+supplement) must be the Ferric Ammonium Citrate for Bacteriology (Ref. kept away from light, since it helps the produc- 06-112CASE) and Listeria Supplement for Secondary tion of acriflavine oxidised photocomplexs that Enrichment UVM II/Fraser (Ref. 06-111CASE) to each repress Listeria growth. flask and homogenize well. Note: Prepared medium (broth+supplement) must be Description kept away from light, since it helps the production This medium according to Lovett et cols. formulation has of acriflavine oxidised photocomplex that repress been adopted by the FDA for the analysis of food, and it Listeria growth. is recommended by the IDF/FIL for the selective enrichment of Listeria in milk samples, due to its good results Description in the recovery of stressed bacteria, with only an enrich- This broth base for Listeria enrichment is made accord- ment stage. ing to the modifications of Fraser and Sparber over the UVM medium, which have been adopted by the USDA- Technique FSIS. The inclusion of lithium chloride inhibits the devel- Mix the sample (25 mL or 25 g) with 225 mL of complete opment of enterococci which also may hydrolyze esculin enrichment broth and incubate at 30ºC for 7 days. Make in the same way of Listeria. Thus, any darkness in the subcultures after 24 hours, 48 hours and 7 days in the medium produced by the reaction of esculetin coming following way: from esculin hydrolysis with iron present in the medium Inoculate 0,5 mL of enrichment culture in a solid medium can be taken as a presumptive presence of Listeria. for the Listeria isolation (Oxford Agar Base, Ref. 01-471, Moreover, it seems the ferric citrate helps L. monocy- or Palcam Agar Base, Ref. 01-470, with their respective togenes development. selective supplements). Alkalinize 0,5 mL of enrichment culture by mixing with Technique 4,5 mL of 0,5% sterile KOH solution and inoculate on a Although some authors use the Fraser Broth as the only solid medium for Listeria isolation. enrichment, it has been verified than better results are obtained if it is employed as secondary enrichment, according the following methodology: Inoculate the sample to examine in a primary enrichment broth (UVM I, Ref. 02-472 or Lovett Broth, Ref. 02-498) and incubate for 18-24 hours.

88 Listeria Enrichment Media

Take aliquots of 0,1 mL, inoculate them in tubes with 10 yellow colonies and halos, contrasting with the cherry mL of Fraser Broth and incubate for 24-28 hours. red colour of medium. Tubes that become dark are considered presumptively However, when there are many Listeria colonies, the positive and must be subcultured over isolation and entire medium turns dark, and the differentiation can be confirmation solid media, such as Oxford Agar Base interfered. In these cases it is advisable to perform a (Ref. 01-471) or Palcam Agar Base (Ref. 01-470). Tubes more diluted inoculation. that remain clear are considered negative and can be discarded or kept in incubation for 24 hours more to Technique clear the doubts if any. Seed the Palcam Agar with a growth from a primary enrichment broth (UVM I, Ref. 02-472 or Lovett, Ref. Palcam Agar Base 02-498) or a secondary enrichment broth (UVM II, Ref. 02-472 or Fraser, Ref. 02-496). Incubate in a microaer- Ref. 01-470 ophile atmosphere for 48 hours at 37ºC. In these conditions, Listeria colonies have a size approx. 2 mm diameter, green-grey coloured with black core Specification and halo. Enterococcus and Staphylococcus colonies Solid, selective and differential medium for the detection, are bigger, grey with green-brown halo if they do not enumeration and isolation of Listeria spp. according ISO use mannitol or form yellow colonies with yellow halo if standards 11290-1 and 11290-2. they do. Anyway, suspicious colonies must be confirmed biochemically and serologically. Formula (in g/L) Tryptone ...... 23,00 Oxford Agar Base Lithium chloride ...... 15,00 Mannitol ...... 10,00 Xn Sodium chloride ...... 5,00 Ref. 01-471 R-22-36/38 Yeast extract ...... 3,00 S-26-37/39-46 Starch ...... 1,00 Specification Esculin ...... 0,80 Solid, selective and differential medium for the detection, Ferric Ammonium citrate...... 0,50 enumeration and isolation of Listeria sp. according ISO Dextrose ...... 0,50 standards 11290-1 and 11290-2. Phenol red ...... 0,08 Agar ...... 13,00 Formula (in g/L) Final pH 7,2 ± 0,2 Tryptone ...... 10,00 Lithium chloride ...... 15,00 Directions Proteose peptone ...... 10,00 Suspend 72 g of powder in 1 L of distilled water and Sodium chloride ...... 5,00 let it soak. Heat to boil and distribute 500 mL per flask. Yeast extract ...... 3,00 Sterilize by autoclaving at 121ºC for 15 minutes. Cool Starch ...... 1,00 to 50ºC and aseptically add the Palcam Agar Selective Esculin ...... 1,00 Supplement (Ref. 06-110CASE) to each flask. Mix well Ferric Ammonium citrate...... 0,50 and pour into sterile plates. Agar ...... 13,00 Note: Prepared medium (broth+supplement) must be Final pH 7,2 ± 0,2 kept away from light, since it helps the production of acriflavine oxidised photocomplex that repress Directions Listeria growth. Suspend 58,5 g of powder in 1 L of distilled water and let it soak. Heat to boil and distribute 500 mL per flask. Description Sterilize by autoclaving at 121ºC for 15 minutes. Cool to Palcam Agar is based in the formulation described initial- 50ºC and aseptically add the Oxford Agar Selective Sup- ly by van Netten et cols., which has a high selectivity and plement (Ref. 06-109CASE) to each flask . Mix well and a good colonial differentiation. Selectivity is achieved by pour into sterile plates. the inclusion of lithium chloride, acriflavine, polymixin B Note: Prepared medium (broth+supplement) must be and ceftacidine, since they inhibit the growth of almost kept away from light, since it helps the production all the gramnegative bacteria and most of grampositive of acriflavine oxidised photocomplex that repress companion bacteria. Listeria growth. Listeria hydrolyze esculin to esculetin, which reacts with ferric ammonium citrate producing a dark precipitate colouring the colonies to green-grey with beige halos. The colonies of enterococci or staphylococci that may overpass the high selectivity of this medium can be easily recognized, since they use mannitol and produce

89 Listeria Enrichment Media

Description References Oxford Agar is derivated from the original formulation by McCLAIN, D., W.H. LEE (1988) Development of USDA- Curtis et al, which worked a medium with a high nutritive FSIS Method for isolation of Listeria monocytogenes ability as the Columbia Agar and added inhibitor agents from raw meat and poultry. JAOAC 71:3:660-664. to remove all the undesirable companion bacteria. ATLAS, R.M. (1993) Handbook of Microbiological Media. The current formulation keep the high capacity to sup- CRC Press. Boca Raton, Florida. port growth and restrain all the gramnegative ﬂora and VANDERZANT, C., D.F. SPLITTSTOESSER (1992) most of grampositive, including yeast. Thanks to the Compendium of methods for the microbiological exami- inhibitors incorporated in the selective supplement: nation of food. APHA, Washington D.C. cycloheximide, acriﬂavine, colystin, phosphomicyn LOVETT, J., D.W. FRANCIS, J.M. HUNT (1988) Listeria and ceftacidine. These inhibitors in addition to lithium monocytogenes in raw milk; Detection, incidence and chloride restrain the growth of all other bacteria except pathogenicity. J. Food Protect. 50;188-192 Listeria. LOVETT,J., A.D. HITCHINS (1989) Listeria isolation. However, selectivity is not total, but Listeria colonies are FDA Bacteriological Analytical Manual. 6th Ed. Supp. easily recognizable since as they hydrolyze esculin. Free Sept. 1987 (2nd Print):29.01 esculetin that reacts with the ferric ions and produce FRASER, J.A., W.H. SPERBER (1988) Rapid detection a dark precipitate around the colonies, which typically of Listeria sp. in food and environmental samples by present a grey-blue colour with a very dark core. esculin hydrolysis. J. Food Prot. 51:762-765. van NETTEN, P., J.PERALES, A. van de MOODSDUCK, Technique G.D.W. CURTIS, D.A.A. MOSSEL (1989) Liquid and Although the selectivity of the medium is enough to allow solid selective differential media for the detection and the isolation and differentiation by direct surface inocula- enumeration of Listeria monocytogenes. Int. J. Food tion, a previous dilution of the inoculum is advisable, or Microbiol. 8:299-316. even more when the sample is highly polluted. CURTIS, G.D., R.G. MITCHELL, A.F. KING, E.J. GRIF- In anycase, most authors prefer one or two previous FIN (1989) A selective differential medium for the isola- cultures in any of the primary enrichment media (UVM I, tion of Listeria monocytogenes. Letters Appl. Microbiol. Ref. 02-472 or Lovett, Ref. 02-498) or secondary enrich- 8:95-98 ment media (UVM II, Ref. 02-472 or Fraser, Ref. 02-496) ISO 11290 standard (1996) Microbiology of food and before inoculating in Oxford Agar. animal feeding stuff. Horizontal method for the detec- Incubation is carried out at 37ºC, and after 24 hours tion and enumeration of Listeria monocytogenes. Part 1 typical colonies of Listeria monocytogenes are visible. - Detection method. Part 2 - Enumeration method. However, it is recommended to extend incubation for more 20-24 hours in order to evidence the slow growing strains even though this could allow staphylococci or streptococci development, since they would grow weakly.

Ref. 02-496 Listeria Enrichment Broth acc. Fraser. Left: control; right: positive.

90 Lysine Media

Lysine Iron Agar (LIA) Technique From some suspicious colonies in the isolation media, Ref. 01-094 and with a needle, inoculate a Kligler’s medium tube, and without recharging the inoculation loop, pass it to Specification LIA by surface streaking and depth stab. Incubate them closed, but at the same time sufficiently ventilated, at Differential medium for Enterobacteria, recommended by 35-37°C for 24 hours. Edwars and Ewing for Salmonella identification. Microorganisms that decarboxylate the lysine, quickly, produce a strong alkalinization in all the medium, that Formula (in g/L) is observed by the indicator turning to purple. Whereas, Gelatin peptone ...... 5,00 those that have no lysine decarboxylase activity, acidify Yeast extract ...... 3,00 the medium at the bottom producing a yellow coloura- Dextrose ...... 1,00 tion, meanwhile the surface of the medium remains with L-Lysine ...... 10,00 the same original colour or with alkaline reaction. Ammonium Ferric citrate ...... 0,50 Proteus type members are distinguished easily, since, Sodium thiosulfate ...... 0,04 over the yellow bottom, they produce a typical red or Bromocresol purple ...... 0,02 orange colour on the surface, due to oxidative deamina- Agar ...... 15,00 tion of lysine. The microorganisms which produces of Final pH 6,7 ± 0,2 hydrogen sulfide blacken the medium because of iron sulfur precipitates. Directions Although the gas production may be observed, generally Suspend 34,5 g of powder in 1 L of distilled water and this medium does not offer optimal conditions for this, heat to boiling . Dispense in tubes and sterilize by and gives very irregular results, even giving total inhibi- autoclaving at 121°C for 15 minutes. Allow to solidify in tion in some cases. slanted position, with copious depth and short slant. References Description EDWARDS, P.R. and M.A. FIFE (1961) Lysine-Iron Agar Lysine and Iron medium has been widely used for the in the detection of Arizona cultures. Appl. Microbiol. 99, differentiation among different biotypes of Salmonella, 478-480. above all those corresponding to S. arizona, which, on EWING, J. (1982) Edwards and Ewing’s identification of usual selective isolation in plate media, like MacConkey Enterobacteriaceae. 4th. Ed. Elsevier Sci. Pub. Co. Inc. or Deoxycholate, may give colour or colourless colonies N.Y. due to the fact that their lactose fermentative capacity is McFADDIN, J.F. (1985) Media for the isolation, cultiva- quite variable. tion, identification and maintenance of medical bacteria. If it is considered that these microorganisms, when William & Wilkins. Baltimore cultured in tubes with Kligler Iron Agar (Ref. 01-103) or ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Triple Sugar Iron (Ref. 01-192), produce enough acid to biological Media. CRC Press,London avoid sulfide formation, it is comprehensible that they DOWNES, F.P. & K. ITO (2001)) Compendium of Meth- are sometimes not recognized or ods for the Microbiological Examination of Food. 4th ed overlooked as pathogen, and wrong or negative results APHA. Washington are consequently obtained. On the other hand, Salmo- HORWITZ, W. (2000) Official Methods of Analysis. 17th nella is the only genus of enterobacteria that normally ed. AOAC International. Gaitherburg. MD. decarboxylates lysine and produces important amounts MARSHALL R.T. (1992) Methods for the examination of of hydrogen sulfide. dairy products. APHA. Washington. LIA works perfectly verifying these two characteristics, and that is the reason why it is used at the same time of use of Kligler Iron Agar (Ref. 01-103) and/or TSI (Ref. 01-192) in the second phase of isolation of pathogenic enterobacteria.

91 Lysine Media

92 M-17 Media

M-17 Agar M-17 w/o Lactose Broth

Ref. 01-245 Ref. 02-580

Specification Specification Solid selective medium for Streptococcus thermophilus Liquid selective medium for Streptococcus thermophilus in the common examination of yoghurt acc. to ISO 7889: in the examination of yoghurt and other dairy products. 2003 and IDF 117: 2003. Formula (in g/L) Formula (in g/L) Tryptone ...... 2,50 Tryptone ...... 2,50 Meat peptone ...... 2,50 Meat peptone ...... 2,50 Soya peptone ...... 5,00 Soya peptone ...... 5,00 Yeast extract ...... 2,50 Yeast extract ...... 2,50 Meat extract ...... 5,00 Meat extract ...... 5,00 Sodium glycerophosphate ...... 19,00 Sodium glycerophosphate ...... 19,00 Magnesium sulfate ...... 0,25 Magnesium sulfate ...... 0,25 Ascorbic acid ...... 0,50 Ascorbic acid ...... 0,50 Final pH 7,0 ± 0,2 Lactose ...... 5,00 Agar ...... 15,00 Directions Final pH 7,0 ± 0,2 Disolve 37 g of powder in 1 L of distilled water, heating if necessary. Dispense into suitable containers. Sterilize by Directions autoclaving at 121°C for 15 minutes. Suspend 57 g of powder in 1 L of distilled water and let it soak. Heat to boiling and dispense into suitable contain- Description ers. Sterilize by autoclaving at 121°C for 15 minutes. M-17 Agar was developed by Terzaghi and Sandine for Avoid unnecessary overheating and remelting. the screening of bacteriophages in streptococci of dairy industry. Afterward, Shankar and Davies demonstrated M-17 Broth the efficacy of this medium for selective isolation of Streptococcus thermophilus in yoghurt. Medium com- Ref. 02-245 bines a strong buffer, which aids development of streptococci, with a high concentration of glycerophosphate, which inhibits the growth of lactobacilli. Specification Liquid selective medium for Streptococcus thermophilus in the common examination of yoghurt. Technique Suggested technique to enumerate streptococci is to seed in mass or by stabbing, with agar melted and Formula (in g/L) cooled to 50-55°C, and then to incubate them at 42°C for Tryptone ...... 2,50 a 24 hours period. With these conditions, all the colo- Meat peptone ...... 2,50 nies might be streptococci. Longer incubation periods or Soya peptone ...... 5,00 lower temperatures may cause morphological changes Yeast extract ...... 2,50 in the colonies which hinders in the the recognition of the Meat extract ...... 5,00 colonies. Sodium glycerophosphate ...... 19,00 FIL-IDF has adopted this medium for yoghurt examina- Magnesium sulfate ...... 0,25 tion, and uses it simultaneously with MRS Agar (Ref. Ascorbic acid ...... 0,50 01-135). Lactose ...... 5,00 Lactobacilli count is performed analogously in MRS Me- Final pH 7,0 ± 0,2 dium, at 30°C and in CO2 enriched atmosphere. Colonies of lactose positive streptococci are visible after Directions 15 hours, and after 5 days they may reach a diameter of Suspend 42 g of powder in 1 L of distilled water and let it about 3-4 mm, whereas those lactose negatives are 1 soak. Heat if necessary and dispense into suitable con- mm of diameter. However, longer incubation period may tainers. Sterilize by autoclaving at 121°C for 15 minutes. hinder the observations, due to the growth of lactobacilli. Bacteriophages presence is observed by the appearance of characteristic plaques over the bacterial growth.

93 M-17 Media

References TERZAGHI, B.E. and SANDINE, W.E. (1975) Improved medium for lactic streptococcacal phages from cheese factories. Appl. Environm. Microbiol 29:807-813 SHANKAR, P.A. and DAVIES, F.L. (1977) Selective Technique for yogurt Bacteria Enumeration. J. Soc. Dairy Technol. 30:28. FIL-IDF Standard 146A (1998) Identiﬁcation of characteristic micro-organisma of yoghurt. ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press,London DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Food. 4th ed.APHA. Washington. ISO 7889 standard (2003). Yogurt - Enumeration of characteristic microorganisms - Colony- count technique at 37°C.

94 MacConkey Media

In the production program of SCHARLAU you can find Directions the following formulations of the MacConkey media: Dissolve 40 g of powder in 1 L of distilled water. Bring to the boil and distribute into suitable containers fitted with MacConkey Agar ...... Ref. 01-118 Durham tubes. Sterilize by autoclaving at 121°C for 15 (Eur. Phar. Medium H) minutes. MacConkey Broth ...... Ref. 02-118 MacConkey Modified Broth ...... Ref. 02-120 MacConkey G Broth MacConkey Sorbitol Agar ...... Ref. 01-541 MacConkey w/o Salt Agar ...... Ref. 01-320 (Eur. Phar. Medium G) MacConkey WHO Broth ...... Ref. 01-121 Mac Conkey G Broth ...... Ref. 02-611 Ref. 02-611 (Eur Pharm Medium G) Specification MacConkey Agar Liquid medium for the detection and enumeration of (Eur. Phar. Medium H) coliforms by MPN technique. It is a modification of the classic medium, where neutral red is replaced by a less aggressive indicator, according European Pharmaco- Ref. 01-118 poeia.

Specification Formula (in g/L) A selective and differential medium for the detection, Peptone ...... 20,00 isolation and enumeration of coliforms from a variety of Lactose ...... 10,00 samples according European Pharmacopoeia and ISO Bile Salts ...... 5,00 standard. Bromocresol purple ...... 0,01 Final pH 7,4 ± 0,2 Formula (in g/L) Peptone ...... 20,000 Directions Lactose ...... 10,000 Dissolve 35 g of powder in 1 L of distilled water. Heat Bile Salts #3 ...... 1,500 only if necessary to help the dissolution. Distribute into Sodium chloride ...... 5,000 suitable containers fitted with Durham tubes. Sterilize by Neutral red ...... 0,030 autoclaving at 121°C for 15 minutes. Crystal violet ...... 0,001 Agar ...... 15,000 Final pH 7,2 ± 0,2 MacConkey Modified Broth

Directions Ref. 02-120 Suspend 51,5 g of powder to 1 L of distilled water. Bring to the boil and sterilize by autoclaving at 121°C for 15 Speciﬁcation minutes. Liquid medium for the detection and enumeration of coliforms by MPN technique according ISO standard. MacConkey Broth Formula (in g/L) Ref. 02-118 Peptone ...... 20,00 Lactose ...... 10,00 Bile Salts ...... 5,00 Speciﬁcation Sodium chloride ...... 5,00 Liquid medium for the detection and enumeration of Bromocresol purple ...... 0,01 coliforms by MPN technique. Final pH 7,4 ± 0,2

Formula (in g/L) Directions Peptone ...... 20,000 Dissolve 40 g of powder in 1 L of distilled water. Heat Lactose ...... 10,000 only if necessary to help the dissolution. Distribute into Bile Salts ...... 5,000 suitable containers ﬁtted with Durham tubes. Sterilize by Sodium chloride ...... 5,000 autoclaving at 121°C for 15 minutes. Neutral Red ...... 0,075 Final pH 7,4 ± 0,2

95 MacConkey Media

MacConkey Sorbitol Agar ing colonies and some times there is some colony of the o157:H7 serotype that begins to ferment sorbitol. Ref. 01-541 Some gramnegative bacteria like Pseudomonas, Pro- teus and Klebsiella can growth on the MacConkey Agar with Sorbitol but their colonies are diverse and easy to Specification differentiate from E. coli. Selective and differential solid medium for the detection Because the failure in the fermentation of sorbitol by of Enterohaemorrhagic Escherichia coli (EHEC O157: some strains on E. coli no-enterotoxigenic and the atypi- H7) according ISO standard. cal colony production by some enterohaemorrhagic ones it is recommended the use of some others media in con- Formula (in g/L) comitance with the MacConkey Agar with Sorbitol and to Peptone ...... 20,000 confirm the suspect colonies by serological, biochemical Sorbitol ...... 10,000 or molecular techniques. Bile salts ...... 1,500 Sodium chloride ...... 5,000 MacConkey w/o Salt Agar Neutral Red ...... 0,030 Crystal violet ...... 0,001 Agar ...... 15,000 Ref. 01-320 Final pH 7,1 ± 0,2 Specification Directions A selective and differential medium for the detection, Suspend 51,5 g of powder into 1 L of distilled water and isolation and enumeration of enterobacteria, especially heat to boiling. Sterilize in the autoclave at 121ºC for 15 Proteus sp. from the clinical specimens. minutes. If the medium is used the same day of preparation autoclaving is not necessary but the boiling must be Formula (in g/L) for 3 minutes at least. Peptone ...... 20,000 Lactose ...... 10,000 Description Bile Salts #3 ...... 1,500 The substitution of lactose by sorbitol for the isolation of Neutral red ...... 0,075 the enteropathogenic serotypes O111 y O55 of Es- Agar ...... 15,000 cherichia coli was proposed in 1952 by Rappaport and Final pH 7,4 ± 0,2 Hening. The usefulness of the medium was showed by March and Ratman (1986) and Adas (1991) for the de- Directions tection, differentiation and isolation of the enterohaemor- Add 46,5 g of powder to 1 L of distilled water. Bring to rhagic (EHEC) and the verocytoxin-producing (VTEC) the boil and distribute in suitable containers. Esterilize by strains of the serotype O17:H7 of E. coli. autoclaving at 121°C for 15 minutes. The only modification on the typical MacConkey Media formulations is the replacement of lactose with sorbitol. MacConkey WHO Agar The enterohaemorrhagic strains do not use this substrate and produce colourless colonies, but the other Ref. 01-121 serotypes can ferment sorbitol and produce red colonies In all others aspects, MacConkey Agar with Sorbitol works similarly as the other media in the MacConkey Specification group. Peptone supply the nitrogen and sodium chloride Differential medium with moderate selectivity, without the osmotic environment. Crystal violet and bile salts crystal violet, according to WHO recommendation, for inhibits the growth of grampositive bacteria. Neutral red the isolation of enterobacteria. acts as the pH indicator. Formula (in g/L) Technique Peptone ...... 20,000 Spread the inoculum on the dry surface of the medium Lactose ...... 10,000 and incubate at 35±2ºC for 24 hours. Usually, the O157: Bile Salts mixture ...... 2,500 H7 serotype forms colourless colonies and the other Sodium chloride ...... 5,000 strains of E. coli red colonies. The results must be Neutral Red ...... 0,075 recorded at 24 hours because an extended incubation Agar ...... 15,000 produce a decreasing colouring in the sorbitol-ferment- Final pH 7,4 ± 0,2

96 MacConkey Media

Directions layer of another 5 mL of sterile medium is poured into Suspend 53 g of powder in a litre of distilled water. Bring every plate to seal the surfaceand improve enumera- to the boil, distribute in suitable containers and autoclave tion of the colonies. at 121°C for 15 minutes. For the enumeration, after an incubation of 24 h. at 35°C, select plates with 30-300colonies. The character- Description istic colonies must be confirmed for coliform identity by The MacConkey media are well known and popular gas production from lactose in a broth culture. enrichment system for coliform bacteria. The MacConkey broth (code 2-118) can be used for the At the beginning of the last century, MacConkey made enumeration of coliforms by the MPN technique, select- the original formulation and included ox bile as inhibitor ing the positive tubes that shows turbidity, colour turned of the gram-positive bacteria and the litmus as the indi- to red purple and with gas production. cator of the acid production from lactose sugar. Lately The same above characteristics, but turning into a yellow the litmus has been substituted by phenol red indicator color are valid if the MacConkey broth code 2-120 is to make interpretations easier and more precise. used. The media have been adapted to facilitate the coliform If the MUG medium is used a supplementary reading detection with the advancement of knowledge of the under UV illumination must be carried out. bacterial physiology. The most significant modification to the original formulation has been the substitution of the References ox bile by purified bile salts that improve the selectivity ADAMS, S. (1991) Screening for verotoxin-producing E. and avoid the inherent turbidity which is due to the fat coli. Clin Lab. Science 4:1:19-20. material of the bile. The effectivity of the inhibition of the ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- bile salts is variable and depends on the relative concen- biological Media. CRC Press, Inc. London. tration of cholate and taurocholate. CLESCERI, L.S., A.E. GEENBERG & A.D. EATON Another important modification was the inclusion of (1998) Standard Methods for the Examination of Water supplementary inhibitors such as crystal violet and/or and Wastewater. 20th ed. APHA-AWWA-WEF. Washing- brilliant green, that are the most popular formulations in ton DC. USA America, but not in Europe where the lower selectivity is DOWNES, F.P. & K. ITO (2001) Compendium of Meth- prefered. ods for the Microbiological Examination of Foods.4th. ed. In the 60’s the toxicity of neutral red on the stressed APHA. Washington cells of coliforms was demonstrated, especially on EUROPEAN PHARMACOPOEIA (2007) 5th ed. some strains of E. coli, and then the pH indicator was (Supp.5.7) EDQM. Council of Europe. Strasbourg. changed to the Bromcresol purple, being less aggressive HITCHINS, A.D., P. FENG, W.D. WATKINS, S.R. RIPEY than the neutral red. y C.A. CHANDLER (1998). E. coli and coliform bacteria. However the most extensive use is in the liquid form, Bacteriological Analytical Manual. 8th ed. AOAC Interna- nevertheless there are solids formulations with agar, for tional. Gaitherburg. MD. USA every HORWITZ, W. (2000) Official Methods of Analysis. modification. AOAC Intl. Gaithersburg. MD. USA At the moment, the several formulations available of the ISO 9308-2:1990 Standard. Water Quality. Detection and MacConkey media offer a wide range from a low to high enumeration of coliforms, thermotolerant coliforms and selectivity and the lactose positive bacteria grown on this presumptive E. coli. MPN Method. medium form red colonies due to acid production due to ISO 21150:2006 Standard. Cosmetics – Detection of the lactose fermentation and thus E. coli colonies can be Escherichia coli easily distinguished as they also form a small precipita- ISO 21567. Standard (2004) . Horizontal method for the tion zone of bile salts around them. detection of Shigella ssp. Eventually,some enterococci can also grow, but they MARCH, S.B. y S. RATMANN (1986) Sorbitol-McConkey are easy to distinguish from the coliforms, as they form Medium for detection of E. coli O157:H7 associated with smaller colonies and the absence of precipitation zone. hemorrhagic colitis. J. Clin. Microbiol. 23:869-872 Medium can be used as Presumptive medium for E.coli McCONKEY, A.T. (1905) Lactose-fermenting Bacteria in (by fluorescent reaction) if before sterilization MUG (Ref. faeces. J. Hyg 5:333. 06-102CASE) is added. In the medium with MUG the E. MURRAY, P.R., E.J. BARON, M.A. PFALLER, F.C. TEN- coli colonies show a light blue fluorescence under the OVER, y R.H. YOLKEN (Eds) (1995) Manual of Clinical UV illumination. The formulation without salt offers a low Microbiology 6th ed. A.S.M. Washington D.C. USA electrolyte content that almost suppresses the swarming RAPPAPORT, F. y E. HENING (1952) Media for the iso- growth of Proteus. lation and differentiation of pathogenic E. coli (serotypes O111 and O55) J. Clin. Pathology 5:361-362 Technique USP 29-NF 25. (2006) <61> Microbial Limit Tests US From a decimal dilution bank, 1 mL samples are inocu- Pharmacopoeial Corp. Inc. Rockville. MD, USA lated into empty sterile petri dishes in duplicate. Then ,15 VANDERZANT, C. y D.F. SPLITTOESSER (Eds) (1992) mL of molten medium at 45°C is poured into every plate Compendium of methods for the microbiological exami- and mixed carefully . After the solidification, a second nation of foods. 3rd ed. A.P.H.A. Washington D.C.

97 MacConkey Media

VARNAM, A.H. y M.G. EVANS (1991) Foodborne pathogens. Manson Publishing Ltd. London. U.K. WHO (1963) International Standards for Drinking Wa- ters, 7th ed., Churchill Ltd. London WINDLE-TAYLOR, E.(1958) The Examination of Water and Wastewater Supplies, 7th ed. Churchill Ltd. London

Malachite Green Broth

Ref. 02-467 by the German StandardizationInstitute (DIN) and other legal bodies for the microbiological analysis of water and Specification food. Liquid culture medium for the selective enrichment of Pseudomonas aeruginosa in water, according the DIN Technique 38411 standard. If the product to be examined is not restricted to specific standard (as DIN 38411 for water), it is suggested that Formula (in g/L) the final malachite green concentration for enrichment Meat peptone ...... 15,00 should not exceed 0,01 g/L. Thus the concentrated or Meat extract ...... 9,00 diluted broth should be used in function of the size or Malachite green (Oxalate) ...... 0,03 volume of the sample. Dipotassium phosphate ...... 1,10 Carry out the incubation at 35±2ºC for 24-48 hours. Final pH 7,0 ± 0,2 Cultures that show turbidity due to growth should be selected for the later confirmation for the presence of Pseudomonas aeruginosa. Directions To prepare concentrated broth: Dissolve 25 g of powder in 1 L of distilled water and distribute into suitable References containers. BUNDESGESUNDHEITSAMT: Amtliche Sammlung von To prepare diluted broth: Dissolve 8,3 g of powder in 1 L Untersuchungverfahren nach 35 LMBG Beuth Verlag of distilled water and distribute into suitable containers. Berlin Köln. In both the cases, sterilize by autoclaving at 121ºC for DEUTSCHE EINHEITSVERFAHREN sur Wasser-, 15 minutes. Abwasser- und Schlammuntersuchung. VCH Verlags- gesellschaft D-6940 Weinheim. DIN 38411; Teil 6: Mikrobiologische Verfahren (Gruppe Description K):Nachweis von Pseudomonas aeruginosa (K8) Habs and Kirschner first described this medium in 1948 HABS, H., K.H. KIRSCHNER (1943) Der Pyocyaneus based on the resistance of Pseudomonas aeruginosa Meerschweinchenhautversuch zur Prüfung von Haut to malachite green as a selective factor. Lately, in 1974 desinfektionsmiteln. Z. Hyg. 124:557-578. Schubert and Blum modified the medium composition SCHUBERT, R., U.BLUM (1974) Zur Frage der Eignun and proposed it as an enrichment step of Pseudomonas der Malachitgrün-Bouillon nach Habs u. Kirschner als in very polluted water since malachite green oxalate Anreicherungsmedium fur Pseudomonas aeruginosa at the concentration as above formulation inhibited the aus dem Wasser. Zbl. Bakt. Hyg. I Orig. B 158:583-587 growth of almost all gramnegative microorganisms but it did not affect Pseudomonas growth. Years after the proposal of Schubert and Blum, the medium was adopted officially as an enrichment medium

98 Malt Extract Media

Media based on malt extract may be considered as gen- then this will make the solidification of agar more difficult. eral growth substrates due to their richness and nutrient When acidification is below pH 5,0 do not remelt the balance. They are very suitable for the cultivation of agar since the solidifying agent will be hydrolized. fastidious microorganisms. Classically, with acidic pH, they are used for the isolation, cultivation and mainten- Malt Extract Agar No. 2 ace of moulds and yeast, but with pH near to neutrality, they support bacterial growth of bacteria with special or fastidious nutritional needs. Ref. 01-573

The SCHARLAU manufacturing program covers all the Speciﬁcation published formulations, and included in this manual are Solid medium for the isolation and enumeration of fungi. some of the most commonly used. We propose that the technicians choose the formulation most suited to their Formula (in g/L) requirements, or, alternatively, ask us for more details or Malt extract ...... 30,0 possible modiﬁcations. Soy peptone ...... 3,0 Agar ...... 15,0 See also YM Media (Refs. 1-219, 2-219) and Wort Agar Final pH 5,6 ± 0,2 (Ref. 01-132) Directions Malt Extract Agar No. 1 Suspend 48 g of powder in 1 L of distilled water and let it soak. Bring to the boil. Distribute into suitable containers Ref. 01-111 and sterilize in the autoclave at 121°C for 15 minutes.

Speciﬁcation Description Culture medium for moulds and yeast. Malt Extract Agar may support the growth of almost all of the fungi very well, because of its balanced composition, and restrains most of the bacteria due to the strong Formula (in g/L) acidity. Malt extract ...... 13,0 Should more selection against the bacterial growth be Dextrine ...... 2,5 desired, readjust the pH to 3,5 by adding a sterile solu- Gelatin peptone ...... 5,0 tion of 10% lactic acid or 5% tartaric acid to the molten Agar ...... 15,0 medium. After the additions do not reheat the medium. Final pH 5,5 ± 0,2 Malt Extract Agar No. 3 Directions Suspend 35,5 g of powder in 1 L of distilled water and heat gently with constant stirring until the boiling. Dis- Ref. 01-574 pense in suitable containers and sterilize by autoclaving at 115°C for 15 minutes. Avoid overheating since the low Speciﬁcation pH of the medium may hydrolize the agar. Solid medium for the isolation and enumeration of fungi.

Description Formula (in g/L) Malt Extract Agar is a classic culture medium for moulds Malt extract ...... 30,0 and yeast. Malt extract has enough sugar (maltose, glu- Mycological peptone ...... 5,0 cose, sucrose) to allow a copious growth, and in extreme Agar ...... 15,0 cases, additional necessary growth factors are provided Final pH 5,4 ± 0,2 by the gelatin peptone. Directions Technique Suspend 50 g of powder in 1 L of distilled water and let it Malt Extract Agar has been widely used in maintenance, soak. Heat to the boil and distribute into suitable contain- isolation and identiﬁcation of fungi, and it is also pro- ers. Sterilize in the autoclave at 121°C for 15 minutes. posed in several pharmacopiea as a medium for the control of sterility in pharmaceutical products, though it is mostly used for comparative morphological studies. Should more selectivity be desired, you can add a few millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but

99 Malt Extract Media

Description Description The balanced and rich nutrient composition of the me- This formulation of classic Malt Extract Broth is ac- dium makes it suitable for morphogenetic and structural cording to Reiss’ modification in order to achieve better studies of fungi. Due to its low pH it restrains the bacte- results for the cultivation of Aspergillus flavus. rial growth to a greater extent, but the total supression can be achieved by adding to the melted medium at Technique 55°C, 20 mL of sterile solution of 10% Lactic acid or 5% Malt Extract Broth has been widely used in mainte- tartaric acid, making the pH reduces to 3,5. In these nance, isolation and identification of fungi, and it is also conditions, do not heat the medium to avoid the hydroly- proposed in several pharmacopeia as a medium for the sis of agar. control of sterility in pharmaceutical products, though it is mostly used for comparative morphological studies. Malt Extract Broth No. 1 Should more selectivity be desired, you can add a few millilitres of 10% Lactic Acid, or 5% Tartaric Acid, but this Ref. 02-111 makes the solidification of agar very difficult. When acidification is below pH 5,0, do not remelt the agar since the solidifying agent is hydrolized below pH 5,0. Specification Liquid culture medium for the moulds and yeasts. References FDA (1998). Bacteriological Analytical Manual. 8th ed. Formula (in g/L) Revision A AOAC International Gaithersburg) MD. Malt extract ...... 13,0 DOWNES, F.P & K. ITO (2001) Compendium of Meth- Dextrine ...... 2,5 ods for the Microbiological Examination of Food. 4th ed. Gelatin peptone ...... 5,0 APHA. Washington. Final pH 5,5 ± 0,2 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press,. Directions BALLOWS, HAUSLER, HERMAN, ISENBERG & Dissolve 20,5 g of powder in 1 L of distilled water, heat- SHADOMY (eds.) (1991) Manual of Clinical Microbiol- ing up if necessary. Distribute into suitable containers ogy. ASM. Washington. and sterilize in the autoclave at 121°C for 15 minutes. REIS, J. (1972) Ein selektives kulturmedium für der Nachweiss von Aspergillus flavus. Zbl. Bakt. Hyg. I. Abt. Description Orig. 220:564-566. Malt Extract Broth is a classic culture medium for the RAPP, M. (1974) Indikator-Zusätze zur Keimdifferen- moulds and yeasts. Malt extract has sufficient sugar zierung auf Würze und Malzextrakt Agar Milchwiss. (maltose, glucose, sucrose) to allow a copious growth, 29:341-344. and in more demanding cases, the necessary growth factors are provided by the gelatin peptone.

Malt Extract Broth No. 2

Ref. 02-491

Speciﬁcation Liquid culture medium for the moulds and yeasts.

Formula (in g/L) Mycological peptone ...... 3,0 Malt extract ...... 17,0 Final pH 5,4 ± 0,2

Directions Dissolve 20 g of powder in 1 L of distilled water, heating up if necessary. Distribute in suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Do not overheat, since a browning by Maillard reaction can be produced.

100 Mannitol Salt Agar (Chapman Agar)

Ref. 01-116 References Speciﬁcation ATLAS, R.M. & L.C.PARKS (1993) Handbook of Micro- Selective medium for the isolation of staphylococci biological Media. CRC Press. BocaRaton. Fla. USA according USP and ISO standard. CHAPMAN (1945) The signiﬁcance of sodium chloride in studies of staphylococci. J. Bact 50:201 DOWNES, F.P. & K. ITO (2001) Compendium of Meth- Formula (in g/L) ods for the Microbiological Examination of Foods.4th ed. Meat extract ...... 1,000 APHA. Washington DC. USA Casein peptone ...... 5,000 FDA (1995) Bacteriological Analytical Manual. 8th ed. Meat peptone ...... 5,000 Revision A. AOAC Internacional Inc. Gaithersburg. Md. Sodium chloride ...... 75,000 USA D-Mannitol ...... 10,000 ISO 22718:2006 Standard. Cosmetics – Detection of Phenol red ...... 0,025 Staphylococcus aureus. Agar ...... 15,000 USP 29- NF 25(2005) <61>Microbial Limit Tests. US Final pH 7,4 ± 0,2 Pharmacopoeial Convention Inc. Rockville. Md. USA

Directions Suspend 111 g of powder in 1 L of distilled water and bring to the boil. Dispense in tubes or ﬂasks and sterilize by autoclaving at 121°C for 15 minutes.

Description Mannitol Salt Agar is a classical medium for the detection and enumeration of staphylococci. It was described by Chapman and has been adopted by many ofﬁcial organisations. Several modiﬁcations have been developed from it with more or less similar effectivity. This medium uses the advantage of high tolerance of staphylococci to salinity, to use sodium chloride as a selective agent, since only the staphylococci and halophilic enterobacteria are able to grow freely at this concentration of salt employed in this medium while other bacteria are inhibited. It also exploits the correlation between the pathogenic and fermentative capacity of mannitol of staphylococci, to establish a presumptive diagnosis. Mannitol fermentation with an accumulation of acid products is shown by the phenol red indicator turning yellow, Staphylococcus aureus ATCC 25923 that produces a yellow halo surrounding the presumptive pathogen colonies, meanwhile the rest of the medium remains orange in colour.

Technique A massive surface inoculation and an incubation at 37°C for 36 hours or at 32°C for 3 days is recommended. The typical appearance of the colonies after the correct incubation is as follows: Presumptive pathogenic staphylococci (coagulase +) are mannitol positive and are big colonies with a yellow halo. Non-pathogenic Staphyloco- cci (coagulase -) are usually mannitol negative and are small colonies without halo or change in colour. In any case, coagulase presence must be tested by the classical technique, after a pure culture in the liquid medium is obtained, in order to establish its true pahogenic potential.

101 Maximum Recovery Diluent

Ref. 02-510 Technique According to the ISO method, the sample is diluted in a Speciﬁcation ratio 1:10 with the Maximum Recovery Diluent and ho- Isotonic diluent for the maximal recovery of stressed mogenized by a vortex mixer or stomacher. After a short microorganisms according ISO standard. period (10-15 minutes) of rest, a decimal dilution bank with the same diluent is released following the standard Formula (in g/L) procedures. Plates are inoculated from the different concentration of the dilution bank. Peptone ...... 1,00 Sodium chloride ...... 8,50 Final pH 7,0 ± 0,2 Reference ISO/DIS 6649 Meat and Meat Products. Detection and Directions Enumeration of Clostridium perfringens ISO 21149:2006 Cosmetics – Enumeration and detec- Disolve 9,5 g of powder in 1 L of distilled water and dis- tion of aerobic mesophilic bacteria. tribute into suitable containers. Sterilize by autoclaving at ISO 21150:2006 Standard. Cosmetics – Detection of 121ºC for 15 minutes. Escherichia coli ISO 22717:2006 Standard. Cosmetics – Detection of Description Pseudomonas aeruginosa. This formulation combines the osmotic pressure of the ISO 22718:2006 Standard. Cosmetics – Detection of physiological saline solution with the protective action Staphylococcus aureus. of the peptone to obtain a good recovery of stressed microorganisms. The sodium chloride assures the isotonic conditions and the low concentration of the peptone does not allow the cellular growth in the short period (2-4 hours) of time required for the preparation of the dilution bank of the sample.

102 Mayeux Agar

Ref. 01-223 Sodium azide avoids the growth of undesired flora, and at the same time, hampers the colonial development of Specification lactic streptococci. Other authors state that the addition Solid culture medium for the detection of Leuconostoc in of little amounts of tetracycline (15 mcg/mL) produces fermentation starters of mixed flora. the inhibition of lactic streptococci without affecting Leuconostoc. Formula (in g/L) Peptone ...... 10,000 Technique Yeast extract ...... 5,000 The plates are inoculated by surface inoculation, and Sucrose ...... 100,000 then incubated at 21°C for 4 days. Most of the strepto- Dextrose ...... 5,000 coccal strains, including Streptococcus lactis, S.cremoris Sodium citrate ...... 1,000 and S.diacetilactis do not grow or grow a little after the Gelatine ...... 2,500 third day of incubation. In those cases, their colonies are Sodium azide ...... 0,075 small, opaque and cream or yellow coloured. Leucon- Agar ...... 15,000 ostoc colonies have a bigger and earlier growth. Leu- Final pH 6,0 ± 0,2 conostoc citrovorum form the colonies of 0,5 to 1 mm diameter, which are translucent and iridescent. L. dextranicum form big colonies (1-5 mm), which are Directions transparent and mucosal. Suspend 138,5 g of powder in 1 L of distilled water and heat up in boiling water bath at 50-55°C, till the complete liquefaction of the gelatine is obtained. Heat to boiling References and dispense into suitable containers. Sterilize in the MAYEUX and COLMER (1961) J. Bact. 81:1.009. autoclave at 121°C for 15 minutes. Avoid overheating, MAYEUX, SANDINE and ELLIKER (1962) J. Dairy Sci. since it may affect the solidification. 45:665. McDONOUGH, HARDGROVE and TITSLER (1962) J. Dairy Sci. 45:656. Description FIL-IDF Standard 149A (1997) Dairy starters of lactic This differential and selective medium for Leuconostoc acid bacteria culture. Composition standard. was originally described by Mayeux in 1961, and was later modified by the same author to this formulation. This allows a very specific separation of microorganisms in the lactic fermentation starters with mixed flora. Citrate, glucose and gelatine helps for the growth of Leu- conostoc, and the large amount of sucrose allows a copious production of dextrane polymer by L.dextranicum.

103 Meat Liver Agar

Ref. 01-562 reduced to H2S by some Clostridium spp and reacts with the iron from ammonium iron citrate producing a dark Specification precipitate that blackening the medium. Solid medium for the cultivation of anaerobic microorganisms Technique The meat liver agar can be used in petri dishes or Formula (in g/L) in tubes. When the inoculum is in poured plates, the Meat Extract ...... 10,00 reducing power of the medium permits the growth of the Liver Extract ...... 10,00 anaerobes. If spreading plates are used the incubation in Dextrose ...... 0,75 an anaerobic environment is compulsory, but some times Soluble starch ...... 0,75 is enough cover the plate with Sealing Anaerobic Agar Sodium sulfite ...... 1,20 (Ref. 01-174). Temperature and time of the incubation Ammonium Iron (III) citrate ...... 0,50 must be suitable to the sample, but it is recommended Agar ...... 13,00 more than 48 hours at 35-37ºC Final pH 7,6 ± 0,2 References Directions ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- Suspend 36,2 g of powder in 1 L of distilled water and biological Media. CRC Press Inc. London. had to boiling with constantly stirring. Distribute in suit- CORETTI, C. (1962) Prüfung eines leberpulvers auf able containers and sterilize in the autoclave at 121ºC Eignung zur Herstellung von Leberbrühe und Leberagar for 15 minutes. zur Anaerobenzuchtung. Berl. Münch. Tierärztl. Wissch. 75:205. VANDERZANT, C. & D.F. SPLITTSTOESSER (1992) Description Compendium of methods for the microbial examination The mixture of meat and liver extract is a highly reduc- of Foods. 3rd Ed. APHA. Washington. ing nutrient basis that provides the supply of nitrogen for the growth of anaerobes. The energy source is provided by the dextrose and the starch acts only as a metabolic detoxifier. The sulphite present in the culture medium is

104 Methyl Red Voges Proskauer Media (MRVP Media)

Methyl Red Voges Proskauer Methyl Red Voges Proskauer Broth (MRVP Broth) Modiﬁed Broth for Bacillus (Clarks Lubs Medium) (MRVP Modiﬁed Broth)

Ref. 02-207 Ref. 02-572

Speciﬁcation Speciﬁcation Classic liquid medium for differential tests (Voges Liquid culture media to perform the Voges-Proskauer Proskauer and Methyl Red) in Enterobacteria according test in Bacillus cultures according FIL-IDF 181 standard. ISO standards 6579 and 6585 and FIL - IDF 93 standard. Formula (in g/L) Tryptone ...... 7,00 Formula (in g/L) Dextrose ...... 5,00 Peptone ...... 7,0 Dipotasium phosphate ...... 5,00 Dextrose ...... 5,0 Sodium chloride ...... 5,00 Potassium phosphate ...... 5,0 Final pH 7,0 ± 0,2 Final pH 7,0 ± 0,2 Directions Directions Dissolve 22 g of powder in 1 L of distilled water, heating Dissolve 17 g of powder in 1 L of distilled water, heating if it is necessary. Distribute in suitable containers (5 mL up only if necessary. Dispense in tubes and sterilize by in test tubes of 16 x 160 mm) and sterilize in autoclave autoclaving at 121°C for 15 minutes. at 121ºC for 15 minutes

Description Description The classical Clark and Lubs medium which is used to This medium is produced according the FIL-IDF formula- perform the tests of Methyl Red and Voges Proskauer, tion to perform the test of acetil-metil-carbinol production that together with Indole and Citrate tests (IMViC) allow in Bacillus cereus and other species of Bacillus. the differentiation within the coliform group of bacteria. The fundamentals of these reactions are as follows: Methyl Red Voges Proskauer

Methyl Red Test (M.R. test) Saline Broth Among the Enterobacteriaceae, the E.coli biotype fer- (MRVP Saline Broth) ments glucose by the mixed acid pathway, accumulating acid, which reduces the initial pH. It can be detected by Ref. 02-456 the methyl red indicator, which turns yellow above the pH 5,1 and becomes red below pH 4,4. Specification Liquid culture medium for the Mehyl Red and Voges Voges Proskauer Test (V.P. test) Proskauer tests. Enterobacteria of Klebsiella-Enterobacter biotype ferment the glucose by the 2-3-butanediol pathway. Although the acids are produced in this way, the neutral Formula (in g/L) or alkaline products are also formed and at the end the Peptone ...... 7,00 reaction is neutral or alkaline. Due to this, the incubation Dextrose ...... 5,00 must be extended up to 3 days. After this period, the Sodium chloride ...... 30,00 methyl red reaction is negative. Dipotassium phosphate ...... 5,00 Final pH 7,4 ± 0,2 Nonetheless, Voges Proskauer test is complementary to Methyl Red test in some ways. It shows the 2-3-bu- Directions tanediol and acetoin production, that are substances Dissolve 47 g of powder in 1 L of distilled water, heating difficult to find in the mixed acid pathway. It exploits the up only if necessary. Distribute into suitable containers fact that these two products, in alkaline medium, oxidize and sterilize by autoclaving at 121ºC for 15 minutes. themselves to diacetyl, which reacts with guanidine and produces visibly coloured compounds.

105 Methyl-red Voges Proskauer Media (MRVP Media)

Description Voges Proskauer test may be performed in a quicker This medium is used to perform the tests of Methyl Red way, using very little volumes of medium and massive in- and Voges Proskauer, that, together with Indole and Cit- oculum. This allows the readings with short incubations rate tests allow the differentiation within the enteric bac- (18-20 hours), and also the readings may be accelerated teria. The fundamentals of these reactions are described by gently heating the culture almost to boiling point after in the Methyl-Red Voges-Proskauer Broth (Ref. 02-207) adding the reagents. However, erroneous results are more likely by using this method. Technique There are several techniques to carry out these tests. Refer to the FIL-IDF Standard for the specific technique One of them is as follows: of MRVP Modified Broth. The tube with medium is inoculated with the microorganism to be studied and incubated at 30°C for at least 3 References days and up to 5 days maximum. Just before reading, VOGES, O, B. PROSKAUER (1898) Beitragzur culture is separated in two portions, one for each test. Ernährungsphysiologic und zur Differentialdiagnose der hämorrhagischen Septicäemie. Z. Hyg. 1) Methyl Red Test. CLARK, W., H. LUBS. (1915) The differentiation of bac- Add 4-5 drops of Methyl Red Reagent (Ref. 06-007) to teria of the colon-aerogenes family by the use of indica- the culture, and shake in order to homogenize. Observe tors. J. Inf. Dis. 17:160-173 for the colour development in the medium. The test is BARRIT, M. (1936) The intensification of the Voges considered positive if it turns to red and negative if it Proskauer reaction by the addition of alpha-naftol. J. remains yellow. Path. Bact. 42:441-452 O’MEARA, R. (1931) A simple delicate and rapid Positive (red colouration): E.coli, Edwardsiella, Shigella, methods of detecting the formation of acetylmethyl/car- Salmonella, Citrobacter, Proteus, Klebsiella ozoe- binol by bacteria fermenting carbohidrats. J. Path. Bact. nae, Klebsiella rhinoscleromatis, Yersinia. 34:401-406 Negative (yellow colouration): Enterobacter, Hafnia, Ser- MOLLÄNDER, R., J. BÖHMANN, B. GREWING (1982) ratia, Klebsiella pneumoniae. Die Verstörkung der Voges-Proskauer Reaktion durech With Erwinia, this reaction has no significance since it fumarat. Zbt. Bakt. Hyg. I Alet. Orig. A 252:316-323. gives variable reactions. SCHWEIZERISCHES LEBENSMITTELBUCH 5th Ed. Ch. 56A. Berna. 2) Voges Proskauer Test FDA (1998) Bacteriological Analytical Manual. 8th ed. Add Barrit’s Reagent to the medium (Ref. 06-027) until it Rev. A. AOAC International. Gaithersburg. MD. gets a milky appearance and then add O’Meara’s Rea- ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- gent (Ref. 06-006) until milky appearance disappears. biological Media. CRC Press, Inc.,London. Shake vigorously. PASCUAL ANDERSON, MªRª (1992) Microbiología Test is positive if the medium acquires a pink-violet col- Alimentaria. Diaz de Santos, S.A.,Madrid,. our, forming at the top of the tube. If the test is negative, ISO Standard 6579 (2002) Microbiology of Food and ani- there is no colour formation. Relative amounts of each mal feeding stuffs- Horizontal method for the detection of reagent depend on initial volumes of the medium. Never Salmonella species. incubate above 30°C. FIL-IDF (1998) International Provisional Standard 181. Dried Milk Products: Enumeration of Bacillus cereus: Positive (pink-intense red) : Enterobacter, Hafnia, Kleb- Most Probable Number Technique. siella pneumoniae, Serratia. FIL-IDF (2001) Milk and Milk products Detection of Negative (no colour change): Escherichia, Edwardsiella, Salmonella. Citrobacter, Salmonella, Shigella, Yersinia, Kleb- ISO 6585 standard (2001) Milk and Milk products - De- siella ozonae, Klebsiella rhinoscleromatis. tection of Salmonella. With Proteus and Erwinia types, this reaction has no significance since it gives variable reactions.

106 Microbial Content Test Agar (TSA Lecithin Polysorbate)

Ref. 01-613

Speciﬁcation Solid medium for sampling of surfaces of sanitary importance with RODAC plates technique.

Formula (in g/L) Tryptone ...... 15,00 Soy peptone ...... 5,00 Sodium chloride ...... 5,00 Lecithin ...... 0,70 Polysorbate 80 ...... 5,00 Agar ...... 15,00 Final pH 7,3 ± 0,2

Directions The dehydrated medium has a characteristic “brown sugar” appearance and may seem moist. Suspend 45,7 g of powder in 1 L of distilled water and let it soak. Bring to the boil and dDistribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

Description This medium is a modiﬁcation of the classical TSA for the surface sampling by the RODAC (Replicate Organ- ism Detection and Counting) plate technique. Collection of samples from identical areas (replicate) “before and after” treatment with disinfectant yields data useful in evaluating cleaning procedures in environmental sanita- tion. Lecithin is incorporated to neutralize quaternary ammonium compounds and polysorbate 80 is used to neutralize phenolic disinfectants, hexachlorophene, formalin and, with lecithin, ethanol.

References HICKEY, P.J., C.E. BECKELHEIMER, & T. PARROW (1992) Microbiological tests for equipment, containers, water and air. In R.T. Marshall (Ed.) Standard Methods for the examination of Dairy Products 16th ed. APHA Washington. EVANCHO, G.M., W.H. SVEUM, LL. J. MOBERG & J.F. FRANK (2001) Microbiological Monitoring of the Food Processing Environment. In Downes & Ito (Eds) Compen- dium of Methods for the Microbiological Examination of Foods. 4th ed. APHA. Washington DC. ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- biological Culture Media. CRC Press. Boca Ratón, Fla.

107 Milk Agar

Ref. 01-514

Speciﬁcation Solid culture medium for the plate count test in dairy products.

Formula (in g/L) Peptone ...... 5,00 Yeast extract ...... 3,00 Powdered milk ...... 1,00 Agar ...... 15,00 Final pH 7,2 ± 0,2

Directions Suspend 24 g of powder in 1 L of distilled water and let it soak. Bring to the boil and distribute into suitable containers. Sterilize by autoclaving at 121ºC for 15 minutes.

Description Milk Agar is approved by the European Commission and it is formulated according to the recommendations of the European Association of Ice-Cream Producers (EuroGla- ce) for the microbiological examination of ice-creams.

Technique Suggested technique is the standardized count on mass- inoculated plates. Inoculum is obtained from a decimal dilution bank of the sample. Once inoculated, the plates should be left undisturbed for 1-3 hours and then incubated at 30ºC for 3 days.

References KLOSE, J. (1968) Harmonisierung des speisesrechtes under EWC-Sübwaren 14:778-780. KLOSE, J. (1968) Entwarf einer Riehlinie zar Auﬂeichung der Rechtvorschiften fur Speiseeis unden Mitﬂiedsstaaten der EWG. Sübwaren 14:780-782 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press. Boca Raton. Fla.

108 Motility Media

Motility Indole Ornithine Fluid Motility Nitrate Medium Medium (MIO) Ref. 03-612 Ref. 03-422 Specification Specification Medium for the motility determination and nitrate reduc- Medium for the demonstration of motility, indole production of clostridia in food products acc. ISO 7937. tion and ornithine decarboxylase activity of enterobacteria. Formula (in g/L) Meat extract ...... 3,0 Formula (in g/L) Peptone ...... 5,0 Yeast extract ...... 3,00 Potassium nitrate ...... 1,0 Dextrose ...... 1,00 Disodiun phosphate ...... 2,5 Gelatin peptone ...... 10,00 Galactose ...... 5,0 Casein peptone ...... 10,00 Agar ...... 5,0 L-Ornithine HCl ...... 5,00 Final pH 7,3 ± 0,2 Bromocresol purple ...... 0,02 Agar ...... 2,50 Directions Final pH 6,6 ± 0,2 Suspend 21,5 g of powder in 1 L of distilled water containing 5 mL of glycerol and heat to boiling. Dispense Directions into suitable containers and sterilize by autoclaving at Suspend 31,5 g of powder in 1 L of distilled water and let 121°C for 15 minutes. it soak. Bring to the boil and distribute in tubes. Sterilize by autoclaving at 121°C for 15 minutes. Description This semisolid medium has been made according to the Technique rules suggested by the US Food and Drug Administra- Remove all the dissolved air in the medium by heating tion for the identification of Clostridium perfringens in up the tubes in boiling water bath and cooling them upto food. room temperature. Taking the growth of the primary isolation as the inoculum, inoculate the tubes by a single Technique deep stab. Incubate aerobically at 35±2°C for a 18-24 Prepared tubes regenerate themselves if kept in boiling hours period. water bath for 10 minutes to eliminate the dissolved oxy- Motility can be observed by the diffuse growth at the gen. Let them get cooled to 70-80°C and then inoculate upper side of the stab, meanwhile the immotile bacteria them by stabbing the centre. Take a black colony grown grow along the stab, producing a clear streak. on TSN Agar (Ref. 01-195) as the inoculum. Incubate Ornithine decarboxylation is indicated by the presence of the tubes at 37°C for 18-20 hours without sealing nor in a dark purple colour throughout the tube. Negative reac- anaerobic chamber. tion produces only a single purple band at the top, and In this medium, if the growth is stopped at 5-7mm. from the rest of the tube changes to yellow. the surface, signifies that there is anaerobiosis. Non-mo- Indole production is verified after the addition of a few tile is evident as the growth is observed only inside the drops of Kovac’s Reagent (Ref. 06-018) (shake gently). stab. The presence of a red ring signifies the positive reaction, To verify the nitrate reduction to nitrite, pour a few drops and if the colour is yellow, then the reaction is negative. of Nitrate A Solution (Ref. 06-003) and Nitrate B Solution (Ref. 06-004) on the surface of the medium. If a pink or References red colour appears, reaction is positive. ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Clostridium perfringens is an anaerobic , non-motile and biological Media. CRC Press,Boca Raton,Fla. reducer of nitrate to nitrite microorganism. EDERER, G.M. and M. CLARK (1970) Motility-Indol-Or- nithine Medium. Appl. Microbiol. 2:849. References FDA (1998) Bacteriological Analytical Manual 8th ed. FDA (1998) Baceriological Analytical Manual. 8th ed. REvision A. AOAC International. Gaithersburg. MD. Revision A. AOAC International. Gaithersburg. ISO 7937 Standard (2004). Microbiology of food and animal feeding stuffs. Horizontal methods for the enumeration of Clostridium perfringens. Colony count technique.

109 MRS Media

MRS Agar Directions Suspend 52 g of powder in 1 L of distilled water. Heat up Ref. 01-135 to complete dissolution and dispense into suitable containers. Sterilize by autoclaving at 121°C for 15 minutes. Specification Solid culture medium for lactobacilli, according to de Description Man, Rogosa and Sharpe and ISO standards 9332 and MRS Agar and Broth are a modification of the previously 15214. used media for the cultivation of lactobacilli, all of them based on tomato juice’s nourishing properties. The ad- Formula (in g/L) dition of magnesium, manganese and acetate, together with the Polysorbate, has provided an improved medium Peptone Proteose ...... 10,00 for the growth of lactobacilli, including that of very fastidi- Meat extract ...... 8,00 ous species such as Lactobacillus brevis and Lactoba- Yeast extract ...... 4,00 cillus fermenti. D(+)Glucose ...... 20,00 On the other hand, the quality of the peptones in addition Sodium acetate ...... 5,00 to the meat and yeast extracts, combine together all the Triammonium citrate ...... 2,00 necessary growth factors that make the MRS media one Magnesium sulfate ...... 0,20 of the best media for the cultivation of lactobacilli. Manganese sulfate ...... 0,05 Nevertheless, these media selectivity is low and the con- Dipotassium phosphate ...... 2,00 taminants tend to grow in these media, which signifies Polysorbate 80 ...... 1,00 a higher selectivity is needed. We therefore suggest the Agar ...... 14,00 use of subculture in solid medium, on double layer and Final pH 6,2 ± 0,2 broth. In many cases,the growth is encouraged by a CO2 enriched atmosphere in the medium. Directions MRS media is particularly recommended for the enumer- Suspend 66 g of powder in 1 L of distilled water. Bring to ation and maintenance of lactobacilli either by the MPN the boil slowly with gentle stirring until complete dissolu- technique (in broth) or on the plate by massive inoculation. Dispense into suitable containers and sterilize by tion, overlaying it with a second layer of molten medium. autoclaving at 121°C for 15 minutes. This technique overcomes the need of a CO2 enriched atmosphere. MRS Broth References Ref. 02-135 DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Foods.4th Ed. Specification APHA. Washington DC. USA Liquid culture medium for lactobacilli, according to de FIL-IDF Standard 146 (2003) Yoghurt. Identification of Man, Rogosa and Sharpe and ISO standards 9332 and characteristic micro-organisms. 15214. IFU Method No 5 (1996) Lactic Acid Bacteria Count Pro- cedure. Schweizerischer Obstverband. CH-6302 Zug Formula (in g/L) IFU Method No 7 (1998) Sterility testing of aseptic filled products, commercial sterile products and preserved Peptone Proteose ...... 10,00 products. Schweizerischer Obstverband. CH-6302 Zug Meat extract ...... 8,00 IFU Method No 9 (1998) Microbiological examination of Yeast extract ...... 4,00 potential spoilage micro-organisms of tomato products. D(+)-Glucose ...... 20,00 Schweizerischer Obstverband. CH-6302 Zug Sodium acetate ...... 5,00 ISO Standard 9232 (2003) Yoghurt – Identification of Triammonium citrate ...... 2,00 characteristic microorganisms (Lactobacillus delbrueckii Magnesium sulfate ...... 0,20 subsp bulgaricus and Streptococcus thermophilus) Manganese sulfate ...... 0,05 ISO Standard 15214 (1998) Horizontal method for the Dipotassium phosphate ...... 2,00 enumeration of mesophilic lactic acid bacteria – Colony Polysorbate 80 ...... 1,00 count technique at 30ºC Final pH 6,2 ± 0,2 MAN, J.C. de, ROGOSA, M. y SHARPE, M. Elisabeth (1960) A médium for the cultivation of lactobacilli. J. Appl. Bact.; 23:130.

110 Mueller Hinton Media

Mueller Hinton Agar In this medium, and in its solid version, presence of the starch is very important, since it acts as a detoxifying Ref. 01-136 agent against the toxic substances if present in the sample and it also acts as the cell regenerator. Speciﬁcation Widely recommended medium for antibiotic and sulfona- Technique mide susceptibility testing, according to the Kirby-Bauer For the culture of Neisseria the best results are obtained and the Ericsson methods. if incubation is carried out in a humid chamber with a CO2 enriched atmosphere, if an anaerobic jar is not available. This environment can be obtained by placing Formula (in g/L) the plates in a hermetically sealed air-tight container, Peptone ...... 17,5 a dessicator for instance, together with a cotton swab Beef infusion solids ...... 2,0 soaked in water and a lighted candle end. Once the con- Starch ...... 1,5 tainer is full the ﬂame consume oxygen and by the time it Agar ...... 17,0 is extinguished, the atmosphere inside the container has Final pH 7,3 ± 0,2 got 5 to 8% CO2 enrichment.

Directions The Mueller-Hinton Agar has proved to be one of the Add 38 g of powder to 1 L of distilled water and let it most efficient medium in the anti-bacterial susceptibil- soak. Bring to the boil to dissolve the medium complete- ity testing. Without the addition of blood it can even ly. Sterilize by autoclaving at 121°C for 15 minutes. be used for sulfonamide sensitivity testing since it is free from most of its antagonists (nucleotides, etc.). If Description this type of assay is conducted, the zones of inhibition The Mueller Hinton Agar was originally designed for the should be examined just after 12-18 hours, before the primary isolation of meningococci and gonococci. With usual overgrowth occurs, since after 24 hours it tends to the addition of blood it becomes an optimal medium interfere with the examination of sulfonamides sensitivity. for the growth of Neisseria. It is also more effective if For this purpose, a small inoculum will help the early reheated and turned into a Chocolate Agar. It should formation of zones of inhibition. It should amount to a never be remelted or reheated once the blood has been 100 to 300 times smaller inoculum than that of the cor- added to it. responding strain which is used in the antibiotic sensitivity testing. Mueller Hinton Broth In 1970 the WHO proposed this medium for antibacterial sensitivity testing, and it has been widely used since Ref. 02-136 then. Sensitivity testing can be conducted by a variety of Specification techniques, both on solid and liquid media. The most Liquid version of the agar with the same name, recom- commonly used method in routine work is that derived mended for the studies about MIC of antibiotic. from Kirby-Bauer and recommended by the American Association of Clinical Pathologists. It provides informa- Formula (in g/L) tion on growth around a disk impregnated with antibacte- Peptone ...... 17,5 rial substance. Starch ...... 1,5 Solids of meat infusion ...... 2,0 The Bauer-Kirby method is more precise and is semi- Final pH 7,3 ± 0,2 quantitative by category. It uses the Mueller-Hinton Agar and disks with high antibiotic concentration. The inocu- Directions lum is first standardized with a Mac-Farland nephlom- Add 21 g of powder to 1 L of distilled water and dissolve eter. Then the plate is inoculated with a swab dipped in it completely. Distribute in suitable containers. Sterilize the standardized suspension, and finally the disks are by autoclaving at 121°C for 15 minutes. arranged properly and at the equidistance from each other on the plate and then incubated. Description Some authors suggest that the inoculum should be Mueller Hinton Broth is the liquid version of the agar with modified by introducing a double layer of mass inoculat- the same name, and can be used in parallel with the ed medium. This system undoubtedly provides sharper agar when comparative studies are desired as well as and more defined zones of clearing or inhibition. Plates when a broth with a high nutritive capacity is required. are incubated at 37°C It is especially suggested for inoculum preparation for sensitivity assays.

111 Mueller Hinton Media

overnight and then the zones of inhibition are measured. Sensitive, Resistant or as Minimum Inhibitory Concentra- Results are reported in terms of Resistant, Moderately tion (MIC). Resistant and Sensitive strains (Table above). This latest technique undoubtedly offers more precision The Ericsson technique, which has been adopted in and reliability than the previous ones. Nevertheless, the most European countries has already standardized the Kirky method, which is semiquantitative, is much more culture medium (Mueller-Hinton) and the quantity per simple and easy to adopt in everyday practice. On the plate (25 mL on 9 cm diameter plates). It has also stand- other hand, the Ericsson technique is highly recom- ardized the inoculum concentration. mended for the effectivity and the sensitivity studies. The fresh culture suspension to be examined (incubated for 18 hours in liquid medium) must be diluted enough, The Mueller-Hinton medium plates can be stored refrig- so as to ensure the presence of confluent growth on the erated in plastic bags for a month without affecting their agar. results of sensitivity testing. However, they should not be Suggested Dilutions: used if the medium shows any dehydration. Enterobacteria- Pseudomonas: dilution of 1/300. The Mueller Hinton Agar Scharlau fulfills the WHO re- Staphylococcus - Enterococcus: dilution of 1/300. quirements for the conducting microbial sensitivity tests Streptococcus - Haemophilus: dilution of 1/10. and the basic characteristics are verified in every batch. The plate is seeded by flooding its surface. The ex- Nevertheless some variation in the results between cess inoculum is removed with a sterile pipette and the batches can be observed and technicians claims about antibiotic disks are arranged properly on the plate. Allow the origin of these variability. At this point must keep in a pre-diffusion period of 30-60 minutes before incuba- mind a lot of factors that are a source of variability: tion so that the antibiotic can slowly diffuse before the 1. Since the nutritional requirements of organisms vary, growth. After the incubation at 37°C for 12-18 hours, some strains may be encountered that fail to grow or measure the zones of inhibition and refer to the Assay grow poorly on these media. Regression Curves. Results are reported in terms of

112 Mueller Hinton Media

2. Numerous factors can affect results: inoculum size, References rate of growth, medium formulation and pH, length of BAUER A.L., W.M.M. KIRBY, J.C.SHERRIS & incubation and incubation environment, disk con- M.TURCK (1966) Antibiotic susceptibility testing by a tent and drug diffusion rate, and measurement of standardized single disc method. Am. J. Clin. Pathol 45: endpoints. Therefore, strict adherence to protocol is 493. required to ensure reliable results. BARRY, A.L., M.D. COYLE, C. THORNBERRY, E.H. 3. Disk diffusion susceptibility testing is limited to rapidly GARLACH & R.W. HAWKINSON (1979) Methods of growing organisms. Drug inactivation may result measuring zones of inhibition with Bauer-Kirby disk sus- from the prolonged incubation times required by slow ceptibility test. J. Clin. Microbiol. 10:885-889. growers. ERICSSON & SHERRIS (1971) Antibiotic sensitivity 4. Media containing excessive amounts of thymidine or testing. Report of an International Collaborative Study. thymine can reverse the inhibitory effects of sulfona- Acta Pathol. Microbiol. Scand Suppl. 217 p: 90. mides and trimethoprim, causing zones of growth HINDLER, J. (1998) Antimicrobial Susceptibility Testing inhibition to be smaller or less distinct. In Essential Procedures for Clinical Microbiology. ASM 5. Variation in the concentration of divalent cations, Press. Washington D:C. primarily calcium and magnesium, affects results of MUNRO, S. (1995) Disk Diffusion Susceptibility Testing. aminoglycoside, tetracycline, and colistin tests with In Clinical Microbiology Procedures Handbook. H.D. Pseudomonas aeruginosa isolates. A cation content Isenberg (Ed) APHA Whasington D.C. that is too high reduces zones sizes, whereas a MILLER, J.M., C. THORNBERRY & C.N. BAKER (1984) cation content that is too low has the opposite effect. Disk diffusion susceptibility test troubleshooting guide. 6. When Mueller Hinton Medium is supplemented with Lab. Med. 15:183-185. blood, the zone of inhibition for oxacillin and methicil- NCCLS Standard M2-A6 (1997) Performance standards lin may be 2 to 3 mm smaller than those obtained for antimicrobial disk susceptibility tests. 6th ed. National with unsupplemented agar. Conversely, sheep blood Committee for Clinical Laboratory Standards. Vilanova. may markedly increase the zone diameters of some PA. cephalosporins when they are tested against ente- THORNSBERRY, C., W.G. GAVAN, E.H. GERLACH & rococci. Sheep blood may cause indistinct zones or J.C. SHERRIS (1977) Cumitech 6. ASM. Washington. a ﬁlm of growth within the zones of inhibition around WHO (1977) Requeriments for antibiotic susceptibility sulfonamide and trimethoprim disks. tests. Technical Report Series No 610. Geneva. 7. Mueller Hinton Medium deeper than 4 mm may cause WOODS, G.L. & J.A. WASHINGTON (1995) Antibacteri- false-resistant results, and agar less than 4 mm deep al susceptibility tests: dilution and disk diffusion methods. may be associated with a false-susceptibility report. In P.R. Murray, E.J. Baron, M.A. Pfaller, P.C. Tenover 8. A pH outside the range of 7,3±0,1 may adversely and R.H. Yolken (Eds.) Manual of Clinical Microbiology. affect susceptibility test results. If the pH is too low, 6th ed. APHA. Washington, D.C. aminoglycosides and macrolides will appear to lose CFR (1972) Rules and Regulations. 37: 20525. potency; others may appear to have excessive activ- NEUMAN, M.A., D.F. SAMM, C. THORNSBERRY, I.E. ity. The opposite effects are possible if the pH is too McGOWAN (1991) New developments in antimicrobial high. agent susceptibility testing: A practical guide. ASM. 9. When Mueller Hinton Medium is inoculated, no drop- Washington, D.C. lets of moisture should be visible on the surface or on the petri dish cover. 10. Mueller Hinton Medium should be inoculated within 15 minutes after the inoculum suspension has been adjusted. 11. The zone of inhibition diameters of some drugs, such as the macrolides, aminoglycosides and tetracy-

clines, are signiﬁcantly altered by CO2. Plates should

not be incubated in increased CO2 atmosphere.

For further information on the performance of the antibiotic disk susceptibility test refer to the M2-A6 NCCLS Monograph.

113 Mycological Agar

Ref. 01-131 Description Mycological Agar is a general medium that provides Speciﬁcation enough nutrients for the development of most yeasts Solid culture medium for the maintenance, enumeration and moulds. and chromogenesis of fungi. It is employed in plates for the colonial isolation and characterization, as it aids chromogenesis. Formula (in g/L) In the slants it is used for the maintenance of strains, because its low content of glucose yields very slow acid Soy peptone ...... 10,0 formation. Dextrose ...... 10,0 Agar ...... 17,0 Final pH 7,0 ± 0,2 References AJELLO, GEORG, KAPLAN and KAUFFMAN (1963) Directions CDC Lab Manual for Medical Mycology. PHS Pub. N° 994, Washington DC. Suspend 37 g of powder in 1 L of distilled water and heat ATLAS, M.R., L.C. PARKS (1993) Handbook of Micro- to boiling. Dispense in ﬂasks or tubes and sterilize in the biological Media. CRC Press, London. autoclave at 121°C for 15 minutes. Should a selective VANDERZANT & SPLITTSTOESSER (1992). Compen- medium by the acidic pH be desired, adjust the pH to dium of Methods for the Microbiological Examination of 4,0 with a sterile solution of 10% lactic acid, and do not Food. 3rd. Ed. APHA. Washington. reheat the medium afterwards. CTFA Microbiological Guidelines (1993) The Cosmetic Toiletry and Fragance Association. Washington DC.

114 Neutralizing Fluid Eur. Phar.

Ref. 02-512 Description Neutralizing Fluid is formulated according to the Euro- Specification pean Pharmacopeia formulation for the microbiological Liquid medium for neutralizing the antimicrobials accord- examination of non sterile products. Its composition is ing to the European Pharmacopoeia. the same as the general diluting solution for biological assays with the addition of polysorbate and lecithin as Formula (in g/L) non toxic neutralizing agents. However, the European Pharmacopoeia lets the tech- Peptone ...... 1,00 nician to increase the concentration of polysorbate if L-Histidine HCl ...... 1,00 the original is not enough or to add other agents when Lecithin ...... 3,00 thepreservative type to be neutralized is not known. Monopotassium phosphate ...... 3,60 This way, the European Pharmacopeia suggests the Disodium phosphate ...... 7,20 compounds shown in the table below, which have to be Sodium chloride ...... 4,30 always aseptically added to the fluid once sterilized and Final pH 7,0 ± 0,2 cooled to 50ºC or below. Directions Dissolve 20,1 g of powder in 1 L of distilled water con- References European Pharmacopoeia (2002) 4th ed. Supplement taining 30 mL of Polysorbate 80 (Ref. 06-088). Distribute 4.2.2.6.13. Tests for specified microorganisms. Council into suitable containers and sterilize by autoclaving at of Europe.Strasbourg. 121ºC for 15 minutes. Cool to 50ºC and homogenize the solution.

115 Nickerson Agar (BiGGY)

Ref. 01-137 The appearance of the colonies in this medium after an incubation of 48-72 hours at 30-35°C is as follows: Specification Candida albicans: Creamy colonies, very convex, circu- Solid medium for the isolation and identification of Can- lar with very slight mycelial border and black or dida sp. dark brown colour. Neither it has metallic sheen nor the diffused pigment, even after 72 hours of incubation. Formula (in g/L) Candida tropicalis: Acuminated colonies, creamy, irregu- Yeast extract ...... 1,0 lar and with slight mycelial borders. Dark brown Dextrose ...... 10,0 with black centre. After 72 hours of incubation it Glycine ...... 10,0 may take on a metallic sheen and produce a dif- Sodium sulfite ...... 3,0 fussed zone of pigment. Ammonium Bismuth Citrate ...... 5,0 Candida krusei: Big and plain colonies, with irregular Agar ...... 15,0 borders. Brown colour, darker in the centre. A yel- Final pH 6,8 ± 0,2 low halo appears around the colony. Candida parakrusei: Plain colonies, average size, irregu- Directions lar. Dark red centre and light red borders. Yellow Suspend 44 g of powder in 1 L of distilled water and mycelial border. heat to boiling. Dispense in tubes or dishes, stirring the Candida pseudotropicalis: Big and plain colonies, dark precipitate before pouring. Do not autoclave. Avoid red colour. Mycelial border. overheating. Candida stellatoidea: Average size plain colonies, dark brown colour, without mycelial development. Description Rhodotorula: Creamy convex colonies, with irregular Nickerson Agar is suitable for the isolation and identi- border and colours ranging from pink to orange. fication of yeast of the Candida type. Medium is made Moulds in general: Restricted colonial growth and cot- according to the general principles of Bismuth-Sulfite tony appearance. Agar, as inhibitor and differential agent, and completely selective with the high concentration of glycine. This To maintain these colony characteristics it is important medium is highly inhibitory, and does not allow bacterial that the medium is freshly prepared and not reheated or growth, however most Candida grow freely and quickly. overheated. In some occasions, tiny colonies of the bacteria or highly repressed moulds may appear. Bacterial development References may be totally prevented by adding neomycin sulfate to NICKERSON, W.J. (1953) Reduction of inorganic the medium before pouring it into Petri dishes and its substance by yeast I. Extracellular reduction of sulfite by concentration in the medium in this case must be around species of Candida. J.Inf.Dis 93:43. 2 mcg/mL, so that the antibiotic will not affect the development or appearance of yeast.

116 Nitrate Broth

Ref. 02-138 Technique Inoculate 2-3 tubes of broth with one loop of pure culture Specification and incubate at 37°C, reading after 18-24 hours, 2 days A liquid culture medium, according to ISO 7932 stand- and 5 days in each tube, adding some drops of Nitrate ard, to determine the ability of enterobacteria to reduce A Reagent (Ref. 06-003) and of Nitrate B Reagent (Ref. the nitrate to nitrites or free nitrogen gas. 06-004). If the first two readings are negative, it is recommended to investigate with the third one for the pres- Formula (in g/L) ence of nitrate by the method of zinc powder in order to have quick nitrate reduction reaction . Meat extract ...... 3,0 Peptone ...... 5,0 Potassium nitrate ...... 1,0 References Final pH 7,0 ± 0,2 DOWNES, F.P. & K. ITO (2001). Compendium of Meth- ods for the Microbiological Examination of Food.4th ed. Directions APHA. Washington. F.D.A. (1998) Bacteriological Analytical Manual 8th ede. Dissolve 9 g of powder in 1 L of distilled water, heating Rev. A. AOAC International, Gaithersburg.MD up only if necessary to help the dissolution. Distribute ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- into final containers and sterilize by autoclaving at 121°C biological Media. CRC Press, Inc.,London. for 15 minutes. ISO standard 7932 (1993) General guidance for the enumeration of B. cereus. Colony count at 30ºC. Description The Nitrate Broth is prepared according to classical formula for the assay of nitrate reduction by enterobacteria, although it can also be used with aerobic bacilli and other bacterial types.

Nutrient Media

Nutrient Agar (APHA) Nutrient Agar (Brit. Phar.)

Ref. 01-144 Ref. 01-140

Speciﬁcation Speciﬁcation Solid culture medium for the general purposes according Solid culture medium for general purposes and less ISO standard. fastidious organisms acc. EN 12780:2002

Formula (in g/L) Formula (in g/L) Peptone ...... 5,0 Meat extract ...... 1,0 Meat extract ...... 3,0 Yeast extract ...... 2,0 Agar ...... 15,0 Peptone ...... 5,0 Final pH 7,0 ± 0,2 Sodium chloride ...... 5,0 Agar ...... 15,0 Directions Final pH 7,4 ± 0,2 Suspend 23 g of powder in 1 L of distilled water and heat to boiling. Dispense into suitable containers and sterilize Directions in the autoclave at 121°C for 15 minutes. Suspend 28 g of powder in 1 L of distilled water and bring to the boil to dissolve completely. Sterilize by autoclaving at 121°C for 15 minutes.

Description The Nutrient Agar is a simple medium in the range of meat infusions, complemented by a formulation which reinforces its nutrient qualities as well as its growth factors by adding yeast extract. It is most suitable for general routine work and can support the growth of common

117 Nutrient Media organisms, even those considered mildly fastidious with Directions regard to nutrient elements. Besides this, by incorporat- Dissolve 13 g of powder in 1 L of distilled water, heating ing sodium chloride it allows the addition of blood, even if necessary to help dissolve the medium. Distribute into though it is not an optimal medium for it. final containers and sterilize by autoclaving at 121°C for 15 minutes. Nutrient Broth (APHA) Description Ref. 02-144 The Nutrient Broth is the liquid version of the solid medium which bears the same name. It is a classical broth Specification in the range of meat infusions. It is useful for the routine laboratory purposes since its yeast extract supplement Liquid medium for the cultivation of non fastidious micro- allows the growth of most common organisms. It is also organisms according ISO standard. suitable for the preparation of inocula and for the efficiency testing of bactericides, as well as for determina- Formula (in g/L) tion of the Phenol Coefficient and others. Peptone ...... 5,0 Meat extract ...... 3,0 Final pH 7,0 ± 0,2 Nutrient Broth No. 2

Directions Ref. 02-561 Dissolve 8 g of powder in 1 L of distilled water heating up only if necessary. Dispense into suitable containers and Speciﬁcation sterilize by autoclaving at 121°C for 15 minutes. Liquid medium for general purposes.

Description Formula (in g/L) Nutrient Broth is a modern version of the classical general culture medium based on meat infusion. It is a Meat Extract ...... 10,00 simple medium that may be used in general purposes Peptone ...... 10,00 (i.e. maintenance of strains) as well as a base for other Sodium chloride ...... 5,00 specialized media. However, in this way, there are other Final pH 7,5 ± 0,2 media with more nutrient capacity and better performance. Directions Nutrient broth is the liquid version of the Nutrient Agar, Dissolve 25 g of the powder in 1 L of distilled water, and it is a classical medium for normal tasks with non heating if necessary. Distribute in suitable containers and fastidious microorganisms. It is the ideal medium for sterilize in autoclave at 121ºC for 15 minutes. the subculture of general bacteria, especially staphylococci, to carry out later the coagulase and other biochemi- Description cal tests. It may also be used to determine the Phenol This medium in the classical way of the meat infusions, Coefficient by following the technique and microorgan- presents a specially rich nutritional characteristics that isms suggested by the AOAC. facilities the growth of very low inocula, even with fastidious microorganisms. Its formulation is according the Nutrient Broth (Brit. Phar.) BSI for the determination of Rideal-Walker Coefficient of disinfectants, where it is used at double concentration. Ref. 02-140 Nutrient DEV Agar Specification A general purpose liquid culture medium for the less Ref. 01-451 fastidious microorganisms. Specification Formula (in g/L) Solid culture medium for the total enumeration of micro- Meat extract ...... 1,0 organisms in water, according to the German legislation Yeast extract ...... 2,0 and ISO standard. Peptone ...... 5,0 Sodium chloride ...... 5,0 Final pH 7,4 ± 0,2

118 Nutrient Media

Formula (in g/L) DOWNES F.P. & K. ITO (2001) Compendium of Meth- Meat peptone ...... 10,0 ods for the Microbiological Examination of Food.4th ed Meat extract ...... 10,0 APHA. Washington. Sodium chloride ...... 5,0 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Agar ...... 18,0 biological Media. CRC Press, Inc. London. Final pH 7,3 ± 0,2 EUROPEAN STANDARD EN 12780:2002 Water Quality. Detection and enumeration of Pseudomonas aeruginosa Directions by membrane filtration Suspend 43 g of powder in 1 L of distilled water. Bring BUNDESGESUNDHEITAMT: Amtliche Sammlung von to the boil with constant stirring . Distribute into suitable Untersuchungsverfahren nach §35 LMBG. Beuth Verlag containers and sterilize in the autoclave at 121°C for 15 Berlin- Köln. minutes. VERORDNUNG von 12/12/1990 über Trinkwasser und über Wasser fur Lebensmittelbetriebe. Bundesgesetz- Description blatt: Teil I:2613-2629. This medium is formulated according to the German DEUTSCHE EINHEITSVERFAHREN zur Wasser-, legislation but it differs from the other Anglo-Saxon Abwasser- Und Schlammuntersuchung. VCH Verlags- media with the same name in the concentration of its gesellchaft, D-6940 Weinheim. compounds. This change intends to aid the recovery ISO 8523 standard (1991) General guidance for the and growth of damaged microorganisms. detection of enterobacteriaceae with pre-enrichment. ISO 6785 standard (2001) Milk and milk-products - De- Technique tection of Salmonella spp. German standards state a deep inocule of the water ISO 6340 standard (1995) Water Quality - Detection of sample, following the mass seed technique, directly in Salmonella species. the Petri plate. Incubation is performed at 20±2°C for ISO 6579 standard (2002) Horizontal method for the 44±4 hours in most of the cases, but incubations at detection of Salmonella spp. 37±1°C for the same period of time are also allowed. If ISO 10273 standard (1994) General guidance for the the water is chlorinated, the incubation time must last up detection of presumptive pathogenic Yersinia enteroco- to 72 hours. litica. ISO 21567. Standard (2004) . Horizontal method for the References detection of Shigella ssp. APHA (1948) Standard Methods for the Examination of ISO 16266:2006 Standard. Water Quality.– Detection Dairy Products. Washington. and enumeration of Pseudomonas aeruginosa. Method BRITISH PHARMACOPOEIA (1968), 357. by membrane filtration BRITISH STANDARD 541 (1934). Determining the Ri- deal-Walker Coefficient of Disinfectants. BSI London 9.

Nutrient Gelatin Media

Nutrient Gelatin Description Gelatin Nutrient is used, essentially, to identify pure Ref. 03-088 cultures that have no specific nutritional requirements. On the other hand, gelatin liquefaction is considered Specification very important to differentiate enteric bacilli on the basis of their proteolysis. Gelatin was one of the first solidify- Culture medium for determination of gelatin liquefaction. ing agents employed in bacteriology, and helped in the development of the Plate Count Technique, performed Formula (in g/L) by Koch. Nonetheless, nowadays it is not used in that Meat extract ...... 3,0 way, since it was replaced by Agar, which bears incuba- Gelatin peptone ...... 5,0 tions at higher temperatures and was not so attacked or Gelatin ...... 120,0 degraded as gelatin. Final pH 6,7 ± 0,2 However, the Plate Count Method is still used with Directions gelatin. Standard Methods still recommend it for aerobic Suspend 128 g of powder in 1 L of cold distilled water counting at 20-22°C. and heat gently in boiling bath up to 50°C. Keep at that temperature until total dissolution of gelatin. Dispense in tubes or flasks and sterilize in the autoclave at 121°C for 15 minutes.

119 Nutrient Gelatin Media

Technique Nutrient Gelatin DEV The gelatin liquefaction test may be performed in plates or tubes, that are inoculated by stabbing,and incubated Ref. 03-453 at 20-22°C or at the optimum temperature for the microorganism to assay. Because gelatin is liquid above 20°C, Specification it is necessary to put the plates or tubes in the refrigera- Culture medium for the enumeration of total bacteria tor before reading. in not very polluted waters, according to the German High temperature assays (35-37°C) eliminate wrong legislation. results from bacteriolysis, but they require more care in readings and also inclusion of a control (uninoculated) in order to verify effect of heat on the medium. Formula (in g/L) Gelatin ...... 120,00 Gelatin tube readings must be carried out carefully, since Meat extract ...... 10,00 often they have diagnostic value. In the case of using Meat peptone ...... 10,00 several plates, Stone’s reaction may be used to include Sodium chloride ...... 5,00 different strains in the same dish, seeding them by paral- Final pH 7,3 ± 0,2 lel streak. After incubation period, cool in the refrigerator and then, over each streak, put a few drops of saturated Directions solution of Ammonium sulfate or Sulfosalicilic acid 20% Suspend 145 g of powder in 1 L of cold distilled water solution (freshly prepared). and heat up, in boiling water bath, to 50-60°C. Maintain this temperature until total dissolution. Distribute into Liquefaction (positive test) is shown by the presence containers and sterilize in the autoclave at 121°C for 15 of a clear halo or zone around the growth, 10 minutes minutes. after reagent addition. Incubation periods for gelatinase activity assay vary, ranging from a few days to weeks, Description at 20-22°C. Some Klebsiella and Enterobacter strains This medium has the same applications as the Gelatin take up to 3 weeks before they show activity. Though Nutrient (Ref. 03-088) recommended by the APHA, incubation times are not standarized, it is recommended AWWA and Standard Methods. The only difference is the a maximum of 14 days with regular intermittent readings concentration of nutrients and the inclusion of sodium every 3 days. chloride. A modern version of this methodology is the usage of photographic film strips, not exposed, for the liquid me- Technique dia, but this te chnique has a drawback that sometimes German legislation states that water samples are inocu- photosensitive particles included in the film are toxic to lated by deep inoculum (in mass) in Petri plates. Incuba- the microorganisms, and then they give false - negative tion is performed at 20±2°C for 44±4 hours. After the results. incubation, carry out the counting of total bacteria. If water is chlorinated, incubation must be 24 hours more References in order to let damaged cells recover and form visible APHA/AWWA (1995) Standard Methods for the Exami- colonies. nation of Water and Wastewater. 19th. Ed. APHA Inc. New York. 596-597. References ASM (1981) Manual of Methods for General Bacteriol- DEUTSCHE EINHEITSVERFAHREN zur Wasser-, ogy, ASM, Washington, D.C. Abwasser- Und Schlammuntersuchung. VCH Verlags- gesellschaft D-6940 Weinheim.

120 Oxidation-Fermentation Fluid Medium (O/F Medium)

Ref. 03-037 low colouration of the bromothymol blue indicator. The bacteria following the oxidative metabolism carry out this Specification reaction only in the tube without the vaseline but in the Fluid medium according to the Hugh and Leifson formu- other one, which is closed, they change insignificantlly lation, for determining the oxidative and/or fermentative or simply do not grow. Inactive strains do not use sugars metabolism of gram-negative bacilli. and therefore do not induce any change in both the tubes. However, some times there is a slight blue colouration in the open tube, probably due to alkalinization Formula (in g/L) by peptone degradation. Casein peptone ...... 2,00 Some authors have proposed the usage of just one Sodium chloride ...... 5,00 tube for this assay, but in that case the medium must be Dipotassium phosphate ...... 0,20 modified to be the solid (with 1,5% Agar) and with yeast Bromothymol Blue ...... 0,08 and/or cystine extract. In these tubes the stab must be, Agar ...... 2,50 at least, 8 cm. Final pH 7,1 ± 0,2 Hugh and Leifson recommend simultaneous assay with glucose, lactose and sucrose of 1% concentration, add- Directions ing the sterilized sugars to the medium by filtration. Suspend 9,8 g of powder in 1 L of distilled water and bring to the boil. Add sugar in the desired concentration References and distribute in fermentation tubes. Add the vaseline HUGH, R. and E. LEIFSON, (1953) The taxonomic seals or vaspar to half of them. Sterilize by autoclaving significance of fermentative vs. oxidative metbolism of at 121°C for 15 min. cabohidrates by various gram negative bacteria J.Bact. 66:24 Description ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- Hugh and Leifson obtained a clear differentiation of biological media. CRC Press, Inc.London gram-negative bacteria with this medium. They classi- FDA (1998) bacteriological Analitycal Manual. 8th ed. fied them into three categories: fermentative, oxidative Rev. A. AOAC International. Gaithersburg. MD and inactive. The strain to be studied is inoculated in two DOWNES, F.P. & K. ITO (2001) Compendium of meth- long narrow tubes (12x120 mm) by deep stab and one ods for the microbiological examination of food. 4th ed. is covered with a vaseline layer to induce an anaerobic APHA Washington environment that forces the strain to carry out the fer- ISENBERG, H.D. (1992) Clinical Microbiology Proce- mentative metabolism. dures Handbook Vol I ASM Press. Washington Fermentative organisms give a copious production of acid in both the tubes, and it is indicated by the yel-

P Medium Agar

Ref. 01-500 Directions Suspend 30,36 g of powder in 1 litre of distilled water Speciﬁcation and bring to the boil. Distribute in suitable containers and Solid culture medium used as seed agar in the inhibitory sterilize in autoclave at 121ºC for 15 minutes. substances test in milk. Description Formula (in g/L) The present formulation is according the Food and Drug Meat extract ...... 3,00 Administration Bacteriological Analytical Manual for the Peptone ...... 5,00 investigation of Inhibitory substances (Antibiotics and Soy peptone ...... 0,30 preservatives) in milk. This medium that in previous edi- Dextrose ...... 5,25 tions of the BAM was called “PM Indicator Agar”, is used Polysorbate 80 ...... 1,00 as seed agar with B. stearothermophilus spores in the Sodium chloride ...... 0,50 qualitative method or disk assay. Dipotassium phosphate ...... 0,25 Bromcresol purple ...... 0,06 References Agar ...... 15,00 MATURIN, L.J. (1998) Inhibitory substances in milk. Final pH 7,8 ± 0,2 Qualitative Method II: B. stearothermophilus disk assay. In FDA Bacteriological Analytical Manual. 8th Ed. Revi- sion A. AOAC International Inc. Gaithersburg MD.

121 Peptone Agar

Ref. 01-570 milk, the medium will be adjusted to 8.00 with 0,1N NaOH sterile solution after the sterilization. Speciﬁcation Solid culture medium used for the enumeration of Technique contaminants in dairy products, according the FIL-IDF The inoculum or its dilution (in duplicate) is deposited standard over the surface of the medium in the plate in volumes of 0,1 mL. The inocula are spreaded quickly with a Formula (in g/L) Drigalski rod (Ref. 5-010) and let stand 15 minutes to Casein Peptone ...... 7,50 be absorbed in the medium. The plates are incubated Gelatine Peptone ...... 7,50 at 30ºC for 72±2 hours. Select plates with less than 150 Sodium chloride ...... 5,00 colonies to the count. In the counting the needle-bite Agar ...... 15,00 colonies are not considered because they are probably Final pH 7,5 ± 0,2 lactic bacteria. The contaminant must be conﬁrmed by its active catalase. For the sampling, processing and dilution of the products Directions refer to the corresponding FIL-IDF standard. Suspend 35 g of powder in 1 L of distilled water and bring to the boil. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes. References FIL-IDF (1991) Provisional Standard 13: Butter, fresh cheese and fermented milk. Enumeration of non-lactic Description contaminants. Plate Count at 30ºC technique. Peptone Agar is produced according the formulation of the FIL-IDF for its use in the detection and enumeration of non-lactic contaminants in butter, fresh cheese and fermented milk. All the components of the medium are sugar-free and the pH is adjusted to 7,5 that’s is the used for cheese and butter. If the sample is fermented

Peptone Water Phosphate-Buffered

Ref. 02-568 References BEKERS, H.J. (1987) Studies with salmonellae. J. Appl. Speciﬁcation Bact. 62:97-112 Liquid non-selective pre-enrichment medium for entero- SCHWEIZERISCHES LEBENSMITTELBUCH (1992) 5th bacteria ed. Chapter 56A

Formula (in g/L) Meat Peptone ...... 10,00 Sodium chloride ...... 5,00 Disodium phosphate ...... 3,50 Monopotasium phosphate ...... 1,50 Final pH 7,2 ± 0,2

Directions Dissolve 20 g of powder in 1 L of distilled water, heating if necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

Description This medium is produced according the formulation of the Schwezerisches Lebensmittelbuch and is recommended as non-selective pre-enrichment for sub-lethally damaged cells of the enterobacteria group in food or in others samples.

122 Phenol Red Broth Base

Ref. 02-032 Sugar addition can be done in sterile solution after autoclaving the medium, or by adding impregnated discs to 10 mL of Speciﬁcation medium. Addition of some sugars may cause the acidiﬁca- Liquid culture media,suitable for the sugar and other tion of the medium, in which case the original pH must be substrate fermentation studies according ISO 10273 maintained by adding a few drops of 0,1 N NaOH. standard. Should you be working with anaerobics, it is advisable to use a freshly prepared medium, or put the medium in boiling water bath for a few minutes, in order to eliminate dissolved Formula (in g/L) oxygen. Many authors recommend the addition of 0,04% Casein peptone ...... 10,000 agar for these purposes to avoid convection streams and Sodium chloride ...... 5,000 subsequent incorporation of the air. Phenol red ...... 0,018 To study the sugar fermentations of enterobacteria, Brom- Final pH 6,8 ± 0,2 cresol Purple Base Broth (Ref. 02-031) is more suitable, as it is a better indicator of choice which is less toxic than the Directions phenol red. Dissolve 15 g of powder in 1 L of distilled water. Add sugar in the desired concentration and distribute into References suitable containers with Durham’s tubes. Sterilize in ISO 10273 Standard (1994) General guidance for the detec- the autoclave at 121°C for 10 minutes. Heat up the tion of presumptive pathogenic Yersinia enterocolitica. autoclave before putting the tubes into it to avoid sugar ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- caramelization. Addition of some kinds of sugars may biological Media. CRC Press,Boca Raton,Fla. need a pH adjustment. DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Food. 4th ed. Description APHA. Washington. Phenol Red Base Broth is a liquid version of the agar FDA (1998) Bacteriological Analytical Manual 8th ed. base for the fermentation studies, which is preferred by Rev. A. AOAC International. Gaithersburg. MD many authors to use with Durham’s tubes inclusion, to verify the gas production.

Phenylalanine Agar (PPA)

Ref. 01-083 the presence of a characteristic greenish colour in the medium when it reacts with iron. Nowadays, this test and Specification the urease production test, have a lot of importance in Culture medium for Enterobacteria, according Ewing et the taxonomy of Proteus type . al. formulation. Technique Formula (in g/L) A recommended technique is the following: Yeast extract ...... 3,0 Inoculate the slant surface with plenty of inoculum, and DL-Phenylalanine ...... 2,0 incubate it for 12-16 hours. Add 0,2 mL of 10% ferric Di-sodium phosphate ...... 1,0 chloride solution so that the solution floods all over the Sodium chloride ...... 5,0 growth. Agar ...... 15,0 Phenylpiruvic acid presence (positive test) is shown by Final pH 7,3 ± 0,2 the presence of a characteristic green-blue colour on the surface, after approximately 1 minute. Directions Suspend 26 g of powder in 1 L of distilled water and heat References to boiling. Dispense in tubes or flasks and sterilize in the ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- autoclave at 121°C for 15 minutes. biological Media. CRC Press, Inc.London BUTTIAUX,R., R. OSTEUX, R. FRESNOY & J. MO- RIAMEZ (1954) Les propietés biochimiques du genre Description Proteus.Ann.Inst. Pasteur 87:357-386 This formulation corresponds to the solid form, proposed EDWARDS and EWING (1973). Identification of Entero- by Ewing et al. which is a modification of the medium bacteriaceae. Burges Pub.Cod. Minneapolis. developed by Buttiaux et al. in order to achieve the ISENBERG, H.D. (1992) Clinical Microbiology Proce- green colour that confirms the positive reaction which dures Handbook. Vol I ASM Press Washington. lasts longer. Capacity to deaminate the phenylalanine oxidatively to convert it in phenylpiruvic acid is property of the Proteus type in enterobacteria. Phenylalanine is revealed by

123 Plate Count Media

Plate Count Agar (PCA) Directions Dissolve 17,5 g of powder in 1 L of distilled water. Heat Ref. 01-161 to the boiling by constant stirring. Distribute in the suitable containers and sterilize in the autoclave at 121°C Specification for 15 minutes. Medium for the aerobic plate count by surface inoculation method (Standard Plate Count Agar) according ISO Description 4833 and 17410 standards. Plate Count Modified Agar follows the same specifications as Plate Count Agar, with the exception that of the Formula (in g/L) agar concentration has been reduced. This modification provides a better growth of colonies if massive inocula- Casein peptone ...... 5,0 tion method is used, as the medium is softer and hence Yeast extract ...... 2,5 the colony expansion is improved. Dextrose ...... 1,0 Agar ...... 15,0 Final pH 7,0 ± 0,2 Plate Count Skim Milk Agar

Directions Ref. 01-412 Suspend 23,5 g of powder in 1 L of distilled water. Dissolve by bringing to the boil with frequent stirring. Dis- Speciﬁcation tribute into ﬁnal containers and sterilize by autoclaving at Solid medium for the plate counts of milk and dairy prod- 121°C for 15 minutes. ucts, according to DIN and FIL/IDF standards.

Description Formula (in g/L) The Plate Count Agar follows the directions given by Casein peptone ...... 5,0 Buchbinder et al. in their study about media for the plate Yeast extract ...... 2,5 count of microorganisms. Skimmed milk ...... 1,0 The original formulation of the standardized agar for Dextrose ...... 1,0 dairy microbiology has been modified in order to avoid Agar ...... 10,5 the addition of milk. This new composition allows the Final pH 7,0 ± 0,2 growth of most microorganisms without any further additions. Directions This medium’s formulation is equivalent to that pre- Suspend 20 g of powder in 1 L of distilled water and let scribed by the ‘Standard Methods for the Examination it soak . Bring to the boil with constant stirring. Distribute of Dairy products’, the USP’s ‘Tryptone Glucose Yeast into suitable containers and sterilize in the autoclave at Agar’, the ‘Deutsche Landswirtchaft’ and to the APHA 121°C for 15 minutes. and AOAC’s Plate Count Agar. Nowadays this is the medium selected for the plate count of any type of the Description sample. This medium, with the added milk, has a major nutrient richness than other standard media, however, the Plate Count Modified Agar opalescence of the medium makes early observations sometimes difficult. Ref. 01-329 Due to its lesser agar concentration, it may be used by the pouring plate method or by the surface inoculation Specification method. Modification of Plate Count Agar (Ref. 01-161), with a lesser amount of agar, especially recommended for the Technique aerobic enumeration in plates, by the poured plates Prepare a decimal dilution bank of the sample and take method. 1 mL in duplicate from each dilution and put them in sterile Petri plates. Pour 20 mL approx. of sterile cooled Formula (in g/L) medium (around 47°C) in each of the plates. Mix gently Casein peptone ...... 5,0 by moving the plate in eight (8) shape. Leave the plates Yeast extract ...... 2,5 undisturbed to solidify and incubate in inverted position. Dextrose ...... 1,0 Time and temperature of incubation depend on the type Agar ...... 9,0 of microorganism under study. For a general aerobic Final pH 7,0 ± 0,2 count, incubate for 3 days at 30°C, by observing also after 24 and 48 hours. The plate count method proposed by the APHA consists of a massive inoculum by pouring the molten agar at 50°C on plates containing the diluted samples. The final

124 Plate Count Media count is carried out after 48 hours of incubation at 32- DIN 10192 Standard. Prüfungesbestimmungen für Milch 35°C. und Milcherzeugnisse (Deutsche Landwirtsachft, Fach- As for the microorganisms with other temperature bereit und Ernahrung). 1971. requirements, the following incubations have been sug- FIL/IDF Standards 3 (1958), 100 (1981), 101 (1981), gested: 2 days at 32-35°C, 2-3 days at 45°C, 2 days at 109 (1982) and 132 (2004). 55°C, 3-5 days at 20°C, 7-10 days at 5-7°C. PASCUAL ANDERSON, MªRª (1992) Microbiología Sample dilutions are prepared with solutions of 1/4 of Alimentaria. Diaz de Santos, S.A.,Madrid,SPAIN. Ringer solution (Ref. 06-073), 1% of Peptone Water(Ref. ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- 03-156) or 0,1M of phosphate buffer at pH 7,0; depend- biological Media. CRC Press, Inc.,Boca Raton,Fla. ing on their nature. BUCHBINDER, L., Y. BARIS, L. GOLDSTEIN (1953) The poured plate count method is preferred to the Further studies on new milk-free media for the stand- surface inoculation method, since it gives higher results. ard plate count of dairy products. Am. J. Public Health Nevertheless, the latter gives a more appropriate isola- 43:869-872. tion of the colonies. ISO 4833 Standard (2003) Microbiology of food and animal feeding stuffs. Horizontal method for the enumera- References tion of microorganisms. Colony count technique at 30°C. MARSHALL, R.T. (1992) Standard Methods for the ISO 17410 Standard (2001) Horizontal Method for the Examination of Dairy Products, 16th Ed. APHA. Wash- enumeration of psychrotrophic microorganisms. ington. ISO 8552 Standard (2004) Milk - Estimation of psychro- CLESCERI. L.S., A.E. GREENBERG, and A.D. EATON trophic microorganisms - Colony-count technique at (1998) Standard Methods for the Examination of Water 21°C (Rapid method). and Wastewater, 20th ed.. APHA, AWWA, WEF. Wash- ington. DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Food. 4th. Ed. APHA. Washington. HORWITZ, W. (2000) . Ofﬁcial Methods of Analysis. AOAC International. Gaithersburg

125 Potato Dextrose Media

Potato Dextrose Agar Potato Dextrose Broth

Ref. 01-483 Ref. 02-483

Specification Specification Solid culture medium for the detection and enumeration Liquid culture medium for the maintenance and multipli- of yeast and moulds in food, specially recommended in cation of yeast. butter and other dairy products. Formula (in g/L) Formula (in g/L) Potato peptone ...... 4,0 Potato peptone ...... 4,00 Glucose ...... 20,0 Glucose ...... 20,00 Final pH 5,6 ± 0,2 Agar ...... 15,00 Final pH 5,6 ± 0,2 Directions Dissolve 24 g of powder in 1 L of distilled water, heating Directions up only if necessary. Distribute into suitable containers Suspend 39 g of powder in 1 L of distilled water and and sterilize by autoclaving at 121ºC for 15 minutes. bring to the boil. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes. Do not Description overheat. Potato Dextrose Broth is the liquid version of the agar with the same name. This broth is mainly used to detect Description and enumerate yeast and moulds, since it does not Potato Dextrose Agar is a weakly selective medium for contain any solidifying agent it may be acidified without fungi due to its high sugar content and acidic pH. The altering its physical properties. pigment production and aerial mycelium development At pH 3,5, the bacterial growth is totally inhibited without is enhanced by the potato peptone, specially in the significant influence on fungi. This acidification may be Fusarium, Aspergillus and Penicillium species. achieved by the aseptic addition of an adequate amount The selectivity can be increased by adding antibacterial of organic acid to the medium after sterilization:10-15 antibiotics like chloramphenicol or tetracyclines, or by mL/L of a 10% sterile solution of tartaric or lactic acid. simply decreasing the pH to an acidic level. At pH 3,5 This addition may also be made before sterilization, but the bacterial growth is almost totally inhibited without it must be considered that in acidic conditions Maillard significant effect on fungi. This acidification can be reactions are strong and hence the medium may turn obtained by the aseptic addition of an adequate amount slightly brownish. of organic acid to the medium after sterillization: 10-15 mL/L of a 10% sterile solution of tartaric or lactic acid is References usually sufficient. ATLAS, R.M. & PARKS,L.C. (1995) Handbook of After its acidification the medium should not be over- Microbiological Media for the Examination of Food. CRC heated or reheated since it can hydrolyze the agar and Press, London. hence there can be a loss in solidification property of the RICHARDSON, G. H. (1985) Standard Methods for the medium. examination of dairy products.15th Ed. APHA Washing- ton. Technique DOWNES, F.P. & K. ITO (2001) Compendium of meth- Distribute the diluted samples into sterile petri plates. ods for the microbiological examination of food. 4thEd. Pour the molten agar melted cooled to 45-50°C and gen- APHA Washington tly mix to homogenize the mixture. After the solidification, US PHARMACOPOEIA (2002) 25th ed. <61> Microbial plates are incubated for 5-7 days at 20-25°C to permit Limit Test. US Pharmacopoeial Convention Inc. Ltd. the complete development of the fungal colonies. Rockville. MD The weak consistency of the agar due to its original FDA (1998) Bacteriological Analitycal Manual. 8th ed. acidity makes this medium inadequate for streaking. Rev. A. AOAC International. Gaithersburg. MD.

126 R2A Agar (Eur. Phar. Medium S)

Ref. 01-540 Technique The water sample must be processed as quickly as Specification possible. If it is no possible within the first 6 hours, the Solid medium for the enumeration of heterotrophic micro sample must be refrigerate, but not for more than 30 organisms in treated waters hours: then the sample is rejected. R2A Agar is used with pour plates, streak plates or Formula (in g/L) filtration but must be keep in mind that the pour plates method can affect the recovery capacity of the medium Yeast Extract ...... 0,500 because the thermal shock. The incubation period at Proteose peptone ...... 0,500 35ºC is of 3-5 days but is more effective a incubation Casein hidrolysate ...... 0,500 temperature of 20-28ºC an a time of 5-7 days. In any Glucose ...... 0,500 case the plates must be protected against an excessive Starch ...... 0,500 drying. Dipotassium hydrogen phosphate ...... 0,300 The fast-growing or non-stressed microorganisms in Magnesium sulphate, anhydrous ...... 0,024 these conditions of incubation produce different and Sodium pyruvate ...... 0,300 minute colonies than in the rich media. Agar ...... 15,000 Final pH 7,2 ± 0,2 References Directions ATLAS, R.M. (1995) Handbook of Media for Environ- mental Microbiology. CRC Press. Boca Raton USA. Suspend 18,1 g of powder in 1 L of distilled water and EATON, A.D., A.E. GREENBERG and L.S. CLESCERI bring to the boil with constant stirring. Distribute into suit- (1995). Standard Methods for the Examination of Water able containers and sterilize by autoclaving at 121ºC for and Wastewater. 19ª Ed. APHA Washington D.C. USA . 15 minutes. EUROPEAN PHARMACOPOEIA 4th Ed. Suppl. 4.6 (2004) 2.6.13 Test for specified Microorganisms (pg Description 2621) The R2A Agar was proposed in 1979 by Reasoner and GREENBERG, A.E., R.R TRUSSELL and L.S. Geldenreich and few years later accepted by the APHA CLESCERI (1985). Standard Methods for the Examina- as an alternative medium for stressed cells in treated tion of Water and Wastewater. 16ª Ed. APHA-AWWA- potable water. WPCF Washington D.C. USA The use of nutrient rich media like PCA or TSA allows to REASONER, D.J. and E.E. GELDREICH (1979) A new the growth of normal microbiota, but do not permits the medium for the enumeration and subculture of bacteria recuperation of the stressed or chlorine resistant biota. from potable water. Abstracts of Annual Meeting . ASM By the use of a medium like R2A of low nutrients in com- 79th Meeting. Paper #N7. bination with a lower temperature and longer incubation Van SOETSBERGER, A.A. and C.H. LEE (1969) Pour time it is possible induce the resuscitation of this dam- plates or streak plates? Appl. Microbiol 18:1092-1094. aged cells. In the R2A Agar the source of nitrogen is the peptone and the Yeast Extract supplies the vitamins and growth factors. The source of carbon is the dextrose and magnesium sulphate and potassium phosphate maintains the osmotic pressure. The starch is a detoxifier and sodium piruvate increases the recuperations of stressed cells. The agar acts as gelling agent.

127 Rappaport Vassiliadis Media

Rappaport Vassiliadis Broth VASSILIADIS, P, PATERAKI, EPAPAICONOMOU,N , PAPADAKIS, J.A. A. TRICHOPOULOS, D (1976) Ref. 02-379 Nouveau procédé d’enrichissement de Salmonella. Ann. Microbiol. (inst. Pasteur) 127B (195-200) RAPPAPORT, F. N. KONFORTI & B. NAVON (1956) A Specification new enrichment medium for certain salmonellae. J. Clin Liquid medium for the selective enrichment of Salmo- Pathol. 9:261-266 nella in foodstuffs and other materials. VAN SCHOTHORST M. & A.M. RENAUD (1983) Dynamics of salmonellae isolation with modified Rappa- Formula (in g/L) port’s medium (R10) J.Appl. Bact. 54: 209-215 Soy peptone ...... 4,500 FIL-IDF Standard 93B:1995. Milk and Milk products. Sodium chloride ...... 7,200 Detection of Salmonella. Brussels Monopotassium phosphate ...... 1,260 FDA (1998) Bacteriological Analytical Manual 8th ed. Dipotassium phosphate ...... 0,180 Rev A. AOAC International. Gaithersburg. MD. Magnesium chloride ...... 13,580 HORWITZ, W. (2000) Official Methods of Analysis. Malachite green ...... 0,036 AOAC International. Gaithersburg. MD Final pH 5,2 ± 0,2 DOWNES, F.P. & K. ITO (2001) Compendium of methods for the microbiological examination of foods. 4th ed. Directions APHA Washington. Dissolve 26,8 g of powder in 1 L of distilled water, heating if necessary to help dissolve the powder. Dispense Rappaport Vassiliadis Modified into test tubes or flasks and sterilize by autoclaving at Semi-Solid Medium Base (MSRV) 121°C for 15 minutes.

Description Ref. 03-376 The Rappaport Vassiliadis medium complies with the recommendations of the APHA for the examination of Specification food. Semi-solid medium for the isolation of mobile strains of This culture medium is the modificaction of the R10 me- Salmonella. dium (from Rappaport et cols) or RV broth (from Vas- siliadis et cols.)by van Schothort & Renaud. The modi- Formula (in g/L) fications are an adjustement in the magnesium chloride Tryptose ...... 4,590 concentration and a buffered reaction of the medium. Casein Peptone ...... 4,590 It shows a higher selectivity towards Salmonella and Sodium chloride ...... 7,340 produces better yields than other similar media, espe- Mono-Potassium phosphate ...... 1,470 cially after preliminary enrichment and at an incubation Magnesium chloride ...... 10,930 temperature of 43°C. Malachite green ...... 0,037 Malachite green and magnesium chloride inhibit the Agar ...... 2,700 growth of the microorganisms normally found in the in- Final pH 5,2 ± 0,2 testine but do not affect the proliferation of most Salmo- nellae. Malachite green inhibits the growth of Shigella. Directions Soy peptone improve the growth of Salmonella. The low Suspend 31,6 g of powder in 1 L of distilled water. Heath pH of the medium increases the selectivity. in a water bath until boil and complete dissolution. Cool to 50ºC an add 20 mg/L of Novobiocin. Without autoclav- Technique ing nor reheating, homogenize and pour plates. Keep Inoculate the culture medium with the sample or mate- plates in a fresh place to settle the gel (1 hour minimum) rial from a pre-enriched culture in Buffered Peptone and handle it with care because the medium is only Water (Ref. 02-277) and incubate for up to 18-24 hours semi-solid. It is recommended to keep MSRV plates in a at 41±1°C. Streak the sample material from the resulting cooler at 2-8ºC at the dark. cultures onto selective culture media. Description References The Modified Semi-Solid Rappaport-Vassiliadis Medium ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio- Base is formulated according DeSmedt and cols. That logical Media. CRC Press Inc.,London shows its higher efficiency over the traditional enrich- VASSILIADIS,P (1983) The Rappaport-Vassiliadis(RV) ment methodology. enrichment medium for the isolation of salmonellas: An overview. J.Appl.Bact.54;54, 69-76.

128 Rappaport Vassiliadis Media

The rapid migration of mobile strains of Salmonella in 5. To prevent false negatives results due to the ab- the semisolid medium allows to the early detection by sence of mobile strains of Salmonella is convenient the production of an halo of growth around the inocula- to performs simultaneously a traditional enrichment tion zone. in liquid medium. The other competitive mobile organisms are inhibited by the novobiocin, the malachite green and the high References concentration of magnesium chloride. De SMEDT, J.M., R. BOLDERDJIK, H. RAPPOLD and The low concentration of agar produces a very soft and D. LAUTENSCHLAEGER (1986). Rapid Salmonella fragile gel but, at the temperature of incubation (42ºC), it detection in foods by motility enrichment on a Modi- is an special environment in which the mobile strains of fied Semi-Solid Rappaport-Vassiliadis Medium. J. Food Salmonella moves easy and quickly. Protect. 49:510-514 De SMEDT, J.M. and R. BOLDERJIK (1987) Dynamics Technique of Salmonella isolation with Modified Semi-Solid Rappa- 1. Three drops (~0,1 mL) of a pre-enrichment culture port-Vassiliadis Medium. J. Food Protect. 50:658-661 are inoculated in a three different spots on the dry HOLBROOCK, R., J.M. ANDERSON, A.C. BAIRD- surface of the medium in a room-temperate plate. PARKER, L.M. DODDS, D. SAWHNEY , S.H. STRUCH- 2. Incubate the plates aerobically in an upright position BURY and D. SWAINE (1989) Rapid detection of Salmo- for no longer than 24 hours at 42ºC. nella in food: A convenient two-day procedure. Lett. Appl. 3. The formation of a turbid or opaque halo around the Microbiol. 8:139-142 initial inoculation zone shows the presence of mobile salmonellae. 4. To confirm the purity of the isolation and to follows with the identification tests, samples of the external border of the halo can be used.

Reinforced Clostridial Media

Reinforced Clostridial Agar Reinforced Clostridial Medium (RCM) (Eur. Phar. Medium P) Ref. 01-289 Ref. 03-289 Speciﬁcation Solid medium for the cultivation and enumeration of Speciﬁcation clostridia and other anaerobic bacteria. Fluid medium for the cultivation and enumeration of clostridia by the MPN method. Formula (in g/L) Casein peptone ...... 10,0 Formula (in g/L) Yeast extract ...... 3,0 Casein peptone ...... 10,0 Meat extract ...... 10,0 Yeast extract ...... 3,0 Dextrose ...... 5,0 Meat extract ...... 10,0 Sodium chloride ...... 5,0 Dextrose ...... 5,0 Sodium acetate ...... 3,0 Sodium chloride ...... 5,0 Soluble starch ...... 1,0 Sodium acetate ...... 3,0 L-Cysteine HCl ...... 0,5 Soluble starch ...... 1,0 Agar ...... 15,0 L-Cysteine HCl ...... 0,5 Final pH 6,8 ± 0,2 Agar ...... 0,5 Final pH 6,8 ± 0,2 Directions Suspend 52,5 g of powder in 1 L of distilled water and Directions heat to boiling with constant stirring. Distribute into suit- Suspend 38 g of powder in 1 L of distilled water and heat able containers and sterilize in the autoclave at 121°C to boiling with constant stirring. Distribute into suitable for 15 minutes. containers and sterilize in the autoclave at 121°C for 15 minutes.

129 Reinforced Clostridial Media

Description cin sulfate 0,05 g/L, Sodium iodoacetate 0,025 g/L and Reinforced Clostridial Agar was originally described by triphenyl-tetrazolium HCl 0,025 g/L to obtain a selective Hirsch and Grinstead to initiate the growth of small in- and differential medium for bifidobacteria in water and oculums and get a higher Clostridial count. Later, Barnes wastewater. and Ingram used the medium to develop vegetative cells in assays of Clostridium perfringens. Barnes also used References this medium to count clostridia in food, moreover other ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio- authors used this medium in enumeration assays of Cl. logical Media. CRC Press, Inc.,Boca Raton,Fla. thermoscharolyticum in sugar, study of intestinal flora, INGRAM, M. and BARNES, E.M. (1956) A Simple Modi- and bacterial count in human or animal faeces, etc. fication of the Deep Shake Tube for Counting Anaerobic For the enumeration by the MPN method, the liquid ver- Bacteria. Lab. Practise 5, 4:145. sion is the preferred one. HIRSCH, A. and GRINSTEAD, E. (1954) Methods for the Growth and Enumeration of Anaerobic Sporeformers Technique from Cheese, with Observations on the Effect of Nisin. Material to be examined is ground in a Turmix or Stom- J.Dairy Res. 21:101. acher, and a decimal dilution bank is prepared. From MUÑOA, F.J., R. PARÉS (1988) Selective medium for each of the dilutions, take an aliquote to Petri plates or isolation and enumeration of Bifidobacterium spp. Appl. tubes, and pour the molten medium at 50°C over them. Environm. Microgiol 54:1715-1718. Let it solidify. Incubate at 30-55°C (depending on the EUROPEAN PHARMACOPOEIA,(2002) 4th ed. Suplle- microorganism that is anticipated to be found) for 1-10 ment 4.2 Chap. 2.6.13.Test for specified micro-organ- days. An anaerobic environment can be achieved if isms. Council of Europe. Strasbourg. tubes are used and they are covered with Sealing Anaer- obic Agar (Ref. 01-174) immediately after the Reinforced Clostridial Medium is solidified. If the plates are used, they have to be incubated in the anaerobic jars. Muñoa and Parés added a filter sterilized solution of Nalidixic acid 0,02 g/L, Polymyxin 0,025 g/L, Kanamy-

Rinse Fluid K

Ref. 03-109 Technique After the filtration, wash the membrane by passing 3 Specification volumes of 100 mL of solution through it. Medium for rinsing the membrane filters according to the When the sample has a high concentration of fats or USP and European Pharmacopoeia specifications. sugars it is recommended to double the concentration of polysorbate Formula (in g/L) (2 mL/L). Meat peptone ...... 5,0 Meat extract ...... 3,0 References Final pH 6,9 ± 0,2 US PHARMACOPOEIA (2002) 25th ed.<71> Sterility Tests. US Pharmacopoeial Convention Inc. Rockville. Directions MD European Pharmacopoeia. (2002) 4th Ed. V.2.18 Control Dissolve 8 g of powder in 1 L of distilled water with 10 of microbial contamination in no sterile products mL of Polysorbate 80 (Ref. 06-088). Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Description This nutrient solution which is formulated according to the USP (Rinsing Fluid K) and European Pharmacopoe- ial speciﬁcations, removes all the fat and carobohydrate residues, due to the surfactant effect of Polysorbate, and at the same time, it avoids the osmotic shock to the microorganisms.

130 Rogosa Agar Base

Ref. 01-300 The low pH and high acetate concentration, that can be adjusted to the sample confers to this medium a high Specification selectivity for the lactobacilli. Nevertheless, it is no suit- Selective solid medium for the isolation and enumeration able for the dairy samples, for which is recommended of lactobacilli. the addition of 20% sterile tomato serum (Ref. 06-092) or using the MRS Agar (Ref. 01-135) that offers a best performance with the dairy lactobacilli. Formula (in g/L) Rogosa Agar, due to its high acidity, is not suitable for Glucose ...... 20,000 the maintenance of the microorganisms. Tryptone ...... 10,000 Sodium acetate ...... 10,000 Monopotasium phosphate ...... 6,000 References Yeast extract ...... 5,000 ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951) Ammonium citrate ...... 2,000 A selective medium for the isolation and enumeration of Sorbitan monooleate ...... 1,000 oral and faecal lactobacilli. J. Bacteriol. 62:132. Magnesium sulphate ...... 0,575 ROGOSA, M., J.A. MITCHELL & R.F. WISEMAN (1951) Manganese sulphate ...... 0,120 A selective medium for the isolation and enumeration of Ferrous sulphate ...... 0,034 oral and faecal lactobacilli. J. Dental Res. 30:682 Agar ...... 15,000 DOWNES, F.P. & K. ITO (Eds) (1991) Compendium of th Final pH 5,5 ± 0,2 methods for the microbial examination of foods. 4 ed. APHA. Washington D.C. MACFADDIN J.D. (1985) Media for isolation-cultivation- Directions identification-maintenance of medical bacteria. William & Suspend 69,7 g of powder in 1 L of distilled water and Wilkins, Baltimore. MD. add 1,32 mL of glacial acetic acid and the complemen- ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- tary amount of sodium acetate to fit standard. Heat with biological Media. CRC Press. London. gently stirring until boiling. Pour plates without autoclaving nor overheating.

Description Rogosa Agar was developed in 1951 for the isolation and enumeration of oral and faecal lactobacilli, but with changes in the acetate concentration it was used for several kinds of samples, including foods.

131 Rose Bengal Agar

Ref. 01-301 T Technique

R-45 After making a dilution bank, take 0,1 mL from each dilu- Speciﬁcation S-53-45 tion and inoculate with a Drigalsky Loop (Ref. 5-010) or Solid and selective medium for the isolation of yeast and glass spreader on Rose Bengal Agar plates. Should the moulds from the environment and food products. massive seed method be preferred, take 1 mL from each dilution and put it in an empty plate. Pour the molten Formula (in g/L) medium at 50°C and homogenize it by gently moving the plate in an eight (8) shape. Incubate at 22°C for 5 days Peptone ...... 5,00 and proceed to enumerate the fungi. Dextrose ...... 10,00 Potassium phosphate ...... 1,00 Magnesium sulfate ...... 0,50 References Rose bengal ...... 0,05 ATLAS, R.M., & PARKS, L.C. (1993) Handbook of Micro- Chloramphenicol ...... 0,10 biological Media. CRC Press, Inc.,Boca Raton,Fla. Agar ...... 15,00 DOWNES F.P. & K. ITO (2001) Compendium of Methods Final pH 7,2 ± 0,2 for the Microbiological Examination of Food. 4th Ed. APHA. Washington. Directions MARSHALL, R.T. (1993) Standard Methods for the examination of dairy products. 16th ed. APHA Washington. Suspend 32 g of powder in 1 L of distilled water and heat APHA-AWWA-WEF (1998) Standard Methods for the to boiling with constant stirring. Distribute in suitable examination of water and wastewater. 20th. ed. APHA containers and sterilize in the autoclave at 121°C for 15 Washington. minutes.

Description Rose Bengal Agar is a selective medium to detect and enumerate moulds and yeast in food samples. Apart from the nutritional requirements for moulds and yeast,this medium also contains Rose Bengal, which apart from tainting the yeast with a pink colour, also facilitates their count, avoiding massive growth of moulds such as Rhizopus and Neurospora, therefore it is easier to detect other moulds with slower growth. Chloramphenicol and also the Rose bengal, restrains bacterial growth, but does not interfere with fungi growth.

Yeast and moulds colonies after 5 days at 22ºC

132 Sabouraud Media

Sabouraud Chloramphenicol Description Agar Sabouraud Dextrose Agar is a modification of the classical Sabouraud medium for the cultivation of fungi. This T new formula helps to maintain the morphological aspects Ref. 01-166 R-45 of fungi and thus permits a reliable cultivation and dif- S-53-45 ferentiation. Specification Its selectivity is due to a low pH and a high glucose con- Solid culture medium for the isolation of fungi. centration, which together with incubation at a relatively lower temperature (25-30°C) favours the growth of fungi Formula (in g/L) while discouraging that of bacteria. Besides, the com- Casein peptone ...... 5,0 position of this peptone has been studied to provide the Meat peptone ...... 5,0 fungi with all their nitrogenated nutrient requirements. D(+) Glucose ...... 40,0 Since the Sabouraud medium’s strong acid reaction Chloramphenicol ...... 0,5 partially hydrolyzes the agar, only the required amount Agar ...... 15,0 should be prepared and it should not be remelted. Any Final pH 5,6 ± 0,2 overheating will considerably diminish its gelling capacity. Directions Should a higher selectivity be required, a variety of in- Suspend 65,5 g of powder in 1 L of distilled water and hibitors or selective agents may be added after steriliza- bring to the boil. Distribute into final containers and tion, while the medium is still in the molten form. It can sterilize by autoclaving at 121°C for 15 minutes. Do not even be made differential by adding the indicator agents. overheat or reheat the medium since it will affect the Some of the inhibitory and differential mixtures most solidification. commonly used are listed below: Penicillin: at 20,000 units/litre, encourages the selectivity Description of the medium by inhibiting most of the bacteria. Penicillin and Streptomycin: at 20,000 u/L and 40,000 This culture medium differs from the classical Sabour- u/l each, favours the isolation of Histoplasma in aud Agar only in the addition of Chloramphenicol. This dogs. thermostable antibiotic has a wide antibacterial spectrum Penicillin and Neomycin: at 20,000 u/L and 40 mg/L which ensures the selective isolation of fungi from highly each, is used for the isolation of yeast. contaminated samples, such as eudates, faeces, nails Streptomycin and Chloramphenicol: at 40 mg/L and 500 and hair. mg/L each, for the isolation of Trichophyton ver- rucosum . Sabouraud Dextrose Agar Colistin, Novobiocin and Cycloheximide: at 8 mg/L, 0.1 (Eur. Phar. Agar Medium C) mg/L and 30 mg/l each, for the isolation of Can- dida albicans . Ref. 01-165 Potassium Tellurite: at 150 mg/L, is used for the primary isolation of fungi from scales and scabs. Cupric Sulfate, Crystal Violet and Brilliant Green: at 500 Specification mg, 2 mg and 5 mg each, achieves considerable Medium for the enumeration and cultivation of fungi. bacterial inhibition. Triphenyltetrazolium chloride (TTC): at 100 mg/L, it is the Formula (in g/L) basis of a Pagano-Levin medium for the isola- D(+) Glucose ...... 40,0 tion of Candida albicans, unpigmented, among Casein peptone ...... 5,0 other pathogenic yeast which form pink coloured Meat peptone ...... 5,0 colonies. Agar ...... 15,0 Final pH 5,6 ± 0,2 Sabouraud Broth

Directions Ref. 02-165 Dissolve 65 g in 1 L of distilled water and bring to the boil with frequent stirring. Distribute into ﬁnal containers and sterilize by autoclaving at 121°C for 15 minutes. Do Speciﬁcation not overheat the medium as its acidic pH may partially Liquid medium for the sterility control. hydrolize the agar. Alternatively,if the European Pharma- copoeia formulation is desired, add before sterilization 50 mg/L of chloranphenicol (Ref. 06-118CASE)

133 Sabouraud Media

Formula (in g/L) Casein Peptone ...... 5,0 Sabouraud Oxytetracycline Meat Peptone ...... 5,0 Agar Base (OGYEA) D (+) Glucose ...... 20,0 Final pH 5,8 ± 0,2 Ref. 01-275 Directions Dissolve 30 g of powder in 1 L of distilled water, heating Specification up only if necessary. Dispense into suitable containers Solid culture medium for the total enumeration of moulds and sterilize in preheated autoclave for 15 minutes at and yeasts. 121°C. Avoid overheating, since it may caramelize the glucose. Formula (in g/L) Glucose ...... 20,0 Description Yeast extract ...... 5,0 This medium is especially adapted to fungi and acido- Agar ...... 20,0 philic bacteria culture. Final pH 7,0 ± 0,2 Sabouraud USP Broth is according to the formulations of US Pharmacopeia, US NF and 21 CFR guidelines. In the Directions latest editions of these books it is also mentioned and Suspend 45 g of powder in 1 L of distilled water and let allowed to use Tryptone and Soy Broth for the sterility it soak for a few minutes. Distribute into suitable contain- checking in parenteral pharmaceutical products. This for- ers and sterilize by autoclaving for 10 minutes at 115ºC. mulation is similar to the Antibiotic Medium 13 by Grove Cool to 50°C and then add oxytetracycline (Ref. 06- and Randall and 21 CFR. 115CASE) to reach a 0,1 mg/mL concentration. This medium is not a selective one, but the strong acidic pH notably inhibits the growth of non acidophilic microor- Description ganisms. Nonetheless, special measures must be taken This formulation differs with others as it has no peptone while reconstituting and heating the medium due to this and has a neutral reaction or pH. Unlike the others, it strong acid reaction and the high content of glucose. It is has a high oxytetracycline concentration that makes it important to preheat the autoclave and thereby reach the almost impossible for the growth of bacteria. sterilization temperature as soon as possible and in a regular way, since otherwise, glucose becomes caramel- Technique ized turning the medium dark and effectiveless. Some authors suggests an inoculum of 1 mL in each dilution, in duplicate and in mass. Perform an incubation Technique at 22-25°C for 5 days with the intermittent observations It has been recommended to use this medium in many or readings after 3 days of incubation. tests and assays, but for a long time it has been the medium of choice for the verification of sterility of the sterile References pharmaceutical products. AJELLO, L.(1957) Cultural Methods for Human Patho- Efficacy of the medium and absence of fungistatic prod- genic Fungi J. Chron. Dis. 5:545-551. ucts is verified by checking if there is a growth from a PAGANO, J., LEVIN,J.D. and TREJO, W.(1957-58) loop of inoculum of Candida albicans, from a 1:1000 di- Diagnostic Medium for Differentiation of Species of Can- lution of a fresh 24 hours grown culture. Sterility assay or dida. Antibiotics Annual, 137-143. test is carried out in controlled and verified medium. To SABOURAUD, R.(1910) Les Tignes. Masson, Paris. check the fungistatic activity of any product, prepare an HANTSCHKE, D.(1968) Mykosen, 11:769-778. inoculum as mentioned above and inoculate two series EUROPEAN PHARMACOPOEIA (2002), 2.6.13 Tests of tubes with the same medium as follows: for specified micro-organisms Supplement 4.2, 4th Ed., a) Add to one batch the specified amount of product. EDQM. Council of Europe, Strasbourg. This is the test series. US PHARMACOPOEIA (2002) 25th Ed. <51>Antimicro- b) Add to the another batch only the inoculum and bial efectiveness Testing; <61> Microbial Limit Tests. US simultaneously incubate with the test series. This is Pharmacopoeial Convention Inc. Rockville. MD the control series. ISO 13681 Standard (1995) Enumeration of yeasts and Incubation of both the series must be carried out at 22°C moulds - Colony count technique. for 10 days. After this period compare the results or observations of both the series. If the assay series has a lesser growth than the control one, product has the fungistatic activity. If the growth is equal or more, then it has not any fungistatic properties. For the quantitative assay of the fungistatic activity, perform the assay with several series of different concentrations (one lower than the previous) until reaching an equal growth in both control and test series.

134 Salmonella Shigella Agar (SS Agar)

Ref. 01-171 medium, such as Brilliant Green Agar (Ref. 01-203) or MacConkey Agar (Ref. 01-118). Specification Incubate the inoculated plates at 37°C for 18-24 hours. Solid and highly selective medium for the isolation of The suspicious colonies should then be subcultured Salmonella and some Shigella species. on differential media to be identified biochemically or serologically. Appearance of the colonies after 24 hours on SS Agar: Formula (in g/L) Shigella: Colourless, transparent and flat. Meat extract ...... 5,00000 Salmonella (Non H S producers): Colourless, transpar- Peptone ...... 5,00000 2 ent and flat. Lactose ...... 10,00000 Salmonella (H S producers): Black or black centered, Bile salts ...... 8,50000 2 flat, with transparent borders. Sodium citrate ...... 10,00000 Proteus: Similar appearance as Salmonella colonies, but Sodium thiosulfate ...... 8,50000 smaller in size. Ferric citrate ...... 1,00000 Escherichia coli: If they grow, they are small, convex and Brilliant green ...... 0,00033 pink or red coloured. Neutral red ...... 0,02500 Coliforms (in general): Big, opaque, smooth and col- Agar ...... 15,00000 oured in white or pink shade. Final pH 7,0 ± 0,2 References Directions LEIFSON, E.(1935) New culture media based on sodium Suspend 63,1 g of the dehydrated medium in 1 L of deoxycholate for the isolation of intestinal pathogens and distilled water. Slowly bring to the boil, stirring until com- for the enumeration of colon bacilli in milk and water. J. plete dissolution. Boil for 2 minutes. Do not autoclave. Pathol. Bacteriol., 40.581. Cool to 50°C and pour into sterile Petri dishes.Do not DOWNES, F.P. & K. ITO (2001) Compendium of Meth- overheat. ods for the Microbiological Examination of Food. 4th Ed. APHA. Washington DC. Description HORWITZ,W.(2000). Official Methods of Analysis 17th The SS Agar is a highly selective agar for the isolation of ed. AOAC International. Gaithersburg. MD. Salmonella and Shigella species from very contaminated ATLAS, R.M.,and L.C. PARKS (1993) Handbook of samples. Microbiological Media. CRC Press, London Selectivity is obtained by a high concentration of bile GRAY, L.D. (1995) Escherichia, Salmonella, Shigella salts and brilliant green, which inhibits the growth of and Yersinia. In Murray, Baron, Pfaller Tenover & Yolken gram-positive bacteria. As for the other gram-negative (eds) Manual Clinical Microbiology. 6th ed. ASM Wash- flora, its growth is highly repressed by the presence of ington DC. citrate and thiosulfate. Nevertheless, some coliforms may still grow on this medium. In such case, differentiation between pathogenic species and coliforms becomes evident by the colour change of the pH indicator neutral red. Lactose fermenters produce a pink or red colored medium and colonies, while non-fermenting species form colourless colonies and turn the medium yellow.

Should any species eventually produce H2S, it will be easily detected by the ferrous sulﬁde’s black precipitate, which turns the colonies black. The peptone and the meat extract are usually capable of inducing the growth of most pathogenic species, nevertheless some Shigella are very fastidious and grow poorly.

Technique While using the samples suspected of being exposed to the treatments that might have damaged the viability of microorganisms (processed food, faeces from the patients under antibiotic treatment, etc.) it is advisable to proceed with a previous enrichment in Selenite Cystine Broth Base (Ref. 02-602) or Tetrathionate Base Broth (Ref. 02-033/Ref. 02-335). Afterwards, inoculate SS Agar plates heavily with the specimen and proceed in Salmonella typhimurium ATCC 14028 the same way with other specimens of a less selective

135 Schaedler Media

Schaedler Agar Directions Dissolve 28,3 g of powder in 1 L of distilled water, heat- Ref. 01-231 ing up only if necessary. Distribute into suitable containers and sterilize by autoclaving at 121°C for 15 minutes. Speciﬁcation Solid medium with high reducing and nutrient capacity Description for the cultivation of fastidious anaerobic microorgan- These media viz. Schaedler Agar and Broth, were isms. developed to create the selective conditions to allow the growth of fastidious anaerobic microrganisms from Formula (in g/L) a mixed ﬂora, like gastrointestinal tract, where there are many antagonistic activities Casein peptone ...... 5,60 between fast growing facultatives and the delicate fas- Soy peptone ...... 1,00 tidious anaerobic organisms. For this aspect, the media Meat peptone ...... 5,00 with thioglycolate are widely used, but this compound Yeast peptone ...... 5,00 seems to inhibit some delicate anaerobic organisms. On Glucose ...... 5,80 the other hand, Schaedler media have L-Cystine as a Sodium chloride ...... 1,70 reducing agent, thus some gramnegative do not grow. Dipotassium phosphate ...... 0,80 Effective separation or isolation in several biotypes is Tris buffer ...... 3,00 achieved with the addition of selective agents to the nu- L-Cysteine HCl ...... 0,40 trient base. For example, this medium can be rendered Hemine ...... 0,01 selective for lactic bacteria by adding 10 g/L of sodium Agar ...... 15,00 chloride and 0,002 g/L of neomycin. Final pH 7,6 ± 0,2 For the selection of Clostridium and Bacteroides, it is more advisable to add 2 g/L of placenta powder and Directions 0,002 g/L of neomycin. Should a selective medium for Suspend 43,3 g of powder in 1 L of distilled water and Flavobacterium be desired, add 7 mL of alcoholic solu- heat to boiling. Dispense into suitable containers and tion of tyrothricin 0,5% to 1 L of medium base. In any sterilize in the autoclave at 121°C for 15 minutes. Pour case, incubation must be carried out at 37°C and in an into sterile plates just before the use. anaerobic atmosphere.

Schaedler Broth References SCHAEDLER, R.W., DUVOS, R. and COSTELLO, R. Ref. 02-231 (1965) The development of the bacterial flora in the gastrointestinal tract of mice. J. Exp. Med. 122:59. Specification ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio- Liquid version of the agar with the same name, especial- logical Media. CRC Press, Inc.,London ly suitable for fastidious anaerobic microorganisms. STALONS, D.R., C.THORNSBERRY and V.R. DOWELL (1974) Effect of culture medium and CO2 concentration Formula (in g/L) of growth of anaerobic bacteria commonly encountered in clinical specimens. Appl. Microbiol 27:1098-1104. Casein peptone ...... 5,60 ISENBERG H.D. (1992) Clinical Microbiology Proce- Soy peptone ...... 1,00 dures Handbook. ASM. Washington DC. Meat peptone ...... 5,00 MARSHALL, R.T. (1992) Standard Methods for the ex- Yeast peptone ...... 5,00 amination of Dairy Products. APHA. Washington Glucose ...... 5,80 MacFADDIN, J.F. (1985) Media for Isolation-Cultiva- Sodium chloride ...... 1,70 tion- Identification and Maintenance of Medical bacteria. Dipotassium phosphate ...... 0,80 William & Wilkins. Baltimore, MD, USA. Tris buffer ...... 3,00 WILKINS, T.D. and S. CHALGREN (1976) Medium for L-Cysteine HCl ...... 0,40 use in the susceptibility testing of anaerobic bacteria. Hemine ...... 0,01 Antimicrob. Agents. Chemother 10:926:928. Final pH 7,6 ± 0,2

136 Sealing Anaerobic Agar

Ref. 01-174 Xn Description This solid substrate, without the capacity to support any R-22-43 Speciﬁcation S-24-37-46 growth but with strong reducing power, is made to cover Solid substrate with high reducing capacity to cover and the tubes for anaerobic growth. Once liquiﬁed, let it cool seal the cultures anaerobically. to 50-55°C and pour into the tubes, taking care not to mix it with the medium. Formula (in g/L) A minimum column of 1,5 cm over the medium is recommended to assure an anaerobic cover. L-Cysteine HCl ...... 0,30 Should cultures be stored for a long period of time, it is Sodium Thioglycolate ...... 0,70 advisable to put a sterile vaseline layer over the cap to Resazurine ...... 0,01 avoid Agar ...... 20,00 desiccation. Final pH 7,0 ± 0,2

Directions References SANCHO, J. (1977) Personal communication. Suspend 21 g of powder in 1 L of distilled water and bring to the boil. Dispense into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

Selenite Media

Selenite Brilliant Green avoided. Is not advisable to store the prepared medium Broth Base (SBG Broth Base) for more than eight days, since it loses its selectivity notably.

Ref. 02-603 Selenite Broth Base Speciﬁcation Selective enrichment medium for Salmonella types. Ref. 02-598

Formula (in g/L) Specification Gelatine peptone ...... 5,000 Liquid medium for Salmonella and Shigella enrichment. Yeast extract ...... 5,000 D-Mannitol ...... 5,000 Formula (in g/L) Sodium taurocholate ...... 1,000 Peptone ...... 5,00 Potassium phosphate ...... 3,500 Lactose ...... 4,00 Sodium sulfapyridine ...... 0,500 Potassium phosphate ...... 10,00 Brilliant green ...... 0,005 Final pH 7,0 ± 0,2 Final pH 7,2 ± 0,2 Directions Directions Dissolve 19 g of powder. in 1 L of distilled water and add Dissolve 20 g of powder. in 1 L of distilled water and add 4 g of sodium biselenite (Ref. 06-615). Homogenize and 4 g of sodium biselenite (Ref. 06-615). Homogenize and bring to the boil. Distribute in suitable containers. Termo- bring to the boil. Distribute in suitable containers. Termo- labile medium: Use immediately. Do not autoclave. labile medium: Use immediately. Do not autoclave. Description Description Selenite Broth is formulated according to an original SBG Broth is a modification to the classical Osborne and formulation by Leifson for selective enrichment of Salmo- Stockes medium for enrichment of Salmonella from eggs nellae from very contaminated samples. and egg derivative products. Enrichment is especially effective during the first 12 The medium is maintained at a neutral pH, in spite of the hours of cultivation, since in this period it seems that acid products are liberated from the mannitol fermenta- only Salmonellae, some Proteus and some strains of tion, due to the strong phosphate buffer. On the other Pseudomonas grow easily. For this reason, it is advis- hand, inhibitor effect of sulfamide in gram-negative able not to extend the enrichment phase and go quickly bacteria is helped by the classical selective agents for for the selective medium, either liquid or solid. According salmonellae like brilliant green, selenite and bile salts. to Bänffer, the efficacy of the medium is improved nota- Notwithstanding, presence of these substances, makes bly if enrichment is performed at 43°C. Presence of a red the medium thermolabile and thus autoclaving must be precipitate in the medium before inoculation, indicates

137 Selenite Media that there was a overheating in which case the selective When starting material is urine, the best procedure is to properties of the medium are reduced. use Selenite Cystine Broth in double concentration, and to inoculate it with an equal volume of urine. Anyway, Selenite Cystine Broth Base subculturing must always be done after 6 hours of incubation but before 24 hours. Most authors recommend the simultaneous use of another enrichment broth, such Ref. 02-602 as Tetrathionate Base Broth (Ref. 02-033).

Specification References Liquid enrichment medium for Salmonella sp. acc. USP US PHARMACOPOEIA (2002) 25th ed Chapter <61> and ISO 6785 and 6340 standards “Microbial Limit Tests” The U.S. Pharmacopoeial Con- vention. Rockville MD. Formula (in g/L) DOWNES F.P. & K. ITO (2001) Compendium of Meth- Peptone ...... 5,00 ods for the Microbiological Examination of Food. 4th ed. Lactose ...... 4,00 APHA. Washington. Potassium phosphate ...... 10,00 FDA (1998) Bacteriological Analytical Manual 8th ed. L-Cystine ...... 0,01 Rev. A. A.O.A.C.International.Gaitherburg VA. Final pH 7,0 ± 0,2 LEIFSON, E. (1936) “A new Selenite Selective Enrich- ment media for the Isolation of Typhoid and Paratyphoid Directions (Salmonella) Bacilli” Am.J.Hyg. 24:423-432. Dissolve 19,01 g of powder. in 1 L of distilled water and US FDA (1962) “The determination of Salmonellae in add 4 g of sodium biselenite (Ref. 06-615). Homog- Food”. enize and bring to the boil. Distribute in suitable con- BÄNFFER, J.R. (1971) Comparison of the isolation of tainers. Termolabile medium: Use immediately. Do not Salmonellae from human faeces by enrichment at 37ºC autoclave. and 43ºC Zbl. Bakt. I Orig. 217:(35-40) Description STOCKES, J.L. and OSBORNE, W.W. (1955) A Selenite Selenite Cystine Broth has been developed according Brilliant Green Medium for isolation of Salmonella. Appl. to Leifson’s formulation with the addition of L-Cystine to Microbiol 3-4:217-227. comply with FDA specifications, since it was proved that ATLAS, R.M., LC. PARKS (1993) Handbook of Microbio- logical Media. CRC Press, Inc London the medium was better in reduced CO2 atmosphere. Essencially, it is an enrichment medium for Salmonella DIN - Standard 10160: Untersuchung von Fleisch u. coming from food or pathological materials, such as fae- Fleischerzneugissen. Nachweiss von Salmonella (Ref- ces or urine, as a previous step to isolation in selective erenzverfahren). media plates such as SS Agar (Ref. 01-171) or Hektoen ISO 6785 Standard (2002) Milk and Milk products - De- Agar (Ref. 01-216). tection of Salmonella spp. ISO 6340 Standard (1995) Water Quality Detection of Technique Salmonella spp. For normal assays or experiments, an incubation at 37°C for a period not exceeding 18 hours is recommended, since within this period a good nutrition of coliforms and an enhancement of pathogens is achieved, but after 24 hours this effect seems to disappear and the growth of accompanying organisms may mask the growth of Salmonella. Appearance of red precipitate before inoculation is the indication of overheating the medium, in which case the selective properties are significantly reduced. Presence of abundant sample residues may also inactivate the selective property of the medium, if the sample is e.g. Ref. 02-602 faeces and or egg powder. In those cases, it is better Selenite Cystine to make a dilution of 1:10 and let the bigger particles Broth Base separate by settling down the dilution tube, and then inoculate Selenite Cystine Broth with an aliquot portion of it. Maintain a proportion of 1:10 between the sample and the medium. It has been demonstrated that when it is desired to isolate Salmonella from faeces,the results are better if the enrichment medium is incubated at 43°C. However this procedure does not work with the isolation of Salmonella Left: Salmonella typhimu- typhi. rium ATCC 14028; right: control. 138 Sellers Agar

Ref. 01-175 - Glucose oxidation and pH changes that can be followed with the colour variation of the pH indicators Speciﬁcation included (phenol red and bromo thymol blue) Solid differential medium for gram-negative non ferment- - Nitrogen gas release, indicated by the bubbles which ing coccobacilli. may eventually break the agar.

Formula (in g/L) Technique Gelatin peptone ...... 20,000 The medium is inoculated with a pure culture by streak- Sodium chloride ...... 2,000 ing on the surface and also by stabbing deeply. Incu- Sodium nitrate ...... 1,000 bation is performed at 35-37ºC for 24-48 hours. After Sodium nitrite ...... 0,350 the incubation period, observe the fluorescence under D-Mannitol ...... 2,000 Wood’s light and also observe the change in pH and L-Arginine ...... 1,000 gas production. The yellow band on the surface of the Yeast extract ...... 1,000 medium due to glucose oxidation may disappear within Magnesium sulfate ...... 1,500 24 to 48 hours. Dipotassium phosphate ...... 1,000 On the slant, fluorescence may appear under UV light Bromothymol Blue ...... 0,040 in some species of the fluorescent Pseudomonas group, Phenol red ...... 0,008 especially Ps. aeruginosa. On the surface of the me- Agar ...... 13,500 dium, most of Acinetobacter species produce a yellow Final pH 6,7 ± 0,2 band due to glucose oxidation. This band may disappear after 24 hours, and this phenomenon is very common with some strains of A. calcoaceticus. Directions A blue colour at the bottom of the tube indicates ar- Suspend 43,5 g of powder in 1 L of distilled water and ginine-dehydrolase positive or any other reaction like bring to the boil. Distribute into tubes and sterilize by anaerobic degradation of nitrate or the mannitol utilisa- autoclaving at 121ºC for 15 minutes. Let it solidify in tion by some strains of Alcaligens faecalis. Nitrogen gas slanted position with short slant and good butt. released in the form of small bubbles or splitting of agar Just before the inoculation, allow 2 drops of sterile glu- indicates denitrification reaction from nitrate or from cose 10% solution to slide down on the opposite side of nitrite. In the table below all these typical reactions are the slant. tabulated.

Description References Sellers Agar is formulated to differentiate the gram nega- ATLAS, R.M. and R.C. PARKS (1993) Handbook of tive bacilli that do not produce characteristic acidiﬁcation Microbiological Media. CRC Press, London by the fermentation in the usual diagnostic media, such MacFADDIN, J. (1985) Media for isoltion-cultivation-iden- as TSI (Ref. 01-192) or Kligler Iron Agar (Ref. 01-103). ﬁcation-maintenance of medical bacteria. Vol I. William & Criteria for the differentiation among the microorganisms Wilkins. Baltimore,MD. that can be obtained from this medium are as follows: SELLERS, W. (1964) J. Bact. 87:46 - Fluorescence production enhanced by the magnesium sulfate and mannitol.

139 SF Medium

Ref. 02-072 T cocci to ferment glucose at 44,5°C. Inhibition of accompanying bacteria is achieved by the high concentration of R-45-22-32-52/53 Specification S-53-45-7-61 azide, which does not affect the growth of streptococci. Liquid selective medium for faecal streptococci detection Enterococci growth is indicated by the indicator brom in several materials. cresol purple turning to yellow, when the tubes are incubated at 44,5°C. In this medium, the indicator turning yellow in pres- Formula (in g/L) ence of enterococci is evident after 18-20 hours, but to Casein peptone ...... 20,000 proceed for the isolation, a supplementary incubation in Dextrose ...... 5,000 petri plates is recommended. Di-potassium phosphate ...... 4,000 If the MPN method is used, do not excessively dilute the Sodium azide ...... 0,500 medium with the sample. Sodium chloride ...... 5,000 Brom cresol purple ...... 0,032 Monopotassium phosphate ...... 1,500 References Final pH 6,9 ± 0,2 HAJNA, A.A. and PERRY, C.A. (1943) Comparative study of presumptive and confirmativemedia for bacteria of the coliform group an fecal streptococci. Am.J.Pub. Directions Hlth.,33:550. Dissolve 36 g of powder in 1 L of distilled water. Dis- ATLAS, R.M. and L.C. PARKS (1993) Handbook of pense in tubes or flasks with Durham´s tubes and steri- Microbiological Media. CRC Press, Inc.,London lize in the autoclave at 121°C for 15 minutes. APHA-AWWA-WPCF (1998) Standard Methods for the examination of Water and Wastewater,20th Ed.,APHA, Description Washington. The efficacy of this diagnostic medium for detecting the DOWNES, F.P. and K. ITO (2001)Compendium of Meth- faecal streptococci (enterococci) from several materials ods for the Microbiological Examination of Food,4th Ed. has been widely proved since its publication by Hajna American Public Health Association,Washington D.C. and Perry, who utilized the advantage of the azide effect and combined it with the fermentative ability of strepto-

SIM Medium

Ref. 03-176 fate allow those microorganisms that are able to produce sulfides, and then this reacts with iron and produces Specification black precipitates which in turn make the medium darker. Differential fluid medium for detecting the motility, H S The amount of thiosulfate present in medium does not 2 affect the motility mechanisms, instead it assures H S production and indole formation. 2 production by those microorganisms that are not able to produce it from cystine or cysteine. Formula (in g/L) Finally, the medium allows the production of indole from Yeast extract ...... 10,0 Tryptophan present in the peptone, which can be easily Casein Peptone ...... 10,0 detected with the addition of Kovac’s Reagent (Ref. Meat peptone ...... 6,0 06-018) (directly or with extraction) or with paper stripes Ferric-ammonium sulfate ...... 0,2 impregnated with the reagent. Sodium thiosulfate ...... 0,2 These three characteristics are common for enterobac- Agar ...... 3,7 teria, and on the basis of these properties, more differen- Final pH 7,3 ± 0,2 tial or even selective media can be used.

Directions Technique Suspend 30 g of powder in 1 L of distilled water and let it Recommended technique is to inoculate by deep stab soak for a few minutes. Heat to boiling and dispense into from a pure culture (or from an isolated colony). After an suitable containers. Sterilize by autoclaving at 121°C for incubation period of 16-18 hours at 37°C, observe for the 15 minutes. clarity in stab. Immotile microorganisms produce growth only in the stab, whereas motile ones may be easily Description detected by their displacement which is indicated by the This classical medium was originally developed to dis- turbidity in the medium. tinguish several types of enterobacteria, on the basis of H2S production is indicated by the general blackening of motility test , detection of indole and H2S production. the medium when there is a great amount of FeS pro- It is a semisolid or ﬂuid medium, and so the motile micro- duced or by blackening of the stab when there is a little organisms can move freely. At the same time, richness amount of FeS produced. of sulfur containing amino acids and presence of thiosul-

140 SIM Medium

Indole formation is the last test to be performed if the References soaked or impregnated paper stripes are not used. HARRIGAN, W.F. and McCANCE, M.E. (1966) Labora- Despite the fact that many authors suggest a previ- tory Methods in Microbiology. Academic Press, . ous extraction of indole by stirring on the surface of the BLAZEVIC, D.J. (1968) Improved Motility Indole Me- culture medium with chloroform. If Kovacs’ Reagent dium. Appl. Microbiol. 16, (4),668. (Ref. 06-018) is employed then this is not necessary and BULLMASH, J.M. and FULTON, M (1964). Discrepant the observations can be done by pouring a few drops of Test for Hydrogen Sulﬁde. J.Bact. 88(6)1813. reagent on the surface of the medium. A positive test will produce the interphase in the medium turning to purple red colour, whereas a negative test will produce no colour change. Many a times, chloroform extraction may give erroneous results, since the appearance of colour must be observed immediately after the reagent addition. However if it is delayed by more than 30 seconds, the test must be considered negative.

Simmons Citrate Medium

Ref. 01-177 Technique The techique is simple and one has to take care in us- Specification ing an inoculum as small as possible and the medium Medium for verifying the citrate utilization by enterobac- should be freshly prepared, because if it is very dry, false teria according ISO 10273 standard. turning (colour change) may appear, even before the inoculation, especially at the bottom of the slant. Formula (in g/L) The basis of this medium is in the capacity of microorganisms to use citrate as carbon source and ammonium Magnesium sulfate ...... 0,20 compounds as the nitrogen source for their growth. Monoammonium phosphate ...... 1,00 Among enterobacteria, these properties are possessed Dipotassium phosphate ...... 1,00 by the following genera: Enterobacter, Klebsiella, Ser- Sodium citrate ...... 2,00 ratia, Citrobacter and some species of Salmonella as Sodium chloride ...... 5,00 S.schottumelleri, S.typhimurium, S.arizona etc. whereas Brom thymol blue ...... 0,08 Escherichia, Shigella, Salmonella typhi and S.paratyphi Agar ...... 15,00 are unable to grow. Final pH 6,8 ± 0,2 Although, the test result must be read as the growth produced, the presence of an indicator makes it easy, as the Directions citrate degradation yields an alkaline reaction, which is Dissolve 24 g of powder in 1 L of distilled water. Bring to indicated by the turning of the indicator to intense blue. the boil. Dispense in tubes and sterilize by autoclaving at This is evident even when the growth rate is high. 121°C for 15 minutes. Allow to solidify with long slant. References Description SIMMONS J.S. (1926) A culture medium for differentiat- Simmons Citrate Agar is the solid version of the classical ing organisms of typhoid-colon aerogenes group and for Koser citrate medium, and it can be used in the plates as isolatig certain fungi.J.Inf.Dis. 39:209 well as in slanted tubes. Slanted tubes can be inoculated FDA (1998) Bacteriological Analytical Manual. 8th Ed. by surface streaking or by deep stab. Revision A. AOAC International. Gaithersburg. Although, originally, it was described as an isolation and APHA-AWWA-WEC (1998) Standard Methods for the identification medium for certain fungi, Edwards and examination of water and wastewater. APHA. Washing- Ewing had recommended it for the IMViC test since it ton DC. has the advantage over the Koser’s medium that the HORWITZ, W. (2000) Official Methods of Analysis. 17th readings can be made by the indicator colour change, Ed. AOAC International. Gaithersburg. MD. instead of turbidity of the medium, which is sometimes ISO 10273 Standard (1994) General guidance for the difficult to detect. detection of presumptive pathogenic Yersinia enterocolitica.

141 Slanetz Bartley Media

Slanetz Bartley Agar Description This formulation, without TTC allows its sterilization by Xn Ref. 01-178 autoclaving without the development of the pink colour R-22-32-52/53 due to the formazan which is formed as a result of the S-7-46-61 Specification partial-thermal reduction of TTC. This modification is more tedious in its preparation but provides a colour- Differential and selective medium for the enumeration of less medium, making the results easier to read and the enterococci, colonies are more sharply defined.

Formula (in g/L) Technique Tryptose ...... 20,0 For the membrane filtration technique, take 100 mL of Yeast extract ...... 5,0 water sample which is previously homogenized, and Dextrose ...... 2,0 pass it through a sterile membrane filter. Then wash Potassium phosphate ...... 4,0 it with 30 mL of sterile dilution water to wipe away the Sodium azide ...... 0,4 things left on the funnel of the filtering system. TTC ...... 0,1 By using sterile forceps, transfer the membrane asepti- Agar ...... 12,0 cally on to the culture medium contained in a Petri dish, Final pH 7,0 ± 0,2 making sure that the filter surface faces upwards. Close the lid and invert the plate. lncubate at 37°C for 48 Directions hours. Suspend 43,5 g of powder in 1 L of distilled water and All the developed colonies which appear red or purple bring to the boil. Cool to 50°C and distribute into sterile coloured must be considered as enterococci, since those petri plates immediately. Do not autoclave or overheat. bacteria reduce Triphenyltetrazolium-HCl to an insoluble formazan which is red in colour. The secondary or Description accompanying gram negative bacteria are inhibited by The Slanetz Bartley Agar is recommended for the sodium azide. determination of faecal streptococci by the membrane As regards, food samples from a decimal dilution bank filtration technique. Although originally this medium was of the product to be examined, incoculate 0,1 mL on the described by the authors only for the above technique, it surface of the petri dishes using the Drigalski technique. gives such excellent results that it is now recommended lncubation and examination is carried out as same as in for viable enumeration of enterococci from food samples. the membrane filtration technique.

Slanetz Bartley Agar Base References SLANETZ L.W. & BARTLEY, C.H. (1957). Numbers of Xn Ref. 01-579 Enterococci in Water, Sewage and Faeces Determined R-22-32-52/53 by The S-7-46-61 Speciﬁcation Membrane Filter technique with an lmproved Medium. J.Bact., 74:591-596. Differential and selective medium for the detection and ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro- enumeration of enterococci acc. ISO Standard 7899- biological Media,CRC Press Inc.,London 2:2000 ISO Standard 7899-2:2000 Water Quality. Detection and enumeration of enterococci by membrane ﬁltration Formula (in g/L) method. Tryptose ...... 20,0 LACHICA, L.V.F. & P.A., HARTMAN (1968) Two im- Yeast extract ...... 5,0 proved media for isolating and enumerating enterococci Dextrose ...... 2,0 in certain frozen foods Potassium phosphate ...... 4,0 J. Appl. Bact. 31:151-156 Sodium azide ...... 0,4 Agar ...... 12,0 Final pH 7,0 ± 0,2

Directions Suspend 43,4 g of powder in 1 L of distilled water and Ref. 01-579 Slanetz- bring to the boil. Sterilize by autoclaving at 121ºC for Bartley Agar Base + Ref. 15 minutes. Cool to 50°C and add 10 mL/L of 1% TTC 06-023 TTC 1% Sterile sterile solution (Ref. 06-023). Mix well and distribute into Solution. sterile petri plates immediately. Enterococcus faecalis ATCC 29212.

142 SOB Broth

Ref. 02-523 The addition of 4 g/L of dextrose to this medium produce the SOC Medium that is used in the ﬁnal phase of Speciﬁcation transformation and in the recovery of electroporated E. Liquid medium for the cultivation of recombinant strains coli cells. of Escherichia coli. References Formula (in g/L) HANAHAN, D. (1983) Studies on transformation of Es- Tryptone ...... 20,000 cherichia coli wth plàsmids. J. Mol. Biol. 166:557 Yeast Extract ...... 5,000 SAMBROOK, J., E.F. FRITSCH & T. MANIATIS (1989) nd Sodium Chloride ...... 0,500 Molecular cloning: a laboratory manual. 2 Ed. Cold Magnesium sulphate ...... 2,400 Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Potasium chloride ...... 0,186 Final pH 7,0 ± 0,2

Directions Dissolve 28 g of powder in 1 L of distilled water heating if it is necessary. Distribute in suitable containers and sterilize in autoclave at 121ºC for 15 minutes.

Description This medium was developed by Hanahan in 1983 as a nutritionally rich base for growth preparation and transformation of competent cells that allows the introduction of foreign DNA onto cells..

Sporulating Agar (AK Agar) (USP Antibiotic Medium 32)

Ref. 01-069 in 10 mL of Ringer Solution the growth from a slant. With this suspension, inoculate on the surface a Roux flask Specification (bottle) containing 300 mL of solidified and controlled This medium is according to the Arret and Kirshbaum Sporulation Agar. formulation, adopted by US FDA, for the preparation The roux flask must be incubated at 35°C for 5 days. of spore suspension for antibiotic assay. It is the same Growth is harvested with 50 mL of sterile Ringer So- medium as the Antibiotic Medium 32. lution, helping with a Drigalsky loop or sterile crystal balls (pearls) if necessary. The suspension obtained is carefully centrifuged at 5000 rpm for 15 minutes. Discard Formula (in g/L) the supernatant liquid and suspend the pellet in a fresh Gelatin peptone ...... 6,0 volume of Ringer Solution. Put it in a boiling water bath Casein peptone ...... 4,0 at 70°C for 30 minutes. This suspension is active upto 6 Yeast extract ...... 3,0 months if stored refrigerated. Depending on the objective Meat extract ...... 1,5 of the experiment,it can be standardized by turbidimetry. Dextrose ...... 1,0 Manganous sulfate ...... 0,3 Agar ...... 15,0 References Final pH 6,6 ± 0,2 ARRET and KIRSHBAUM (1959) J.Milk. Food Tech. 22:329 SANCHO, GUINEA, PARES (1980) Microbiología Directions Analítica Básica. Ed. JIM. Barcelona, Dissolve 30,8 g of powder in 1 L of distilled water. Bring US PHARMACOPOEIA (2002) 25th ed. <81> Antibiotic it to the boil with constant stirring . Dispense in suitable Microbial Assays. US Pharmacopoeial Convention Inc. containers and sterilize at 121°C for 15 minutes. Rockville. MD Description Sporulating Agar is made according to the original formulation of Arret and Kirshbaum, and later adopted by FDA for the preparation of Bacillus subtilis ATCC 6633 spores suspension. To prepare this suspension, suspend

143 SPS Agar

Ref. 01-050 The differential system consists of sodium sulfite and ferric citrate. It allows the detection of sulfite-reducing Specification organisms, which form black colonies due to the ferrous Solid medium for the detection of Clostridium perfrin- sulfide precipitate. gens in food. Technique Formula (in g/l) The usual technique for the use of this medium is as Sodium sulfite ...... 0,50 follows: Polymyxin (B) sulfate ...... 0,01 The samples to be examined are ground or homog- Sodium sulfadiazine ...... 0,12 enized with a vortex in a stomacher and then a decimal Casein peptone ...... 15,00 dilution bank is prepared. Take a sample aliquot from Yeast extract ...... 10,00 each one of these dilutions and place in the petri dishes. Ferric citrate ...... 0,50 The medium, molten and cooled to 50°C, is now poured Sodium thioglycolate ...... 0,10 in the dishes and allowed to solidify. The dishes are incu- Polysorbate 80 ...... 0,05 bated in an anaerobic system at 35°C for 24-36 hours. Agar ...... 15,00 Usually, 90% of the black colonies which are formed can Final pH 7,0 ± 0,2 be attributed to the Clostridium perfringens. Neverthe- less, and since the medium is not extremely selective, it is advisable to verify the black colonies formed by Directions gram-positive sporulated immotile organisms incapable Suspend 41,3 g of powder in 1 L of distilled water and of reducing the nitrates to nitrites. bring to the boil. Distribute into tubes or screw-cap The Indole Nitrite Fluid Medium (Ref. 03-101) is suitable containers and sterilize by autoclaving at 121°C for 15 for such purposes, still a small quantity of agar has to be minutes. Cool the sterilized medium quickly by placing in occasionally added in it. the refrigerator or in cold water. Most clostridia are sulfite reductors. Among them are Cl. perfringens and the Cl. botulinum which along with Cl. Description bifermentans are the species most frequently involved The SPS Agar (Sulfite-Polymyxin-Sulfadiazine) is a in food poisoning. modification of the original Wilson & Blair medium for the detection of clostridia. The present medium excels the References formulation of Mossel and also the later modification of DOWNES, F.P. & K. ITO (2001) Compendium of Meth- Angelotti et al. It achieves a higher selectivity for Cl. perf- ods for the Microbiological Examination of Food,4th Ed. ringens with the addition of sulfadiazine and polymyxin. APHA,Washington. On the other hand, the nutritional substrates constituted FDA (1998) Bacteriological Analytical Manual, 8th ed. by the tryptone and the yeast extract are complemented Revision A. AOAC International. Gaithersburg. MD by the Polysorbate, which also allows the recovery of the most delicate cells. The anaerobic conditions are improved by the thioglycolate, which permits the use of the medium on the plates without the Miller-Prichett tubes, used by Mossel and Wilson-Blair.

control

Clostridium perfringens ATCC 13124

144 Staphylococcus 110 Agar

Ref. 01-587 Technique The sample is inoculated in the petri dish by wire loop to Specification obtain isolated colonies, and it is incubated for 48 hours Selective medium for the isolation and presumptive iden- at 30°C. tification of staphylococci. At 37°C early reactions or results are obtained, but pigmentation is not good,as the pigmented colonies form Formula (in g/L) a characteristic golden yellow colour, which in this case are white. Casein Peptone ...... 10,00 Acid production from mannitol is verified by adding a few Yeast extract ...... 2,50 drops of Bromthymol Blue 0,04% solution on suspected Gelatin ...... 30,00 colonies. Positive reaction produces a yellow coloura- Sodium chloride ...... 75,00 tion. Di-potassium phosphate ...... 5,00 Gelatin hydrolysis (Stone’s reaction) is visualised by a Lactose ...... 2,00 clear halo surrounding the colony which forms 10 min- D-Mannitol ...... 10,00 utes after a few drops of 20% aq. soln.of Sulfosalicylic Agar ...... 15,00 acid or sat. aq. solution of Ammonium sulfate are added Final pH 7,2 ± 0,2 over them. These last two reactions are helped by placing Oxford Directions cups around the discrete colonies, isolating them from Suspend 149,5 g of powder in 1 L of distilled water and affecting the other colonies. Identification is completed heat up to 55-60°C in boiling water bath until total dis- by verifying hemolysis tests, subculturing on Blood Agar solution of gelatin. Bring to the boil. Dispense in suitable plates, and verifying plasmacoagulase activity on rabbit containers and sterilize by autoclaving at 121°C for 15 serum. minutes. Before pouring in the petri dishes, gently shake Although the subcultures can be done from isolated to suspend possible precipitate. Should a blood addi- colonies in 110 medium, results are always better if a tion be desired, cool to 45°C and add sterile defibrinated short (4-5 hours) incubation on Tryptic Soy Broth (Ref. blood and homogenize before pouring in the plates. 02-200) is carried out.

Description References This medium combines the basic criteria of staphylococci STONE, R.V. (1935) A cultural method for classifying identiﬁcation i.e.: halotolerance, pigmentation, mannitol staphylococcias of the “food poisoning” type. Proc.Soc. fermentation and gelatin liquefaction as were marked by Exptl.Biol Med.33:185-187 Stone and Chapman. Smucker and Appleman suggests CHAPMAN, G.H. (1945) The signiﬁcance of sodium the addition of sodium azide (5mg/L) to improve the chloride in studies of staphylococci. J. Bacteriol 50:201- inhibition of Bacillus spp. 205 It is widely known that pathogenic staphylococci are co- FDA (1998) Bacteriological Analytical Manual. 6th ed. agulase positive, pigment former, highly halotolerant and Revision A. AOAC International. Gaithersburg able to liquefy gelatin and produce acids by fermenting mannitol. They are also strongly hemolytic. In the 110 medium, the following reactions can be observed: pigmentation, mannitol fermentation, gelatin liquefaction and saline tolerance. From this medium, hemolysis and coagulase production can also be proved afterwards.

145 Starch Media

Starch Agar

Ref. 01-283

Speciﬁcation Solid medium to detect the starch hydrolysis by microorganisms,

Formula (in g/L) Meat extract ...... 3,0 Peptone ...... 5,0 Soluble starch ...... 2,0 Agar ...... 15,0 Final pH 7,2 ± 0,2

Directions Suspend 25 g of powder into 1 L of distilled water and let it soak. Heat to boiling and distribute into suitable containers. Sterilize in the autoclave at 121°C for 15 minutes.

Description Although this medium was initially formulated to perform the test for the identiﬁcation of Bacillus cereus, it can be applied to any kind of microorganism where starch hydrolysis activity is required to be analyzed.

Technique Over the medium plates with Starch Agar, inoculate in straight streaks the strains to be examined, (maximum four per plate). Incubate at 30-35°C for 48 hours if the strains are of Bacillus cereus and up to 5 days for dubious cases. After the incubation, ﬂood the plates with an alcoholic iodine solution 2%. Starch hydrolysis is seen by the appearance of a clear halo surrounding the growth streak, whereas the rest of the medium in the plate acquire a dark blue colour. The bigger the clear zone, the starch activity is considered higher of the strain under study.

References COLLINS, C.H., LYNE, P.M. (1976) Microbiological Methods. 4th Ed. Butterworths,London. ISENBERG H.D. (1992) Clinical Microbiology Proce- dures Handbook. ASM Washington. ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro- biological Media. CRC Press,Inc.,London

146 Stuart-Ringertz Transport Medium

Xn Ref. 03-454 vides a reducing environment which is aided and main- R-22-43 tained by to the low concentration of agar, that avoids S-24-37-46 Specification convection streams and restricts oxygen difussion. Medium for the conservation and transport of pathologi- Progressive oxidation of the medium can be seen by cal specimens or fastidious microorganisms. the change of the methylene blue, which acts as an Eh (redox) indicator. Formula (in g/L) Sodium glycerophosphate ...... 10,000 Technique Sodium thioglycolate ...... 1,000 Sample is placed directly inside the tube, taking care that Calcium chloride ...... 0,100 it is beneath the blue band. If the sample is taken with a Methylene blue ...... 0,002 swab, it is advisable to impregnate it with a suspension Agar ...... 8,000 of active carbon (activated charcoal) before puting it into Final pH 7,4 ± 0,2 the transport medium. The sample must always be in the centre of the medium and beneath the blue band that indicates oxidation. If Directions the depth of the blue band is bigger than the half of the Suspend 19 g of powder in 1 L of distilled water and medium, do not use the tube. bring to the boil. Distribute in tubes or flasks close with air tight cap in such a way that the medium forms a vertical column of 7-10 cm. Sterilize in the autoclave References at 121°C for 15 minutes and cool quickly in the vertical STUART, R.D. (1959) Transport medium for specimens position. in public health bacteriology. Publ. Hlth. Rep. 74:431- 438. RINGERTZ, O. (1960) A modified Stuart medium for the Description transport of gonococcal specimens. Acta Path. Microbiol. The growth of microorganisms in this medium is restrict- Scand. 48:105-112 ed by the total lack of nitrogen, but they remain alive and inactive for a long period. Thanks to the buffering and protective effect of glycerophosphate. Thioglycolate pro-

147 TCBS Media

TCBS Agar Directions Suspend 88 g of powder in 1 L of distilled water and Ref. 01-190 bring to the boil. Boil it for 1 minute. Cool to 45-50°C and pour it into sterile petri plates. Do not autoclave it. Specification TCBS Agar is specially recommended for the selective Description isolation of Vibrio that causes cholera or dysenteric diar- Nowadays TCBS Agar is universally accepted as the rhoea, and for the examination of food that might have medium of choice for differential isolation of enteropatho- Vibrio. genic Vibrio, and it achieves a great inhibition of all the accompanying organisms. This formulation allows a Formula (in g/L) high growth of Vibro cholerae and V.parahaemolyticus. V.alginoliticus and NAG vibrios also grow well. Casein peptone ...... 5,00 Enterobacteria are strongly inhibited by high concentra- Meat peptone ...... 5,00 tions of citrate, thiosulfate, bile and sodium chloride. Yeast peptone ...... 5,00 Although some enteric bacteria may also grow in this Sodium citrate ...... 10,00 medium, their colony morphology is quite different than Sodium thiosulfate ...... 10,00 Vibrios. Ox bile ...... 5,00 The organisms that may be confused with vibrios are Sodium Cholate ...... 3,00 some biotypes of Proteus and Pseudomonas. There are Sucrose ...... 20,00 some resistent enterococci which may form exception- Sodium chloride ...... 10,00 ally small and yellow colonies on this medium. Ferric citrate ...... 1,00 Usually, in this medium, colonies are selected or chosen Thymol blue ...... 0,04 and then identified with primary tests (oxidase reactions Bromo thymol blue ...... 0,04 in Kliger Iron Agar (Ref. 01-103), MRVP Broth (Ref. Agar ...... 14,00 02-207), and antibiogram) before performing serological Final pH 8,6 ± 0,2 identification and phage typing. Due to its high selectivity, the medium allows massive Directions inoculation of pathological material. Once solidified and Suspend 88 g of powder in 1 L of distilled water and cooled, the medium is turbid, but the observations are bring to the boil. Boil it for 1 minute. Cool to 45-50°C and not affected. pour it into sterile petri plates. Do not autoclave it. This medium is very thermolabile and so it must not be autoclaved, overheated or remelted. TCBS Modified Agar Colonial appearance on TCBS Agar after 24 hours at Ref. 01-567 37°C: Vibrio alginolyticus: Big, yellow Specification Vibrio cholerae and sucrose fermentative strains of Vi- brio parahaemolyticus: Average size, dirty yellow TCBS Agar is specially recommended for the selective with yellow halo in the center isolation of Vibrio parahaemolyticus according ISO 8914 Non sucrose fermentative strains standard. of Vibrio parahaemolyticus: Small, yellow, without halo and with a green core. Formula (in g/L) Streptococcus faecalis: Very small and convex, yellow Proteosa Peptone ...... 10,00 with yellow halo Yeast Extract ...... 5,00 Enterobacteria: Small and transparent Sucrose ...... 20,00 Pseudomonas, Aeromonas, Proteus: Average size and Sodium citrate ...... 10,00 blue. Sodium chloride ...... 10,00 Ox bile ...... 8,00 References Sodium thiosulphate ...... 10,00 KOBAYASHI, T., ENOMOTO, S. SAKAZARI, R. and Iron citrate ...... 1,00 KUWAHARA, S. (1963) A new selective medium for Thymol Blue ...... 0,04 pathogenic vibrios TCBS, (modified Nakanishi Agar)Jap. Bromthymol blue ...... 0,04 J.Bact.18:387. Agar ...... 14,00 FDA (1998) Bacteriological Analytical Manual 8th ed. Final pH 8,6 (± 0,2) Revision A. AOAC INTERNATIONAL. Gaithersburg ISO 8914 Standard (1990) General guidance for the detection of Vibrio parahemolyticus.

148 TCBS Media

DOWNES, F.P. & K. ITO (2001) Compendium of Meth- KP DAS (1993). Outbreak of Vibrio cholerae non-01 in ods for the Microbiological Examination of Food,4th Ed. India and Bangladesh. Lancet, 341:1346-1347 American Public Health Association,Washington D.C. HORWITZ, W. (2000) Ofﬁcial Methods of Analysis of PASCUAL ANDERSON, MªRª (1992) Microbiología AOAC International 17 ed. Gaithersburg. MD Alimentaria. Diaz de Santos, S.A.,Madrid,. BHATTACHARYA, M.K., S.K. BATTACHARYA, S. GARG, P.K. SAHA, D.DUTTA, G.B. NAIR, B.C. DEB &

Ref. 01-567 TCBS Modiﬁed Agar

Proteus mirabilis Vibrio alginolyticus ATCC 14273

control

Terriﬁc Broth

Ref. 02-474 Description Terriﬁc Broth was developed by Tartoff and Hobbs to Speciﬁcation improve yield in plasmid bearing E. coli. This medium Liquid medium for the cultivation of recombinant strains supports a high cellular density and mass and maintains of Escherichia coli. the growth in the logarithmic phase for a long time. Due to this fact, it provides greater yields of recombinant Formula (in g/L) proteins and plasmid DNA. On many occasions it substitutes the classical LB Broth (Ref. 02-385, 2-384 and Casein peptone ...... 12,00 2-406) therefore usually increases yields of plasmid DNA Yeast extract ...... 24,00 and recombinant proteins. Dipotassium phosphate ...... 9,40 Monopotassium phosphate ...... 2,20 Final pH 7,3 ± 0,2 References SAMBROC, J., E.F. FRITSCH, T. MANIATIS (1989) Mo- Directions lecular Cloning: A laboratory Manual. 2ª Ed.,Cold Spring Harbor Press. Cold Spring Harbor,USA. Dissolve 47,6 g of powder in 1 L of distilled water, heat- TARTOFF, K.D. & C.A. HOBBS (1987) Improved media ing up only if necessary. Distribute in suitable containers for growing plasmids and cosmid clones. Bethesda Re- and sterilize by autoclaving at 121ºC for 15 minutes. search Laboratoires Focus 9:12

149 Tetrathionate Media

Tetrathionate Broth Base White precipitate is due to calcium carbonate and it must be considered as normal. Ref. 02-033 Description Specification Tetrathionate Broth is a classic medium for the enrich- Medium for the selective enrichment of Salmonellae ment of enteric or intestinal pathogens, and for all the (AOAC 17th, ICMSF 1968, USP 25th) members of Salmonella type, from very polluted samples, like faeces, urine, waste water and others. During the preparation, when iodine is added, tetrathion- Formula (in g/L) ate is produced from the sulfate, and this salt together Meat peptone ...... 2,5 with the bile salts in the medium, provoke a strong inhibi- Casein peptone ...... 2,5 tion to most of the normal intestinal bacteria, except for Bile salts ...... 1,0 those which are capable of reducing tetrathionate, e.g. Calcium Carbonate ...... 10,0 Salmonellae. Reduction reaction liberate sulfuric acid, Sodium Thiosulfate ...... 30,0 which is neutralized by the carbonate, avoiding a decrease of the pH, which is harmful even for Salmonellae. Directions However, many Proteus species resist the bile salts Suspend 46 g of powder to 1 L of distilled water, heat to concentration and moreover, they may even reduce boiling and cool to 40-45°C. Add 20 mL of iodine-iodide tetrathionate. So, many authors recommend the addition solution and 2 vials of the Selective Supplement of Bril- of other inhibitors simultaneously, such as 0,1% Brilliant liant Green-Novobiocin ref. 06-017CASE and distribute Green Solution (10 mL/L) which at the same time inhibits in sterile tubes. gram-positive flora, or Novobiocin in a concentration Do not heat after adding the iodine solution. Medium between 4 and 40 mg/L. must be used immediately. Without the iodine solution, Basal medium can be kept indefinitely in the refrigerator, medium can be stored in refrigeration for some days. but after the addition of inhibitors, efficacy of the medium The appearance of white medium precipitate is normal, decreases with time. and it comes from calcium carbonate. Sorting the inhibitors depending on their stability if they are kept in the refrigerator produce the following list: bril- Description liant green, novibiocine, which is effective for 2 months This is the version originally used, which has been modi- in the refrigerator but only 48 hours at 37°C, and finally fied or improved the Muller-Kauffmann formulation, since iodine solution, which is only effective for 40 hours once the latter one has more efficacy. the inhibitor is added to the medium.

Muller-Kauffmann Medium Base Technique It is recommended to prepare the Base Broth, distribute Ref. 02-335 it into tubes, sterilize and cool it. Add the Brilliant Green

Speciﬁcation Medium for selective enrichment of Salmonellae, acc. ISO standard.

Formula (in g/L) Bile salts #3 ...... 4,78 Meat extract ...... 4,30 Casein peptone ...... 8,60 Sodium chloride ...... 2,60 Calcium carbonate ...... 38,70 Sodium thiosulfate ...... 47,80

Directions Add 107 g of powder to 1 L of distilled water. Heat to boiling and let it cool to 40-45°C. Add 20 mL of iodine- iodide solution and 2 vials of the Selective Supplement of Brilliant Green-Novobiocin Ref. 06-017CASE and distribute into sterile tubes. Do not heat after adding the iodine solution. Complete medium must be used immediately; the base, without iodine, may be stored in the refrigerator for some days.

150 Tetrathionate Media solution and store it in the refrigerator. If you are going FIL-IDF Standard 93. (2001) Milk and milk products: to use the medium before 60 days, you may also add Research of Salmonella. novobiocin. Iodine-iodide solution has to be added just HORWITZ, W. (2000) Ofﬁcial Methods of Analysis. 17th before its use. Do not reheat the medium after one of ed. AOAC International. Gaithersburg. Md.USA these additions. ISENBERG, H.D. (1992) Clinical Microbiology Proce- Usual technique consists of adding the sample to the dures Handbook. Vol. 1. APHA. Washington DC. USA medium (1:10) and then homogenizing it well. Incubate ISO Standard 6579 (2002) Microbiology of food and ani- at 37°C for a period not longer than 48 hours, since mal feeding stuffs – Horizontal method fort he detection after this time the medium loses its selectivity and the of Salmonella spp. suppressed ﬂora may also grow. Some authors suggest ISO Standard 6785 (2001) Milk and Milk Products – De- to incubate at 43°C and perform observations after 18, tection of Salmonella spp. 24 and 48 hours, but one can get better results if the ISO (1975) Standard 3565. Meat Products: Reference sample from the surface of the broth is taken after 30-36 Method for detection of Salmonellae.. hours. U.S. PHARMACOPEIA. (2002) 25th ed. <61> Microbial Take aliquotes with a loop and inoculate on the surface Limits Test. US Pharmacopoeial Convention Inc. Rock- of the selective media like SS Agar (Ref. 01-171) or ville. Md. USA. Hëktoen Enteric Agar (Ref. 01-216), etc... KAUFFMAN, F. (1931) Ein Kombiniertes Anreicherungus verfahren für Typhus und Paratyphus Bazillen.Zblt. Bakt References Microbiol. Hyg Abt. I. Orig. 119:148 DIN Standard 10160 Untersuchung von Fleisch und MARSHALL, R.T. (1993) Standard methods for the ex- Fleischerzeugnissen: Nachweis von Salmonellen. Ref- amination of dairy products. 16th ed. APHA Washington erenzverfahren. DC. USA. DIN Standard 10181 Mikrobiologische Milchuntersuc- MULLER, L. (1923) Un nouveau milieu hung: Nachweis von Salmonellen. Referenzverfahren. d’enrichiessement pour la recherche du bacille typhique DOWNES, F.P. & K.ITO (2001) Compendium of meth- est des partyphyques. Comp. Rend. Soc. Biol. 89:434- ods fort he microbiological examination of foods. 4th ed. 437. APHA. Washington DC. USA FDA (1998) Bacteriological Analytical Manual. 8th ed. Revision A. AOAC International. Gaithersburg. Md. USA.

Thioglycollate Media

Thioglycollate Broth (USP Directions Alternative Thioglycollate Dissolve 29 g of powder in 1 L of distilled water, heating if necessary to help dissolution. Distribute into suitable Medium) containers and sterilize by autoclaving at 121°C for 15 minutes. This culture medium should always be freshly Ref. 02-186 Xi prepared or heated at 100°C for 10 minutes before use. R-43 Speciﬁcation S-24-37 Description A medium for sterility test and the cultivation of microaer- The Thioglycolate broth is a standard medium, named ophilic and anaerobic organisms. It is specially used for also Alternative Thioglycollate Medium, formulated and viscous or turbid samples. recommended by USP, NF, NIH and FDA. It is used for sterility testing of biological products or Formula (in g/L) samples of turbid appearance where Fluid Thioglycol- Peptone from casein ...... 15,0 late Medium (Ref.3-187) is not suitable because of its Yeast extract ...... 5,0 viscosity. Dextrose ...... 5,5 The formula of Thioglycollate broth is the same as Sodium chloride ...... 2,5 Thioglycollate USP Fluid Medium without resazurin and Sodium thioglycollate ...... 0,5 agar. L-Cystine ...... 0,5 Media must be freshly prepared, boiled, sterilised, Final pH 7,1 ± 0,2 cooled and used within 4 hours for its inoculation.

151 Thioglycollate Media

References Description ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- The Thioglycollate Fluid Medium is a standard medium biological Media. CRC Press, Inc. London formulated and recommended by European Pharmaco- DOWNES, F.P. & K. ITO. (2001) Compendium of Meth- poeia, USP, NIH and FDA. ods for the Microbiological Examination of Foods. 4th The reducing agents thioglycollate and L-cystine ensure Ed. APHA. Washington DC. USA an anaerobiosis which is adequate even for fastidious HORWITZ, W. (2000) Official Methods of Analysis. 17th anaerobes. The sulfhydryl groups of these substances ed. AOAC International. Gaithersburg. Md. USA also inactivate arsenic, mercury and other heavy metal US PHARMACOPOEIA (2002) <71> Sterility Test. 25th compounds. The Thioglycollate media are thus suitable ed. US Phamacopoeial Convention Inc. Rockville. Md. for the examination of materials which contain heavy USA. metals or heavy metal preservatives. The higher viscosity of the fluid thioglycollate medium prevents rapid uptake of oxygen. Any increase in the Thioglycollate Fluid Medium oxygen content is indicated by the redox indicator sodium resazurine which changes its colour to pink. Xi Ref. 03-187 R-43 Technique S-24-37 Inoculate the culture medium with the sample material Specification taking care that the sample reaches the bottom of the Fluid medium for sterility testing acc. to Eur. Phar., USP, tubes. FDA, and ISO 7937:2004 for the cultivation of microaer- Incubate for at least 14 days at the optimal temperature. ophilic and anaerobic organisms. It is specially used for Anaerobes grow in the lower part of the culture medium viscous or turbid samples. container.

Formula (in g/L) References Peptone from casein ...... 15,000 ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- Yeast extract ...... 5,000 biological Media, CRC Press Inc., London Dextrose ...... 5,500 BREWER J.H (1940) Clear liquid medium for the Sodium chloride ...... 2,500 “aerobic” cultivation of anaerobes. J. Amer. Med. Assoc. Sodium thioglycollate ...... 0,500 113:598-600 L-Cystine ...... 0,500 DOWNES, F.P. & K. ITO (2001) Compendium of Meth- Resazurine ...... 0,001 ods for the Microbiological Examination of Food. 3rd ed., Agar ...... 0,750 A.P.H.A., Washington D.C. Final pH 7,1 ± 0,2 EUROPEAN PHARMACOPOEIA, (2005) § 2.6.1 Steril- ity. 5th Ed., EDQM Council of Europe. Strasbourg. Directions FDA (1998).Bacteriological Analytical Manual, 8th ed. Dissolve 30 g of powder in 1 L of distilled water, slowly Revision A., AOAC International. Gaithersburg. MD bring to the boil, stirring until complete dissolution. Dis- HORWITZ, W., (2000) Official Methods of Analysis, 17th tribute into final containers and sterilize by autoclaving ed., A.O.A.C. International. Gaithersburg. MD at 121°C for 15 minutes. Store in dark at room tempera- ISENBERG, H.D. (Ed), (1998) Essential Procedures for ture for not more than one month. If after the storage the Clinical Microbiology. ASM., Washington. USA medium is pink coloured (sign of oxidation) for more than MacFADDIN, J.F. (1985) Media for Isolation-cultivation- 1/3 of its depth, recreate anaerobic conditions by heating identification-maintenance of medical bacteria. Vol. I., at 100°C for 10 minutes. Williams & Wilkins. Baltimore. MD. USA US PHARMACOPOEIA (2005) 25th Ed. § <71> Steril- ity Test., US Pharmacopoeial Convention Inc., Rockville MD.

Ref. 03-187 Thioglycollate Fluid Medium

Left: anaerobical growth; center: control; right: fermentative growth

152 Triple Sugar Iron Agar (TSI Agar) (Eur. Phar. Agar Medium M)

Ref. 01-192

Speciﬁcation Differential medium for identiﬁcation of enterobacteria, according ISO standard.

Formula (in g/L) Peptone ...... 20,000 Meat extract ...... 3,000 Yeast extract ...... 3,000 Lactose ...... 10,000 Sucrose ...... 10,000 Dextrose ...... 1,000 Sodium chloride ...... 5,000 Ferric ammonium citrate ...... 0,500 Sodium thiosulfate ...... 0,300 Phenol red ...... 0,025 Agar ...... 12,000 Final pH 7,4 ± 0,2

Directions Dissolve 64,8 of powder in 1 L of distilled water and bring to boiling. Dispense into tubes and sterilize at 121°C for 15 minutes. Leave to solidify with short slant and good butts.

Description TSI Agar is a modification of the classical Kliger’s agar. 1% of sucrose has been added to this medium to differentiate Proteus and Hafnia (sucrose positive) from Salmonella and Shigella (sucrose negative). Sugar degradation with acid formation is detected by the turning of an indicator (phenol red) to yellow, whereas if there is alkalinization, it turns to purple. When there is only glucose degradation, the acid production is weak and is evaporated on the surface, so indicator may be reoxidised producing an alkaline surface (red) and an acid butt (yellow). If lactose or sucrose are degradated, acid production is intense and then all of the medium (surface and depth) turns yellow. Gas production is detected by the formation of bubbles and occasionally cracks in the agar. Hydrogen sulfide production, from thiosulfate or sulfured aminoacids of peptones, is detected by the formation of black FeS precipitate when medium reacts with iron salts. Use the medium in slanted tubes with good depth EDWARD, S.P. and EWING, W.H. (1962). Identification and short slant. Inoculate by streaking on surface and of Enterobacteriaceae. Burgess. Pub. Co. Minneapolis. stabbing deeply. It is advisable to use tubes with cotton EUROPEAN PHARMACOPOEIA.(2005) Supp. 5.8 § plugs, in order to allow a reoxidation of the indicator. If 2.6.13 Test for specified micro-organisms. EDQM. Stras- screw caps are used, they must be loose. boug E.U. Following will find the table of reading (observations) and FIL-IDF (1991) International Standard 93A. Milk and Milk interpretation of results in TSI Agar. Products. Detection of Salmonella species. HAJNA, A.A. (1945) Triple Sugar-Iron medium for the References identification of the intestinal group of bacteria. J.Bact. DOWNES, F.P. & K. ITO (2001) Compendium of Meth- 49:516-517 ods for the microbiological examination of Foods. 4th ed. HORWITZ, W. (2000) Official Methods of Analysis. 17th APHA. Washington DC. USA ed. AOAC International. Gaithersburg. Md. USA.

153 Triple Sugar Iron Agar (TSI AGAR) (Eur.Phar. Agar Medium M)

ISO 3560 Standard. (1975) Reference Method for the Detection of Salmonella in meat and meat products. ISO 6579 Standard. (2002) Microbiology of foods and animal feeding stuffs - Horizontal method for the detection of Salmonella spp. ISO 6785 Stanbdard (2001) Milk and milk Products – Detection of Salmonella spp. ISO 10272 Standard (1995) Microbiology of foods and animal feeding stuffs - Horizontal method for the detection of thermotolerant Campylobacter. ISO 21567. Standard (2004) . Horizontal method for the detection of Shigella ssp. KRUMWIEDE, C. & L. KOHN (1917) A triple sugar modi- ﬁcation of the Russell Double Suigar Medium. J. Med. Res. 37:225-229 US PHARMACOPOEIA (2002)<61> Microbial Limit Tests. 25th ed. US Phamacopeial Convention Inc. Rock- ville. Md. USA

Todd Hewitt Broth

Ref. 02-191 as an alternative type in epidemiologic studies of group A streptotocci as well as pathogenic microorganisms. Speciﬁcation With the addition of 15g/L of agar, medium can be solidi- Liquid culture medium for the propagation of beta-hemo- ﬁed and then it is an excellent substrate for the produc- lytic streptococci and for studies about serologic typing. tion of capsules in streptococci.

Formula (in g/L) References Meat extract ...... 10,0 TODD, E.W. and HEWITT, L.F. (1932) A new culture Casein peptone ...... 20,0 medium for the production of antigenic streptococcol Glucose ...... 2,0 haemolysin.J. Path. Bact. 35:973-974 Sodium bicarbonate ...... 2,0 MacFADDIN, J. (1985) Media for isolation-cultivation- Sodium chloride ...... 2,0 identiﬁcation-maintenance of medical bacteria. Vol. 1. Disodium phosphate ...... 0,4 Williams & Wilkins. Baltimore. USA. Final pH 7,8 ± 0,2 ATLAS, R.M. and L.C. PARKS (1993) Handbook of Microbiological Media. CRC Press Inc.London ISENBERG H.D. (1992) Clinical Microbiology Proce- Directions dures Handbook. ASM. Washington DC. Dissolve 36,4 g of powder in 1 L of distilled water and sterilize by autoclaving at 121°C for 15 minutes.

Description This classical medium formulation has been modiﬁed to achieve optimal results in the growth and production of hemolysins, which are not inhibited due to the high buffer composition of the medium. Many ofﬁcial organisations, such as APHA, have recommended this medium

154 Tryptic Soy Media

Tryptic Soy Agar (TSA) 9.- Chromogenic pseudomonads frequently produce (Eur. Phar. Agar Medium B) pigmentation on the TSA and are therefore easily recognized. (Casein Soybean Digest Agar) 10.- It is widely used for testing contaminated samples. A vast bibliography documents its applications in the Ref. 01-200 food industry. 11.- It has been frequently used in the Health industry to Specification produce antigens, toxins,etc... General purpose solid medium containing animal and 12.- Its simple and inhibitors-free composition makes it plant peptone according ISO 9308-1 standard. suitable for the detection of antimicrobial agents in food and other products. Formula (in g/L) 13.- A balanced and highly nutrient value together with a lack of fermentable carbohydrates make this medium Casein peptone ...... 15,0 one of the most recommended for the strain mainte- Soy peptone ...... 5,0 nance. Sodium chloride ...... 5,0 Agar ...... 15,0 Final pH 7,3 ± 0,2 Tryptic Soy Broth (TSB) (Eur. Phar. Broth Medium A) Directions (Casein Soybean Digest Broth) Mix 40 g of powder in 1 L of distilled water. Let it soak and bring to the boil to dissolve the agar. Sterilize by Ref. 02-200 autoclaving at 121°C for 15 minutes. Specification Description Highly nutrient liquid medium for the general purposes, TSA is a widely used medium containing two peptones formulated according to USP, FDA and Eur. Phar. regula- which support the growth of a wide variety of organisms, tions. even that of very fastidious ones such as Neisseria, Listeria, Brucella, etc. It is frequently used for routine diagnostic purposes due to its reliability and its easily Formula (in g/L) reproducible results. Casein peptone ...... 17,0 The following list includes some of its most common Soya peptone ...... 3,0 applications: Sodium chloride ...... 5,0 Dipotassium phosphate ...... 2,5 1.- Sensitivity testing either by the Kirbky-Bauer system Dextrose ...... 2,5 or by following the WHO guidelines. Both the sys- Final pH 7,3 ± 0,2 tems recommend the use of the Mueller Hinton Agar (Ref. 01-136) for verification purposes. Directions 2.- The medium provides, with added blood,perfectly de- Dissolve 30 g of powder in 1 L of distilled water and fined hemolysis zones, while preventing the lysis of sterilize by autoclaving at 121°C for 15 minutes. the erythrocytes due to its sodium chloride content. 3.- It can be used for the preparation of an exceptionally Description nutrient ‘chocolate´ agar, thanks to the richness of its The Tryptic Soy Broth was initially developed for the peptones. cultivation of very fastidious microorganisms without the

4.- In a reducing environment or with a CO2 enriched addition of serum, blood or any other enrichment agent. atmosphere, its plates provides an excellent medium As a general purpose culture medium it supports the for the isolation of Brucella and Neisseria . It may be growth of most organisms, both aerobes and faculta- made selective by using certain additives. tives, even if their requirements are high. Due to its high 5.- Most streptococci grow in this medium though clear vitamin content the development of Brucella, Pasteurel- differences can be observed from one species to la and Streptococcus is perfectly viable, moreover a another. CO2 enriched atmosphere can further favour it. 6.- The Tryptic Soy Agar is the selective medium for the In anaerobic conditions this broth will easily bear the count of urine samples although the differentiation growth of Bacteroides and Clostridium species. For this must be done on selective differential media. purpose, the best results can be obtained by adding 7.- Several tests for the differentiation and identiﬁcation 0.3% agar and 0.05% sodium azide for Clostridium. of staphylococci can be obtained in this medium, The Tryptic Soy Broth’s superior growth-promoting prop- provided with suitable additives. erties makes it particularly suitable for the tube dilution 8.- Yeast, particularly Candida species, can grow in this method for antibiotic sensitivity testing. It also achieves medium forming very characteristic colonies. good results in the detection of gram-positive cocci. The broth can be used for bile solubility testing in pneumo-

155 Tryptic Soy Media cocci, and also used for catalase and coagulase assays Tryptic Soy Broth w/o Dextrose and for the preparation of hypersaline broths. (TSB w/o Dextrose) It is a most suitable medium for the preparation of antigens and toxins in bacteria, moulds and yeasts. TSB is used as a primary enrichment medium for food Ref. 02-227 examination. In the dairy industry it is employed for testing resazurine reduction. Specification The medium is not suitable for maintenance purposes Liquid culture medium for the massive production of since carbohydrate fermentation liberates many acids spores of B. stearothermophilus for the inhibitory sus- which may threaten the organisms’ viability. Therefore, tances in food acc. FDA-BAM though it allows the growth of streptococci and Neisse- ria, these species tend to die if repeatedly subcultured in Formula (in g/L) this medium. Such fastidious organisms are best main- Tryptone ...... 17,00 tained on Cystine Tryptone Fluid Medium (CTA) (Ref. Soy peptone ...... 3,00 03-045) or even TSA (Ref. 01-200) if it is not suitable or Sodium chloride ...... 5,00 convenient to the use of solid media. Dipotasium phosphate ...... 2,50 Final pH 7,3 ± 0,2 References US PHARMACOPOEIA (2002) 25th ed. <61> Microbial Directions Limit Tests. US Phsarmacopoeial Convention. Rockville. Disolve 27,5 g of powder in 1 L of distilled water, heat- MD. ing if necessary. Distribute in suitable containers and EUROPEAN PHARMACOPOEIA (2002) 2.6.13 Tests sterilize in autoclave at 121ºC for 15 minutes. for Specified Microorganisms. 4th Ed. Suppl.4.2 EDQM Council of Europe Strasbourg. Description ATLAS, R.M., & LC. PARKS (1993) Handbook of Micro- TSB w/o Dextrose is produced according the formulation biological Media. CRC Press, Inc.London from Bacteriological Analitycal Manual of Food and Drug DOWNES, F.P. & K. ITO (2001). Compendium of Meth- Administration for the massive production of spores of ods for the Microbiological Examination of Food, 4th Bacillus stearothermophilus used to determine the pres- ed,ASM,Washington,D.C. ence of inhibitory sustances in milk and dairy products. PASCUAL ANDERSON, MªRª (1992) Microbiologia This medium is not recommended for sugars fermenta- Alimentaria. Diaz de Santos, S.A.Madrid,. tion studies because the great amount of fermentable FDA (1998) Bacteriological Analytical Manual. 8th ed. carbohidrates in the soy peptone. Revision A. AOAC International. Gaithersburg. MD ISO 9308-1 Standard (2000) Water Quality - Detection and enumeration of E.coli and coliform bacteria. Mem- References brane filtration method. MATURIN, L.J. (1998) Inhibitory substances in milk. Qualitative Method II: B. stearothermophilus disk assay. en FDA Bacteriological Analitical Manual. 8th Ed. Revi- sion A. AOAC International Inc. VA.

156 Tryptone Bile Agar

Ref. 01-526 The indol producer microorganisms other than E. coli are inhibited by the bile salts and the incubation tempera- Speciﬁcation ture, but in sugar-rich samples the indol production can Selective solid medium for the rapid enumeration of be inhibited due to the sugar concentration interferes the Escherichia coli according ISO 9308-1 standard. tryptophanase synthesis.

Formula (in g/L) Technique Tryptone ...... 20,00 A cellulose acetate membrane of 0,45 ìm pore is extend- Bile salts #3 ...... 1,50 ed on the surface of the medium. A 0,5 mL of the sample Agar ...... 15,00 dilution (with Tryptone Water Ref. 03-156) inoculum is Final pH 7,2 ± 0,2 spreaded on the membrane and the plates are incubate in upright position at 44±1ºC for 18-24 hours. After incubation the membrane is immersed in indole Directions reactive using the petri dish cover as container and Suspend 36,5 g of powder in 1 L distilled water and bring exposed the direct sunlight for 5 minutes or to the Wood to the boil. Distribute into suitable containers and steri- lamp for 10 minutes. The indole positive colonies turns lize at 121ºC for 15 minutes. to the reddish colours (pink to deep red). The results are expressed as number of Escherichia coli per g or mL of Description sample. This medium is formulated according the Anderson & Baird-Parker Direct Plating Method (DPM) for the rapid References enumeration of Escherichia coli in raw meats. The ANDERSON, J.M. & A.C. BAIRD-PARKER (1975) Appl. method is based on the characteristically production of Bact. 39:111-117 indol from tryptophane when the bacteria growth at 44ºC International Commission on Microbiological Speciﬁca- over a cellulose acetate membrane in the surface of the tions for Foods (1979) Can. J. Microbiol. 25:1321-1327 Tryptone bile agar. ISO 9308-1 Standard (2000) Water Quality - Detection The International Commission on Microbiological Stand- and enumeration of E.coli and coliform bacteria. Mem- ards for Food noticed that he DPM was lesser variable brane ﬁltration method. and offer a better recovery and rapidity than the MPN method for frozen samples of meat. The ISO Standard 6391:1988 also uses this medium for the E. coli enumeration.

157 Tryptone Glucose Extract Media

Tryptone Glucose Extract Agar APHA-AWWA-WEF (1998) Standard Metods for the (TGE Agar) Examinationof Water and Wastewater. 20th ed. APHA. Washington HORWITZ, W. (2000) Ofﬁcial Methods of Analysis. Ref. 01-082 AOAC International. Gaitherburg. MD

Specification Tryptone Glucose Extract Broth Plate count medium for the milk and dairy products, ac- for Membrane Filtration (m-TGE cording to Standard Methods for Dairy Products. Broth) Formula (in g/L) Meat extract ...... 3,0 Ref. 02-082 Casein peptone ...... 5,0 D (+) Glucose ...... 1,0 Specification Agar ...... 15,0 Non-selective liquid medium for enumerating microor- Final pH 7,0 ± 0,2 ganisms by membrane filtration method.

Directions Formula (in g/L) Add 24 g of powder in 1 L of distilled water. Heat to the Tryptone ...... 10,00 boil by constantly stirring. Dispense in suitable contain- Meat extract ...... 6,00 ers and sterilize by autoclaving at 121°C for 15 minutes. Dextrose ...... 2,00 Final pH 7,0 ± 0,2 Description Solid medium Tryptone Glucose Extract was adopted Directions long ago as an alternative to Nutrient Agar acc. APHA Dissolve 18 of powder in 1 L of distilled water, heating (Ref. 01-144) and Nutrient Agar acc. British Pharmaco- if it is necessary. Distribute in suitable containers and poeia (Ref. 01-140) for milk bacteria enumeration, being sterilize in autoclave at 121ºC for 15 minutes. a complement to Plate Count Agar (Ref. 01-161). Description Technique This medium is a liquid nutritive substrate that can be For the enumeration purposes, it is suggested to use the used in the colony count by the membrane filtration poured plate method, and incubation at 30-32°C for 48 method for the absorbent pad impregnation The broth hours. If the dilutions in the plate is more than 10% it is has the same formulation as Tryptone Glucose Extract advisable to add milk to the medium. To do this, prepare Agar , except that the broth contains no agar and the the supension of skimmed milk (Ref. 06-019) separately, ingredients are at twice the concentration. and sterilize it for 10 minutes at 118°C. Autoclaving must be as short as possible. Homogenize with the culture Technique medium which is sterilized and cooled to 50°C. The use The sample is filtered through a membrane. In a plate of natural milk is not recommended due the wide varia- the absorbent pad is impregnated with the medium tion . avoiding any excess. The membrane is transferred on Medium must be quickly poured into Petri dishes be- the pad and the complete system is incubated at 35±2ºC cause if it remains hot for too long, floccules and abnor- for 18-24 hours. After this time total colonies are counted mal precipitates may appear. If the sample under study and results recorded. is not diluted or the volume in the plate is more than 2 mL, it is not necessary to add the skimmed milk (Ref. 06- 019) because it is assumed that the sample provides the References required growth factors. DOWNES, FP. & K. ITO (2001) Compendium of methods for the microbiological examination of foods 4th ed APHA References Washington APHA-AWWA-WEF (1998) Standard Methods for the FDA.(1998) Bacteriological Analytical Manual 8th ed examination of water and wastewater. Revision A. AOAC International,Gaitherburg MD DOWNES, F.P. & K. ITO (2001) Compendium of methods for the Microbiological Examination of Foods, 4th Ed. APHA, Washington. MARSHAL, R.T.. (Ed.) (1992) Standard Methods for the Examination of Dairy Products. 16th Ed. APHA. Washing- ton.

158 Tryptone Phosphate Water (Buffered Peptone Water)

Ref. 02-277 water and the pH change absorbing capacity of the phosphate buffer. Speciﬁcation The composition of this diluent is made according the Dilution and nonselective pre-enrichment liquid medium speciﬁcation of the ISO Standard 6579 for the detection acc. to ISO 6579, 8523, 8261 and 6785 standards. of Salmonella in foods.

Formula (in g/L) References Peptone ...... 10,0 ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro- Sodium chloride ...... 5,0 biological Media,CRC Press, Inc.,London Disodium phosphate ...... 9,0 PASCUAL ANDERSON, MªRª (1992) Microbiologia Potassium phosphate ...... 1,5 Alimentaria. Diaz de Santos, S.A.,Madrid,. Final pH 7,0 ± 0,2 ISO 6579 Standard (2002). Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Salmonella spp. Directions ISO 8523 Standard (1991) General guidance for the Dissolve 25,5 g of powder in 1 L of distilled water. detection of Enterobacteriaceae with pre-enrichment. Distribute into suitable containers and sterilize in the ISO 8261 Standard (2001) Milk and milk products - autoclave at 121°C for 15 minutes. General guidance for the preparation of test samples for microbiological examination. Description ISO 6785 Standard (2001) Milk and milk products - De- This formulation of Tryptone Phosphate Water has the tection of Salmonella spp. advantages of the two classical diluents for food samples: it has the property of revitalization of the peptoned

Tryptone Sulﬁte Neomycin Agar (TSN Agar)

Ref. 01-195 included in the medium restrain the development of Cl.bifermentans and all the accompanying gram nega- Specification tive bacteria. Solid selective medium for Clostridium perfringens isola- The medium is especially suitable for the investigation of tion. food products, and it may be used in tubes as well as in plates. If the incubation is not performed in an anaerobic jar, thioglycolate buffered solution must be added or the Formula (in g/L) inoculated surface must be covered with a sterile layer of Casein peptone ...... 15,00 medium. Sodium sulfite ...... 1,00 Colonies of Cl.perfringens form very characteristic black Neomycin sulfate ...... 0,05 colonies that, if exposed to air, become decolourised by Polymyxin B ...... 0,02 oxidation. TSN have a very short storage period once Yeast extract ...... 10,00 prepared, so it is advisable to rehydrate or reconstitute it Ferric citrate ...... 0,50 in small amounts and use it on the day of its preparation. Agar ...... 13,50 Final pH 7,2 ± 0,2 References MARSHALL, R.S., STEENBERGEN, J.F., MacCLUNG, Directions L.S. (1955) Rapid Technique for the enumeration of Suspend 40 g of powder in 1 L of distilled water and Clostridium perfringens. Appl. Microbiol. 13:559-563. bring to the boil. Dispense in suitable containers and MOSSEL, D.A.A (1959) Enumeration of sulfite reducing sterilize by autoclaving at 121°C for 15 minutes. clostridia occurring in nfoods. J. sci. Food Agr. 10:662- To obtain better results, add 20 mL/L of a solution con- 669 taining 1 g/L dipotassium phosphate, 0,5 g/L sodium car- MacFADDIN, J.F. (1985) Media for Isolation-Cultivation- bonate and 1 g/L sodium thioglycollate just before use. Identification-Maintenance of Medical Bacteria,Williams & Wilkins. Baltimore,USA. Description ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro- This culture medium was formulated by taking the biological Media,CRC Press Inc.,London advantage of the tolerance of Cl. perfringens to the high concentration of sulfite, which apart from being an inhibitor agent, provides a strong reducing environment. Selection of Cl.perfringens is almost complete when it is incubated at 46°C, since neomycin and polymyxin

159 Tryptone Water (Peptone Water)

Ref. 03-156 As an alternative method, the Ehrlich’s Reagent (Ref. 06-024) could also show indole production. After 48 Speciﬁcation hours of incubation at 37°C, take 0,5 mL of growth and Substrate with low nutrient capacity, for the research of mix it with 0,5 mL of Ehrlich’s Reagent. Let them settle indole production in coliform micro-organisms according a few minutes. If it takes on a pink colour,then the test is ISO 7251 standard. positive. Colour appearance is accelerated if a few drops of saturated solution of potassium persulfate is added. Other authors prefer extraction and concentration of in- Formula (in g/L) dole with 1 mL of ether, and then observe on the extract Casein peptone ...... 10,0 with any of the reagents mentioned above. Sodium chloride ...... 5,0 Final pH 7,2 ± 0,2 References ATLAS, R.M.& L.C. PARKS (1993) Handbook of Micro- Directions biological Media,CRC Press Inc.,London Dissolve 15 g of powder in 1 L of distilled water and dis- DOWNES, F.P. & K. ITO (2001). Compendium of Meth- pense into suitable containers. Sterilize by autoclaving at ods for the Microbiological Examination of Food. 4th Ed. 121°C for 15 minutes. APHA,Washington APHA-AWWA-WEP (1998) Standard Methods for the Description examination of water and wastewater. 20th ed. APHA. The standard protocol requires to reinoculate one loop Washington, DC. from each suspected tube in 10 mL of Tryptone Water. ISO 7251 Standard (1993) General guidance for the Incubate for 48 hours at 44°C before investigating the enumeration of E.coli by the MPN technique. indole production with the Kovacs’ Reagent for indole (Ref. 06-018).

Tryptone Yeast Extract Agar

Ref. 01-590 36±2°C for 44±2 hours and the other one at 22°C for 3 days (68±4 hours). Speciﬁcation In order to achieve a good count, select plates with Solid medium for the enumeration of water microorgan- 30-300 colonies. Express the results as number of isms acc. ISO Standard 6222. colony forming units per milliliter (cfu/mL) of the sample for each temperature of incubation. If there are no colonies with the undiluted sample express the results as “no Formula (in g/L) detected in one mL”. If there are more than 300 colonies Tryptone ...... 6,0 in the highest dilution express the results as “>300/mL” Yeast extract ...... 3,0 Agar ...... 15,0 Final pH 7,2 ± 0,2 References ISO Standard 6222 Watewr Quality – Enumeration of culturable microorganisms. Colony count by inoculation Directions in a nutrient agar culture. Suspend 24 g of powder in 1 L of distilled water and ISO Standard 5667-2 (1991) Water Quality-Sampling bring to the boil. Distribute into containers and sterilize – Guidance on sampling techniques by autoclaving at 121°C for 15 minutes. ISO Standard 5667-3 (1996) Water Quality – Sampling. –Guidance on the presevation and handling of samples Description ISO Standard 6887 (1999) Microbiology- General This medium, formulated according to ISO Standard – Guidance for the preparation of dilutions for microbio- 6222, is for the enumeration of heterotrophic microor- logical examination. ganisms from water. ISO Standard 8199 (1988) Water Quality – General guide to the enumeration of microorganisms by culture. Technique From the water sample, obtained according the ISO Standard 5667-2 and 5667-3, make a decimal dilution bank (see ISO Standard 6887) with Ringer Solution (Ref. 6-073) and take aliquots to 2 parallel series of plates. Pour the Tryptone Yeast Extract Agar melted and cooled to 45°C, and homogenize with sample (see ISO Stand- ard 8199). Once solidiﬁed, incubate one of the series at

160 Tryptophan Broth

Ref. 02-418 Technique Medium is inoculated with the previously isolated culture, Specification and then incubated at 30-32°C for 24-48 hours. Liquid medium for the indole production according to Indole production is observed adding a few drops of ISO standard. Kovacs’ Reagent (Ref. 06-018) over the broth (with or without previous extraction) and shaking gently. Forma- Formula (in g/L) tion of a red ring indicates indole presence. Meat peptone ...... 10,0 L-Tryptophan ...... 1,0 References Sodium chloride ...... 5,0 VERORDNUNG über Trinkwasser und über Wasser für Final pH 7,2 ± 0,2 Lebensmittelbetriebe vom 12-12-1990. Bundesgesatbl. I. 2613-2619. Directions BUNDESGESMELHEITSAMT: Amtliche Samnulung von Untersuchungs verfahren nach #35LMBG. Beuth Verlag. Dissolve 16 g of powder in 1 L of distilled water. Distrib- Berlin-Köln. ute into suitable containers and sterilize in the autoclave ISO 9308-1 Standard (2000) Water Quality. Detection at 121°C for 15 minutes. and enumeration of Escherichia coli and coliform bacteria. Part 1: Membrane filtration method. Description ISO 6785 Standard (2001) Milk and milk products. De- This broth allows the indole production from the tryp- tection of Salmonella spp. tophan, and therefore it is suitable for the differentiation ISO 21567. Standard (2004) . Horizontal method for the and identification of coliforms from water and food. detection of Shigella ssp. Its formulation is according to the German standards for waters and foods.

Tryptose Media

Tryptose Agar Tryptose Broth

Ref. 01-197 Ref. 02-197

Speciﬁcation Speciﬁcation Solid medium for isolation, cultivation and differentiation Liquid culture medium for massive culturing of fastidious of Brucella, streptococci and fastidious pathogens. microorganisms.

Formula (in g/L) Formula (in g/L) Tryptose ...... 20,000 Tryptose ...... 20,000 Dextrose ...... 1,000 Dextrose ...... 1,000 Sodium chloride ...... 5,000 Sodium chloride ...... 5,000 Thiamine HCl ...... 0,005 Thiamine HCl ...... 0,005 Agar ...... 15,000 Final pH 7,3 ± 0,2 Final pH 7,2 ± 0,2 Directions Directions Dissolve 26 g of powder in 1 L of distilled water, heating Suspend 41g of powder in 1 L of distilled water and heat up if necessary. Dispense into suitable containers and to boiling. Dispense in tubes or ﬂasks and sterilize it in sterilize by autoclaving at 121°C for 15 minutes. the autoclave at 121°C for 15 minutes. To obtain better results, add to the molten medium, 20 Description mL/L of a solution composed by dipotassium phosphate Tryptose culture media are suitable, essencially for the 1 g/L, sodium carbonate 0,5 g/L and sodium thioglyco- growth of fastidious strains, such as Listeria, Pasteurella, late 1 g/L just before using the medium. Brucella, etc. despite the fact that nowadays it seems there is a trend towards the use of more deﬁned media such as Brucella Agar (Ref. 01-042) and Broth (Ref.

161 Tryptose Media

02-042), which provide better results. Tryptose Agar and Description Broth have been recommended by several National and Tryptose Phosphate Broth is a recommended medium International Organisations for Brucelosis Control and for for the cultivation and propagation of microorganisms the maintenance, propagation and cultivation of stand- that have strong needs, such as streptococci, menin- ardized strains. They may be successfully used to dif- gococci, and Brucella. It has also been also used to ferentiate the several types of Brucella with the addition determinate antibiotic sensitivity testing by the dilution in of suitable indicators. In this case, its use is analogous to tube method. the Brucella Broth (Ref. 01-042). This medium has been used as primary diluent and With the adequate additives (crystal violet, sodium azide emulsifier in dairy products for determination of Brucella, etc.) the media may become very selective and efficient, but it is really effective for the cultivation of many strepto- and their nutrient conditions may be improved if citrate cocci and to test the bile solubility of these organisms. and blood are added, although glucose is not very suit- When it is used to isolate streptococci, it is suggested to able for observing hemolysis. add 0,1% Agar to render it into a fluid medium. Should a very selective medium be desired, add 2,5% of sodium References azide. To get a solid medium, add 1,5% of Agar. CASTAÑEDA, M.R. (1947) A practical method for rutine blood cultures in brucellosis. Proc. Soc. Exp.Biol. Med. References 64:114-115 GINSBERG, H.S. (1955) Tryptose Phosphate Broth as HAUSSLER, W.J. (1976) Standard Methods for the Supplementary Factor for Maintenance of Hella Cell Tis- Examination of Dairy Products,9th Ed. APHA. sue Cultures. Proc. Soc. Exper. Biol. Med. 89(1):66-71. MARSHALL, R.T.. (1992) Standard Methods for the Ex- WAISBREN, B.A. (1951). The Tube Dilution Method of amination of Dairy Products. 16th Ed. APHA. Washington. Determining Bacterial Sensitivity to Antibiotics. Am. J. RENNER, E.D., K.J. McMAHUN (1981) Brucellosis in Di- Clin. Path 21:884. agnostic Procedures for Bacterial Mycotic and Parasitic BALOWS, A., W.J. HAUSSLER (1981) Diagnostic Pro- Infections. 6th Ed.,APHA, Washington.DC cedures for Bacterial Mycotic and Parasitic Infections. ATLAS, R.M. & LC. PARKS (1993) Handbook of Micro- 6thEd,APHA. Washington. biological Media. CRC Press Inc.,London. ATLAS, R.M., & J.W. SNYDER (1995) Handbook of Me- DOWNES,F.P. & K. ITO (2001) Compendium of Meth- dia for Clinical Microbiology. CRC Press. London ods for the Microbiological Examination of Food. 4rdEd. FDA (1998) Bacteriological Analytical Manual. 8th ed. APHA.Washington Revision A. AOAC International. Gaithersburg. MD. FDA (1998) Bacteriological Analytical Manual. 6th ed. Revision A. AOAC International. Gaithersburg. MD MURRAY,P.R., E.J. BARON, M.A PFALLER, F.C. TENO- VER, & R.H. YOLKEN (1995) Manual of Clinical Microbi- ology. 6th ed. APHA. Washington DC .

Tryptose Phosphate Broth

Ref. 02-199

Speciﬁcation Liquid culture medium, with glucose and buffer, for cultivation of fastidious microorganisms and for tissues culture media.

Formula (in g/L) Tryptose ...... 20,0 Dextrose ...... 2,0 Sodium chloride ...... 5,0 Disodium phosphate ...... 2,5 Final pH 7,3 ± 0,2

Directions Dissolve 29,5 g of powder into 1 L of distilled water. Dis- pense into suitable containers and sterilize by autoclaving at 121°C for 15 minutes.

162 Tryptose Lauryl Sulfate Media

Tryptose Lauryl Sulfate Broth References F.D.A. (1998) Bacteriological Analytical Manual. 8th ed. Ref. 02-108 Revision A. AOAC International Gaitherburg, MD. FIL-IDF Standard 73B (1998) Milk and milk products. Specification Enumeration of coliforms. IDF. Brussels. DOWNES, F.P. & K. ITO (2001) Compendium of Meth- Liquid medium for the detection and enumeration of ods for the Microbiological Examination of Food. 4th. ed. coliform bacteria according IDF-FIL 73B and ISO 4831 APHA. Washington. and 7251 standards. MARSHALL R.T. (1992) Standard Methods for the examination of dairy products. 16th ed. APHA. Washington Formula (in g/L) APHA-AWWA-WPCF (1995) Standard Methods for the Tryptose ...... 20,00 examination of water and wastewater. APHA. Washing- Sodium Lauryl sulfate ...... 0,10 ton Lactose ...... 5,00 HORWITZ, W. (2000) Official methods of Analysis.17th Dipotassium phosphate ...... 2,75 ed. AOAC International. Gaithersburg. MD. Monopotassium phosphate ...... 2,75 ISO 4831 Standard (1991) General guidance for the Sodium chloride ...... 5,00 enumeration of coliforms - MPN technique. Final pH 6,8 ± 0,2 ISO 7251 Standard (1993) General guidance for enumeration of E.coli by MPN technique. Directions Dissolve 35,6 g of powder in 1 L of distilled water. Dis- Tryptose Lauryl Sulfate tribute into tubes or containers fitted with the inverted Durham tubes (for gas). Sterilize at 121°C for 15 min- Mannitol Tryptophan Broth utes. As for the double concentration medium, dissolve 71,2 g/L and proceed as indicated above. Ref. 02-460 Preferably store the broth at room temperature, and use screw-capped bottles to prevent evaporation of water. Specification Refrigerated broth generally becomes cloudy or forms Liquid medium for the production of indole and gas in precipitates but clears at incubation at room tempera- a single tube, according to the ISO 9308-1 and 9308-2 ture. However, clarity is not important as only the gas standards. production is significant criterion. Formula (in g/L) Description Tryptose ...... 20,00 Laurylsulfate broth is used for MPN Presumptive Test Mannitol ...... 5,00 of coliforms in water and sewage, confirmatory test of Sodium chloride ...... 5,00 lactose fermentation with gas production for milk and Monopotassium phosphate ...... 2,75 detection of coliforms in food.The high nutrient quality Dipotassium phosphate ...... 2,75 and the presence of phosphate buffer in this medium en- Sodium Lauryl sulfate ...... 0,10 sures rapid growth and increased gas production, even L-Tryptophan ...... 0,20 by slow lactose-fermenting coliforms. Final pH 6,8 ± 0,2 This medium can be used as Presumptive broth for E.coli (by fluorescent reaction) if before sterilization MUG (Ref. 06-102CASE) is added. Directions Dissolve 35,8 g of powder in 1 L of distilled water, heating up if necessary. Distribute in tubes containing Technique Durham´s tubes and sterilize by autoclaving at 121ºC for If the volume of sample to inoculate is substantial, then 15 minutes. Do not overheat. reconstitute the medium at such a concentration which would remain normal, once the sample has been added to it. Description Incubate at 37°C for 24-48 hours. Lactose fermenta- This broth is proposed in the ISO 9308-1 standard as an tion within 48 hours, shown by the appearance of gas in alternative medium for the production of indole and gas the Durham tubes , indicates the presence of coliform in a single tube and to confirm the presence of thermobacteria. tolerant coliforms and the presumptive presence of E.coli Verification can be done by the isolation and identifica- in the water sample. tion of coliforms on an appropiate medium.

163 Tryptose Lauryl Sulfate Media

Technique Tubes with medium are inoculated from suspicious colonies on the already incubated membrane and then are incubated at 44ºC for 24 hours. Gas production, that appears in the Durham tubes, conﬁrms the presence of thermotolerant coliforms. If after the addition of 0,2-0,3 mL of Kovacs Reagent (Ref. 06-018) a cherry red colour appears on the top surface of the medium (Indole +), a presumptive presence of E.coli is considered and it should be conﬁrmed with other tests.

References ISO Standard 9308-1 (1990) Water Quality Detection and Enumeration of coliform organisms, thermotolerant coliform organisms and presumptive E.coli. Part 1. Mem- brane ﬁltration method. Part 2. MPN method.

164 Tryptose Sulﬁte Cycloserine Agar Base (TSC Agar Base)

Ref. 01-278 sufite reduction, growth at 46°C and cycloserine resistance. Specification Nevertheless, it has to be noted that cycloserine does Solid selective and differential medium for isolation and not tolerate temperatures above 100°C and its stability presumptive identification of Clostridium perfringens ac- in a solution is very restrained, even then it is used in cording to ISO 7937 Standard. alkaline media. Therefore, it is advisable to prepare the exact number of plates that are going to be used. Anyway, if it is desired, an active solution of cycloserine Formula (in g/L) in phosphate buffer at pH 8,0 may be prepared (Dipotas- Tryptose ...... 15,00 sium phosphate 16,73 g/L and monopotassium phophate Soya peptone ...... 5,00 0,52 g/L) and if it is maintained refrigerated, it can be Yeast extract ...... 5,00 used for approx. 5 days. Sodium metabisulfite ...... 1,00 Ferric ammonium citrate ...... 1,00 Agar ...... 20,00 Technique Final pH 7,6 ± 0,2 The standard procedure recommends surface inoculation of the samples or their dilutions, and once absorbed, to pour a second layer as cover and seal for anaerobio- Directions sis. After an incubation at 46°C for 18-20 hours, proceed Suspend 47 g of powder in 1 L of distilled water and let it to enumerate the black colonies that appear in the plate. soak . Heat to boiling and distribute into suitable containers, but not more than 250 mL in each one. Sterilize in the autoclave at 121°C for 15 minutes. Let it cool to 60°C References and add 1 flask of CycloserineSelective Supplement SMITH, L.D. (1981) Clostridial Anaerobic Infections, in (Ref. 06-116CASE) to every 250 mL of medium. Mix well Diagnostic Procedures for Bacterial,Mycotic and Para- th and pour it into plates. If it is desired to include egg yolk, sitic Infections. 6 Ed. APHA,Washington. then add Egg Yolk Sterile Emulsion (Ref. 06-016) in a ATLAS, R.M. & L.C. PARKS (1993) Handbook of Micro- concentration of 80 mL/L, simultaneously to the antibi- biological Media. CRC Press Inc.,London otic, DOWNES, F:P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Food. 4rd ed. APHA Washington Description ISO 7937 Standard (2004) Microbiology of food and The medium is a modification of the classical TSN Agar animal feeding stuffs- Horizontal method for enumeration (Ref. 01-195) in which the traditional antibiotics, polymix- of Clostridium perfringens - Colony-count technique. in and neomycin have been replaced by cycloserine. DIN Standard 10165. Referenz Verfahren fur Bes- Cycloserine has been found more selective for Clostrid- timmung von Clostridium perfringens. Fleisch und ium perfringens, and it seems also to reduce the trend to Fleischerzeugnissen. produce diffuse blackening. On the other hand, Clostrid- FDA (1998) Bacteriological Analytical Manual 8th ed. ium perfringens is more resistant to cycloserine than to Revision A. AOAC International. Gaithersburg. MD. sulfadiazine, polymixin and neomycin, which permits a ISO 6461-2:1986 Standard Water Quality - Detection better dosage.The medium has sodium metabisulfite and and enumeration of the spores of sulfite - reducing ferric ammonium citrate to manifest the reducing capac- anaerobes (Clostridia) - Part 2: Method by MF. ity of sulfite, and in this way, three differential characteristics of this anaerobic species may be verified with just one assay. These characteristics are

Left and center: Clostridium perfringens ATCC 13124; right: control.

165 Urea Media

Urea Agar Base ISO 6340 Standard (1995) Water Quality - Detection of acc. to Christensen Salmonella spp. ISO 6579 Standard (2002) Microbiology of food and animal feeding stuffs. Horizontal method for the detection of Ref. 01-261 Salmonella spp. DIN Standard 10160. Untersuchung von Fleisch und Specification Fleischerzeugnissen. Nachweiss von Salmonellen. Ref- Solid medium for detection of urea lysis, according to erenzverfahren. ISO 6579, 6340 and 6785 standards and DIN 10160 FIL-IDF 93 Standard (2001) Detection of Salmonella. standard. Urea Broth Base Formula (in g/L) Gelatin peptone ...... 1,000 Ref. 02-202 Dextrose ...... 1,000 Sodium chloride ...... 5,000 Monopotassium phosphate ...... 2,000 Specification Phenol red ...... 0,012 Liquid diagnostic medium according to Rustigian and Agar ...... 15,000 Stuart formulation. Final pH 7,0 ± 0,2 Formula (in g/L) Directions Monopotassium phosphate ...... 9,10 Suspend 24 g of powder in 950 mL of distilled water and Disodium phosphate ...... 9,50 bring to the boil. Sterilize in the autoclave at 121°C for Yeast extract ...... 0,10 15 minutes. Let it cool to 50-55°C. Add 50 mL of Urea Phenol red ...... 0,01 Sterile Solution 40% (Ref. 06-083) and mix well. Distrib- Final pH 6,8 ± 0,2 ute aseptically in tubes and let them solidify in slanted position. Directions Dissolve 19 g of powder into 950 mL of distilled water Description and sterilize by autoclaving at 121°C for 15 minutes. Let Urea Agar complies with Christensen’s specifications, it cool to 50-55°C and then add 50 mL of Urea Sterile and it is recommended for the detection of urolytic or Solution 40% (Ref. 06-083). Mix well and dispense in urea degrading microorganisms, especially Enterobacte- hemolysis tubes (3,0 mL/tube). riaceae, although it can be used also with gram positive bacteria. Description According to Rustigian and Stuart, this Urea Broth is Technique excellent for diagnosing enterobacteria, since within this Pure culture is inoculated by surface streaking, and then family, only Proteus may alkalinize the medium over pH incubated at 37°C. Generally, organisms with strong 8,1. Despite the fact that some authors prefer a buffer of urease activity can be read after 3-5 hours. potency 10 or 100 times lower to obtain faster results for Reaction is evident as the medium changes its colour. It saving the time (about 2 hours) does not compensate for turns form orange to pink-fuchsia, due to a strong alkali- the instability of the medium. nization produced by ammonia release. Urease production is shown by the indicator turning to dark pink, produced by strong alkalinization by ammonium. With plenty of inoculum (2-3 loops in 3-5 mL of References medium), Proteus produces the colour change after 6-8 CHRISTENSEN, W.B. (1946) Urea decomposition as hours, meanwhile other positive enterobacteria need up means of differentiating Proteus and Paracolon cultures to 24-48 hours. from each other and from Salmonella and Shigella types. J.Bact. 52:461. EDWARDS and EWING (1962) Identification of Entero- References bacteriaceae. Burgess Pub. Co. RUSTIGIAN, R., STUART, C.A. (1941) Decomposition of ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro- urea by Proteus. Proc. Soc. Exp. Biol. Med. 47:108 DOWNES, FP & K ITO (2001) Compendium of Meth- biological Media. CRC Press Inc.London rd DOWNES, F.P. & K. ITO (2001) Compendium of methods for the Microbiological Examination of Food, 4 ed. th APHA,Washington. odscfor the Microbiological Examination of Foods 4 ed. th APHA. Washington FDA (1998) Bacteriological Analytical Manual. 8 ed. MARSHALL, R.T. (1992) Standard Methods for the ex- Revision A. AOAC International. Gaithersburg. MD amination of Dairy Products. 16th ed APHA. Washington PASCUAL ANDERSON, MªR. (1992) Microbiología DC Alimentaria. Diaz de Santos, S.A.,Madrid. ISO 6785 Standard (2001) Milk and milk products - De- tection of Salmonella spp.

166 Violet Red Bile Media

Violet Red Bile Agar (VRB Agar) References DOWNES, F.P. & K. ITO (2001). Compendium of Meth- Ref. 01-164 ods for the Microbiological Examination of Food. 4rd ed. APHA, Washington. DC Specification MARSHALL, R.T. (1992) Standard Methods for the Ex- amination of Dairy Products,16thEd. APHA, Washington. Selective and differential agar medium for the detection DC and enumeration of coliforms in milk and other dairy ICMSF (1978). Microorganisms in Food, University of products, according to APHA, ICMSF, FIL-IDF and ISO Toronto Press. standard. ISO (1986) Standard 5541-1 Milk and Milk Products. enumeration of coliforms. Colony count technique at Formula (in g/L) 30ºC Yeast extract ...... 3,000 FIL-IDF. (1998) Standard 73B. Enumeration of coliform Gelatin peptone ...... 7,000 bacteria. Bile salts #3 ...... 1,500 PASCUAL ANDERSON, MªR. (1992) Microbiología Lactose ...... 10,000 Alimentaria. Diaz de Santos, S.A.,Madrid,. Sodium chloride ...... 5,000 Neutral red ...... 0,030 Violet Red Bile Dextrose Agar Crystal violet ...... 0,002 Agar ...... 13,000 (VRBD Agar) Final pH 7,4 ± 0,2 Ref. 01-295 Directions Suspend 39,5 g in 1 L of distilled water. Bring to the Specification boil and distribute into final containers. Sterilization at Solid medium for the enumeration of enterobacteria ac- 121°C for 15 minutes is optional, but If the medium is cording ISO 8523 standard. to be used on the same day of preparation it need not be sterilized. No sterilization improves the recovery of Formula (in g/L) stressed microorganisms. Yeast extract ...... 3,000 Gelatin peptone ...... 7,000 Description Bile salts #3 ...... 1,500 The Violet Red Bile Agar corresponds to the classic D (+) Glucose ...... 10,000 formulation of standardized media for the screening of Sodium chloride ...... 5,000 coliforms in milk and other dairy products.This medium Neutral red ...... 0,030 has been adopted for the enumeration of coliforms as Crystal violet ...... 0,002 well as for differentiating between lactose fermenting and Agar ...... 13,000 non-lactose fermenting organisms, due to its contents of Final pH 7,4 ± 0,2 crystal violet and bile salts, whose inhibiting or selecting properties have been widely confirmed. Directions This medium can be used as Presumptive medium for Suspend 39,5 g in 1 L of distilled water and let it soak. E.coli (by fluorescent reaction) if before sterilization Bring to the boil and sterilize by autoclaving at 121°C for MUG (Ref. 06-102CASE) is added. 15 minutes. If the medium is to be used on the same day of preparation it need not be sterilized. Prolonged heat- Technique ing in thermostatic bath could cause slight precipitates. The recommended procedure is the massive inoculation directly on Petri dishes, with the molten agar cooled Description to 47°C. Observations can be read after 24 hours of This medium is a modification of the Violet Red Bile Agar incubation at 37°C. (Ref.1-164) and the MacConkey Agar (Ref.1-118) as The size of the colonies ranges from 2 to 5 mm, depend- described by Mossel et al. These authors proved that the ing on the amount per plate. The enterococci that might addition of glucose to the Violet Red Bile Agar favoured eventually develop will appear small in size and pink both the growth of the most fastidious enterobacteria coloured. Lactose fermenting enterobacteria acquire a and the recovery of those having suffered from adverse dark red colour with a clearing zone around them, while conditions. Later on, Mossel himself realized that by lactose non-fermenting ones form colourless colonies. removing the lactose and keeping the glucose, the medium’s efficiency remained stable. Furthermore, an economic improvement occurred since the same amount of product allows the reconstitution of more litres of the medium.

167 Violet Red Bile Media

This medium can be used as presumptive medium for cially used in the recovery of process-stressed bacteria E.coli (by fluorescent reaction) if before sterilization using a progressive enrichment technique. MUG (Ref. 06-102CASE) is added. This medium can be used as presumptive medium for E.coli (by fluorescent reaction) if before sterilization Technique MUG (Ref. 06-102CASE) is added. The Violet Red Bile Dextrose Agar is widely used in the analysis of food, medicines and cosmetics. It is particu- Technique larly indicated for the recovery of bacteria which have Sample is diluted 1:10 in Lactose Broth (Ref. 02-105) been damaged during preparation. In such cases, a and incubate 2-5 hours at 35-37 ºC. Then a volume of progressive enrichment is recommended in TSB (Ref. this pre-enrichment is ten fold dilute in EE Broth (Ref. 02-200) first and in EE Broth (Ref. 02-064) next. Once 02-064) and incubate at 35-37ºC for 18-24 hours. From the enriched culture is ready it can be inoculated by this enrichment the surface of several plates of VRBDL profound inoculation in tubes or by isolation in Violet Red Agar are inoculated. The product passes the test if after Bile Dextrose Agar plates. 18-24 hours of incubation at 35-37ºC there is no growth For the count of enterobacteria, the technique to use will of gram negative bacteria in any plate. be the massive inoculum described for the Violet Red In the surface of the VRBDL Agar the Enterobacteriace- Bile Agar. ae colonies are deep purple in colour surrounded by a Observations can be read after 24 hours of incubation clearing zone. Sometimes are present little colonies from at 31°C. Enterobacterial colonies form an intense purple Pseudmonas or Aeromonas that can be easy differenti- colouring surrounded by a clearer zone . If enterococci ated by the oxidase test. colonies eventually develop, then they will be small and pink coloured. References MOSSEL, D.A.A., MENGERINK and SCHOLTS H.H. Violet Red Bile Lactose (1962) Use of a modified MacConkey Agar medium for Dextrose Agar (VRBLD Agar) the selective growth and enumeration of all Enterobacte- riaceae. J. Bact. 84:381. (Eur. Phar. Medium F) MOSSEL, D.A.A., VISER, M. and CORNELISSEN, A.M.R. (1963) The examination of food for Enterobacte- Ref. 01-220 riaceae using a test of the type generally adopted for the detection of Salmonellae. J. Appl. Bact.(26) 444-452. Specification MOSSEL, D.A.A. (1985) Media for Enterobacteriaceae. Solid selective medium for the detection of Enterobacte- Int. J. Food Microbiol. 2:27-35 riaceae according the European Pharmacopoeia. ISO 5552 Standard (1997) Meat and Meat Products. Detection and enumeration of Enterobacteriaceae Fórmula (in g/L) without resuscitation. MNP technique and colony-count technique. PASCUAL ANDERSON, MªR. (1992) Microbiología Yeast extract ...... 3,000 Alimentaria. Diaz de Santos, S.A.,Madrid. Peptone ...... 7,000 MOSSEL, D.A.A. and M.A. RATTO (1970) Rapid detec- Sodium chloride ...... 5,000 tion of sub-lethally impaired cells of Enterobacteriaceae Bile salts # 3 ...... 1,500 in dried foods Appl. Microbio¡ 20: 273-275. Lactose monohydrate ...... 10,000 EUROPEAN PHARMACOPOEIA 3ª Edición (Suppl. Glucose monohydrate ...... 10,000 1999) Cap. 2.6.13 Microbiological examination of non Neutral red ...... 0,030 sterile products. Tests for specified organisms. Council Crystal violet ...... 0,002 of Europe. Strasbourg Agar ...... 15,000 ISO 8523 Standard (1991) General guidance for the Final pH 7,4 ± 0,2 detection of Enterobacteriaceae with pre-enrichment. Directions Suspend 51.5 g of powder in 1 L of distilled water and heat to the boil. Pour into Petri dishes inmediately. Do not sterilize in autoclave nor overheat.

Description This medium developed in 1962 by Mossel et al. as more effective than MacConkey Agar for the detection of Enterobacteriaceae in foods, has been ofﬁcially adopted by the European Pharmacopoeia for the microbiological examination of non-sterile products. The medium is spe-

168 Ref. 01-164 Violet Red Bile Agar

Salmonella typhimurium Escherichia coli ATCC 25922 ATCC 14028

control

Vogel Johnson Agar (VJ Agar)

Ref. 01-206 lococci may reduce tellurite to tellurium, lithium may perform some action that is compensated by glycine. Specification Moreover a high correlation between tellurite reduction Solid and very selective medium for isolation and identifi- and mannitol fermentation has been proved, and this is cation of staphylococci according ISO 22718 standard. shown in the medium by the indicator turning to yellow due to the amount of acid produced. The medium’s selectivity avoids, in the first 24 hours, the Formula (in g/L) development of any other bacteria, so massive inocu- Casein Peptone ...... 10,000 lation is permited. Nonetheless, after this period, it is Yeast Extract ...... 5,000 possible that other bacteria may appear like micrococci, Mannitol ...... 10,000 which produce tiny colonies, and staphylococci that Dipotassium phosphate ...... 5,000 ferment mannitol and coagulase negative, therefore it is Litium chloride ...... 5,000 recommended to verify this last test separately. Glycine ...... 10,000 Due to reduced tellurite, staphylococci generally appear Phenol Red ...... 0,025 as black colonies over red medium (if they do not fer- Agar ...... 15,000 ment mannitol) or yellow medium (if they do, and these Final pH 7,2 ± 0,2 are presumptive pathogen). Saprophytic staphylococci (S.epidermidis, S.saprophiticus and S.intermedius) have Directions a grey-black colour and are mannitol negative. Complete Suspend 60 g of powder in 1 L of distilled water and medium may be stored up to 1 week in the refrigerator. bring to the boil. Dispense in suitable containers and Do not remelt it after tellurite is added. sterilize at 121°C for 15 minutes. Cool it to 50°C approx. and add aseptically 20 mL of Potassium Tellurite Solu- References tion 1% (Ref. 06-089) or 6,0 mL of Potassium Tellurite VOGEL and JOHNSON (1960) A modification of the Solution 3.5% (Ref. 06-011). Do not reheat after tellurite tellurite-glycine medium for the use in the identification of addition. Staphylococcus aureus. Pub. Health. Lab. 18:131-133. US PARMACOPOEIA (2002) 25th ed. <61> Microbial Limit Description Tests. Pharmacopoeial Convention. Rockville. MD VJ Agar is a selective medium for detection and enu- ATLAS, R.M.,& L.C. PARK (1993) Handbook of Microbio- meration of pathogenic staphylococci. logical Media. CRC Press Inc.,London The medium’s strong selective action is due to lithium FDA (1998) Bacteriological Analytical Manual. 8th ed. chloride, glycine and potassium tellurite presence. They Revision A. AOAC International. Gaithersburg. MD inhibit almost all the accompanying organisms, mean- ISO 22718:2006 Standard. Cosmetics – Detection of while staphylococci are not affected. Although staphy- Staphylococcus aureus.

169 Wilkins-Chalgren CN Modiﬁed Fluid Medium (WCCN Modiﬁed Fluid Medium)

Ref. 03-408 Directions Suspend 87,5 g of powder in 1 L of distilled water. Bring Speciﬁcation to the boil. Sterilize in the autoclave at 121°C for 10 Fluid medium for the cultivation and enumeration of minutes. anaerobic bacteria by the impedometry technique. Prepared medium takes on a dark colour due to the high concentration of sugar. Do not reheat. Formula (in g/L) Tryptone ...... 10,0000 Description Gelatin peptone ...... 10,0000 This medium is a modiﬁcation of the Wilkins-Chalgren Gelatin ...... 8,0000 Anaerobic Medium for impedometric methods. Dextrose ...... 41,0000 Yeast extract ...... 5,0000 References Sodium chloride ...... 5,0000 WILKINS, T.D. and S. CHALGREN (1976) Medium for Ammonium sulfate ...... 5,0000 use in antibiotic susceptibility testing of anaerobic bacte- L-Arginine ...... 1,0000 ria. Antimicrob. Agents Chemorther 10:6:926. Sodium pyruvate ...... 1,0000 Sodium bicarbonate ...... 1,0000 Ferrous sulfate ...... 0,1000 Calcium chloride ...... 0,1000 Hemine ...... 0,0050 Menadione ...... 0,0005 Agar ...... 0,3000 Final pH 7,1 ± 0,2

WL Nutrient Media

WL Nutrient Agar Directions Suspend 80 g of the powder in 1 L of distilled water. Ref. 01-210 Mix thoroughly. Heat with frequent agitation and boil for one minute. If a final pH of 6,5 is desired, the pH may be Specification adjusted with one percent aqueous sodium carbonate, using about 30 mL per litre of medium. Solid medium for the cultivation and enumeration of Dispense and sterilize the medium by autoclaving at yeast and bacteria for microbiological control in brewing 121°C for 15 minutes. and other fermentation industries. The WL Differential Agar has the same formula as the WL Nutrient Agar with the addition of 2 flasks/L of Cy- Formula (in g/L) cloheximide Selective Supplement Ref. 06-022CASE. Yeast extract ...... 4,0000 Tryptone ...... 5,0000 Description Dextrose ...... 50,0000 WL Nutrient Agar was formulated by Green and Gray in Monopotassium phosphate ...... 0,5500 the Wallerstein Laboratory for use in the control of indus- Magnesium sulfate ...... 0,1250 trial fermentations, particularly the processing of beer. It Calcium chloride ...... 0,1250 is recommended for examination of worts, beers, liquids Potassium chloride ...... 0,4250 containing yeast and other materials. Iron (III) chloride ...... 0,0025 WL Nutrient Agar has a pH of 5,5 which is optimal for Manganese sulfate ...... 0,0025 the enumeration of brewersýeast . If bakers or distiller´s Bromcresol green ...... 0,0220 yeast is to be examined, the pH should be adjusted to Agar ...... 20,0000 6,5 (better yields). When cultivating the microorganisms Final pH 5,5 ± 0,2 from an alcoholic mash, tomato juice should be added to

the medium.

170 WL Nutrient Media

WL Differential Agar contains cycloheximide to suppress Description yeast and any other moulds which may be present; this WL Medium was developed in the Wallerstein Labora- medium allows reliable counting of all bacteria which tories for industrial uses, since it allows to differentiate may be encountered in the tests performed in brewery between beer yeast and wild yeasts contaminants. laboratories. Adjusting pH to 6,5 is very advisable to enumerate bread and alcohol yeast. The medium also allows bacterial Technique growth and it is possible then to count the contaminant Dilute the sample material and spread 0,1 mL onto WL bacteria of fermented liquors, but it is recommended to Nutrient Agar or WL Differential Agar. use WL Differential Broth for this assay, since it inhibits The WL Nutrient Agar and WL Differential agar are used yeast growth. together, one plate with nutrient agar and two with the The WL Nutrient Broth is useful to enumerate cells by differential agar. the MPN technique. Alternatively, it can be used as an The WL Nutrient Agar plate is incubated aerobically to enrichment broth previous to the colony plate count. obtain a total count, mainly of yeast colonies. One WL Differential Agar plate is incubated aerobically for growth Technique of acetic acid bacteria, Flavobacterium, Proteus, and Usual the technique is to inoculate one plate of WL Nutri- other organisms; the second plate is incubated anaerobi- ent Broth and two plates of WL Differential. Incubate all cally for detection of such organisms as lactic acid bacilli the plates at 25°C for 5-15 days. It is advisable to incu- and Pediococcus species. bate one of the plates with differential agar anaerobically Plates prepared with both the media are generally incu- to enhance development of contaminants that produce bated at 25°C, if brewing materials are being studied, lactic acid. Green and Gray stated that to perform the and at 30°C for baker´s yeast and alcohol mash sam- observations of viable yeast in bread, WL Nutrient broth ples. Incubation may be continued for a week, or even at pH 5,5 may be used, but to do it in distilleries, the pH for ten days to two weeks, depending upon the flora has to be adjusted to 6,5. present. Counts are made at the intervals during the Analogously, time and temperature of incubation vary incubation period. depending on material to be analysed. Beer samples are incubated at 35°C, but bread, and alcohol fermenta- WL Nutrient Broth tion ones are incubated at 30°C. Incubation time varies between 2 and 7 days, and in some cases, depend- Ref. 02-210 ing on the flora found, it may be up to 14 days. When Differential type (with cycloheximide) is employed for a bacterial count, it should be incubated anaerobically to Specification detect cocci in beer and lactic bacilli, and it should be Liquid medium for the microbial control of industrial fer- incubated aerobically to detect acetic acid bacteria and mentations and massive cultivation of yeast. thermobacteria.

Formula (in g/L) References Yeast extract ...... 4,0000 GREEN, S.R. & GRAY, P.P. (1950) A differential proce- Tryptone ...... 5,0000 dure applicable to bacteriological investigation in brew- Dextrose ...... 50,0000 ing. Wallerstein Lab. Comm. 13:357 Monopotassium phosphate ...... 0,5500 GREEN, S.R. & GRAY, P.P. (1950) Paper read at Magnesium sulfate ...... 0,1250 Am.Soc. of Brewing Chemists Meeting; Wallerstein Lab. Calcium chloride ...... 0,1250 Comm. 12:43 Potassium chloride ...... 0,4250 GREEN, S.R. & GRAY, P.P. (1951) A differential proce- Ferric chloride ...... 0,0025 dure for bacteriological studies useful in the fermentation Manganous sulfate ...... 0,0025 industries. Wallerstein Lab. Comm. 14:289 Bromcresol green ...... 0,0220 GRAY, P.P. (1951) Some advances in microbiological Final pH 5,5 ± 0,2 control for beer quality. Wallerstein Lab. Comm. 14:169 ATLAS, R.M., & L.C. PARK (1993) Handbook of Micro- Directions biological Media for the examination of Food. CRC Press Dissolve 60 g of powder in 1 L of distilled water and dis- Inc. Boca Ratón,Fla. pense into suitable containers. Sterilize by autoclaving at MASTERS BREWERS ASSOCIATION OF THE AMERI- 121°C for 15 minutes. Should a pH 6,5 be desired, ad- CAS (2002) The Practical Brewer 3rd ed. St. Paul. Min- just it by adding 30 mL of Sodium carbonate solution 1%. nesota To obtain 1 L of WL Differential Broth just add, aseptically, 2 ﬂasks of Cycloheximide Selective Supplement (Ref. 06-022CASE) to 1 L of WL Nutrient Broth, after sterilization.

171 Wort Media

Wort Agar Directions Suspend 37 g of powder in 1 L of distilled water and Ref. 01-132 add 2-3 mL of glycerol and bring to the boil to dissolve completely. Distribute into final containers and sterilize Specification by autoclaving at 121°C for 15 minutes. Solid medium for the cultivation, isolation and enumeration or enrichment of fungi, especially of yeast. Description It is especially designed to propagate the multiplication Formula (in g/L) of yeast, and often it has been employed as a semiselective or enrichment medium, due to its high acidity, Malt extract ...... 15,0 which makes it inhibitory for bacteria. This effect may Casein Peptone ...... 1,0 be more enhanced by adding, before sterilization, 10 Maltose ...... 12,5 mL/L of a 10% solution of lactic or tartaric acid. To avoid Dextrine ...... 2,5 precipitate it is recommended to sterilize by filtration. Dipotassium hydrogen phosphate ...... 1,0 Ammonium chloride ...... 1,0 Agar ...... 17,0 References Final pH 4,8 ± 0,2 SCARR, M.P. (1959) Selective media used in the microbiological examination of sugar products. J. Sci. Food Directions Agric. 10:678-681 RAPP, M (1974) Indikator-zusätze zur Keimdifferen- Suspend 50 g of powder in 1 L of distilled water and zierung auf Würze und Malzextrakt-Agar. Milchwiss add 2-3 mL of glycerol and bring to the boil to dissolve 29:341-344 completely. Distribute into final containers and sterilize ATLAS, R.M.,& L.C. PARKS (1993) Handbook of Micro- by autoclaving at 121°C for 15 minutes. Do not over- biological Mediafor the examination of Food. CRC Press heat. Prolonged heating will diminish the gelling strength Inc.London of the medium. MASTERS BREWERS ASSOCIATION OF THE AMERI- CAS (2002) The Practical Brewer. 3rd ed. St. Paul. Min- Description nesota Wort Agar is used for the cultivation, isolation and enumeration of yeast and moulds. It is particulary well adapted for counting osmophilic yeast in butter, sugar and syrups, in lemonade and more generally in sweet or soft drinks. For a more selective utilization it is possible to adjust the pH to 4,5 or 3,5. Never heat the medium after adding acid, in order to prevent the loss of solidifying properties of the agar. The acid pH inhibits the growth of bacteria and favours that of yeast.

Wort Broth

Ref. 02-132

Speciﬁcation This medium is the liquid version of the classical Wort Agar (Ref. 01-132).

Formula (in g/L) Malt extract ...... 15,0 Casein Peptone ...... 1,0 Maltose ...... 12,5 Dextrine ...... 2,5 Dipotassium hydrogen phosphate ...... 1,0 Ammonium chloride ...... 1,0 Final pH 4,8 ± 0,2

172 Xylose Lysine Deoxycholate Media

Xylose Lysine Deoxycholate pH reversion by decarboxylation and even ferrous sul- Agar (XLD Agar) (Eur. Phar. fure precipitate in the first 24 hours. In the table below, typical colonial appearances on XLD Agar Medium K) medium after 24-36 hours of incubation at 37°C are described. Ref. 01-211 References Specification TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose Solid medium for the isolation of enteropathogenic Lysine Agars: New media for isolation of enteric patho- species, especially Salmonella according to ISO 6340 gens. Am. J.Clin. Path 44:471-475 standard. DOWNES, F.P. & K. ITO (2001) Compendium of Meth- ods for the Microbiological Examination of Food,4th ed. Formula (in g/L) APHA,Washington. Xylose ...... 3,50 ICMSF (1978) Microorganisms in Food 1. Univ. Toronto L-Lysine ...... 5,00 Press. th Lactose ...... 7,50 FDA (1998) Bacteriological Analytical Manual. 8 ed. Sucrose ...... 7,50 Revision A. AOAC International Gaithersburg. MD. Sodium chloride ...... 5,00 ATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro- Yeast extract ...... 3,00 biological Mediafor the examination of Food. CRC Press Phenol red ...... 0,08 Inc. London Sodium Deoxycholate ...... 2,50 PASCUAL ANDERSON, MªR. (1992) Microbiología Sodium thiosulfate ...... 6,80 Alimentaria. Diaz de Santos, S.A. Madrid, . Ammonium ferric citrate...... 0,80 EUROPEAN PHARMACOPOEIA, (2002) 2.6.13 Test th Agar ...... 15,00 for specified micro-organisms 4 Ed.,Suppl. 4.2 EDQM Final pH 7,4 ± 0,2 Council of Europe, Strasbourg, US PHARMACOPOEIA (2002) <61> Microbial Limit th Directions Tests. 25 Ed. US Pharmacopoeial Convention. Rocville. MD. Suspend 56,68 g of powder in 1 L of distilled water. Heat ISO 6340 Standard (1995) Water Quality - Detection of up constantly with stirring until boiling. Pour it immediate- Salmonella spp. ly into plates. Do not autoclave and avoid remelting.

Description Xylose Lysine Deoxycholate Xylose Lysine Deoxycholate Agar is a differential me- Modified Agar (XLD Modified dium, slightly selective, very suitable for the detection Agar) of pathogenic enterobacteria, especially Shigella. Gram negative flora is inhibited by the low amount of deoxy- Ref. 01-552 cholate, but Shigella grows easier in this medium than in any other selective media. Xylose, lactose or sucrose fermentation produce the Specification acidification of the medium, and this is seen by an Medium for isolation of enteropathogenic species, espe- indicator turning to yellow, surrounding the colonies. This cially Salmonella in food and animal feeding stuffs, acc. colour disappears after 24 hours, so observations must ISO Standard 6579:2002 be carried out between 18 and 20 hours. Hydrogen sulfide production from thiosulfate is easily de- Formula (in g/L) tected because colonies become darker, due to the ferric Xylose ...... 3,75 sulfure precipitate. Lysine decarboxylation to cadaverine L-Lysine ...... 5,00 may also be observed in the medium, since it produces Lactose ...... 7,50 alkalinization and consequently the indicator turns to red. Sucrose ...... 7,50 All these reactions allow a good differentiation of Shig- Sodium chloride ...... 5,00 ella, which besides Edwardsiella and Proteus inconstans Yeast extract ...... 3,00 are the single enterobacteria that do not ferment xylose Phenol red ...... 0,08 and therefore show negative fermentation reaction. Sodium Deoxycholate ...... 1,00 Salmonella type members do ferment xylose, but it is Sodium thiosulfate ...... 6,80 consumed quickly and then alkalinization of the medium, Ammonium iron(III) citrate ...... 0,80 due to lysine decarboxylation, may mask the reaction. Agar ...... 15,00 The difference between Shigella and Salmonella is that Final pH 7,4 ± 0,2 with the latter colonies become darker due to ferrous sulfure precipitates, and this is a common property with Edwardsiella. The other types of enterobacteria do not suffer this phenomenon, since acid acumulation due to lactose and sucrose fermentation is so high that it avoids

173 Xylose Lysine Deoxycholate Media

Directions precipitates, and this is a common character with Edwar- Suspend 55,43 g of powder in 1 L of distilled water. Heat siella. The other types of enterobacteria do not suffer this up constantly stirring until boiling. Pour it immediately phenomenon, since acid accumulation due to lactose into plates. Do not sterilize and avoid remelting. and sucrose fermentation is so big that it avoids pH reversion by decarboxylation and even ferrous sulfide Description precipitate in the first 24 hours. Xylose Lysine Deoxycholate Agar is a differential medium, slightly selective, very suitable for the detection In the table below, typical colonial appearances on XLD of pathogen enterobacteria in food, especially Shigella, Agar after 24-36 hours of incubation at 37°C are de- with a modification in the original formulation of Tay- scribe. lor to perform the specifications of the ISO standard 6579:2002 References Gram negative flora is inhibited by the low amount of TAYLOR, W.J. (1965) Isolation of Shigella. I. Xylose deoxycholate, but Shigella grows easier than in other Lysine Agars: New media for isolation of enteric patho- selective media. gens. Am. J. Clin. Path 44:471-475 Xylose, lactose or sucrose fermentation produce medium VANDERZANT & SPLITTSTOESSER (1992). Compen- acidification, and this is shown by an indicator turning to dium of Methods for the Microbiological Examination of yellow, surrounding the colonies. This colour disappears Food. 3rd. Ed. APHA. Washington. after 24 hours, so readings must be carried out between ICMSF, ( 1978) Microorganisms in Food 1. University of 18 and 20 hours. Toronto Press. Sulfhydric production from thiosulfate is easily detected FDA (1990) Bacteriological Analytical Manual AOAC because colonies become darker, due to the ferric International Arlington. VA. USA. sulfide precipitate. Lysine decarboxylation to cadaverine ATLAS, R.M., L.C. PARK (1993) Handbook of Microbio- may also be observed in the medium, since it produces logical Media for the examination of Food. CRC Press alkalinization and consequently the indicator turns to red. Inc. Boca Ratón. All these reactions allow a good differentiation of Shig- PASCUAL ANDERSON, MOR. (1992) Microbiología ella, which besides Edwarsiella and Proteus inconstans Alimentaria. Diaz de Santos, S.A. Madrid. are the single enterobacteria that do not ferment xylose ISO Standard 6579 (2002) Microbiology of foods and and therefore show negative fermentation reaction. animal feeding stuffs. Horizontal method for the detec- Salmonella-type members do ferment xylose, but it is tion of Salmonella spp. consumed quickly and the medium alkalinization, due to lysine decarboxylation, may hide the reaction. The difference between Shigella and Salmonella is that with the latter colonies become darker due to ferrous sulfide

Colony appearance Microorganism

Red colonies, transparent Shigella sp., Proteus incontans, Salmonella paratyphi A., sometimes S.cholerasuis and S. Pullorum Red colonies, transparent with black core. Edwardsiella and most of biotypes of Salmonella Orange and slightly opaque colonies Salmonella typhi Red and translucent colonies, without halo. Pseudomonas, Proteus rettgeri. Yellow and opaque Escherichia when it grows, Enterobacter, Aeromonas, Citro- bacter. Yellow, opaque, mucose and with black core. Klebsiella, Citrobacter intermedius when it grows Yellow, transparent and with black core. Most of Proteus mirabilis, P.vulgaris. Yellow, opaque and without halo Serratia, Hafnia.

Ref. 01-552 XLD Modiﬁed Agar

control Salmonella typhimurium ATCC 14028

174 Yeast Extract Media

Yeast Extract Agar Formula (in g/L) Peptone ...... 20,0 Ref. 01-465 Yeast extract ...... 10,0 Dextrose ...... 20,0 Specification Agar ...... 20,0 Final pH 6,8 ± 0,2 Solid medium for the enumeration of microorganisms from water. Directions Formula (in g/L) Suspend 70 g of powder in 1 L of distilled water and bring to the boil. Distribute into suitable containers and Tryptone ...... 5,0 sterilize by autoclaving at 121ºC for 15 minutes. Yeast extract ...... 3,0 Agar ...... 15,0 Final pH 7,2 ± 0,2 Yeast Extract Peptone Dextrose Broth (YPD Broth) Directions Suspend 23 g of powder in 1 L of distilled water and Ref. 02-473 bring to the boil. Distribute into suitable containers and sterilize by autoclaving at 121ºC for 15 minutes. Specification Liquid medium for the cultivation of yeast in molecular Description biology procedures. This medium, formulated according to Windle Taylor, is the most used in the UK for the enumeration of hetero- Formula (in g/L) trophic microorganisms from water. Distinction between Peptone ...... 20,0 bacteria, yeast and filamentous fungi must be carried out Yeast extract ...... 10,0 by morphology after differential incubations at 35º and Dextrose ...... 20,0 20ºC. Final pH 6,8 ± 0,2

Technique Directions From the water sample, make a decimal dilution bank Dissolve 50 g of powder in 1 L of distilled water, heating with Ringer Solution (Ref. 06-073) and take aliquotes to up if necessary. Distribute into suitable containers and 2 parallel series of plates. Pour the Yeast Extract Agar, sterilize by autoclaving at 121ºC for 15 minutes. molten and cooled to 45ºC, and homogenize with sample. Once solidiﬁed, incubate one of the series at 35ºC for 24 hours and the other one at 20ºC for 3 days. Description In order to achieve a good count, select the plates with These media support the growth of most heterotrophic 30-300 colonies. microorganisms, but due to their simple composition they have been adopted as the basal media for the routine cultivation of yeasts for molecular biology studies. References WINDLE TAYLOR, E. (1958) The examination of water and water supplies. 7th Ed. Churchill Ltd. London. References ATLAS, R.M., & L.C. PARKS (1993) Handbook of micro- SHERMAN, F. (1991) Studies on the phenotype switch- biological media, CRC Press, London ing with Candida albicans. Meth. Enzimol 194:3-17. MARTINEZ, J.P., M.L. GIL, M. CASANOVA, J.L. LOPEZ- RIBOT, J. GARCIA DE LOMAS, R. SENTANDREU Yeast Extract Peptone Dextrose (1990) Agar (YPD Agar) Wall mannoproteins in the cells from colonial phenotypic variants. J. Gen. Microbiol. 136:2421-2432. Ref. 01-473 ATLAS, R.M., L.C. PARKS (1993) Handbook of Micro- biological Media. CRC Press. London Speciﬁcation AUSUBEL, F.M., R.BRENT,R.E. KINGSTON, D.D. MOORE, J.G. SEIDMAN, J.A. SMITH & K. STRUHL Solid medium for the cultivation of yeast in molecular (1994) Current Protocols in Molecular Biology. Current biology procedures. Protocols. Brooklyn. N.Y.

175 Yeast Malt Media

Yeast Malt Agar (YM Agar) Yeast Malt Broth (YM Broth)

Ref. 01-219 Ref. 02-219

Speciﬁcation Speciﬁcation Solid medium for the cultivation of fungi and actinomyc- Liquid medium for the cultivation of fungi and actinomycete. etes.

Formula (in g/L) Formula (in g/L) Dextrose ...... 10,0 Dextrose ...... 10,0 Peptone ...... 5,0 Peptone ...... 5,0 Malt extract ...... 3,0 Malt extract ...... 3,0 Yeast extract ...... 3,0 Yeast extract ...... 3,0 Agar ...... 20,0 Final pH 6,2 ± 0,2 Final pH 6,2 ± 0,2 Directions Directions Dissolve 21 g of powder in 1 L of distilled water. Distrib- Suspend 41 g of powder in 1 L of distilled water and ute into suitable containers and sterilize by autoclaving let it soak. Bring to the boil and distribute into suitable at 121°C for 15 minutes. containers. Sterilize in the autoclave at 121°C for 15 minutes. Description This is a classical culture medium for the cultivation of moulds, yeasts and acidophilic actinomycetes. Medium may become selective to one or other group of microorganisms by adding antibiotics when the medium is at 50°C.

References ATLAS, R.M.,& L.C. PARK (1993) Handbook of Micro- biological Mediafor the examination of Food,CRC Press Inc.London. SAMSOM, R.A., E.S. HOEKSTRA, J.C. FRISVAD, O. FILTENBORG (2002) Introduction to food- and airborne fungi. 6th. Ed. CBS. Utrech.

176

Culture Media Ingredients Culture Media Ingredients

Deﬁnitions Production Process EXTRACTS are concentrated preparations of liquid, The general production process is eschematized in the solid or intermediate consistency, usually obtained from following ﬂow-chart. dried vegetable or animal matter. For some preparations, the matter to be extracted may undergo a preliminary treatment, for example, inactivation of enzymes, grind- ing or defatting. Extracts are prepared by maceration, percolation or other suitable, validated methods, using water, ethanol or another suitable solvent. After extraction unwanted matter is removed if necessary.

PEPTONES are complex water-soluble mixes of free amino acids, peptides, sugars, mineral salts and other components obtained by acid, alkaline or enzymatic hydrolysis of protein substrates. Their very variable characteristics depend on: - The nature of the substrate(s). - The nature of the hydrolysis: enzyme(s), alkali(s), acid(s), term of hydrolysis. - The technique of purification (filtration, ultrafiltration, …). - The other operating conditions used in the production process.

The term peptone is more commonly applied to the hydrolisates obtained by enzymatic digestion. The enzymes more frequently used are:

Papain that acts on the links adjacent to arginine, lysine, phenylalaline and glycine. Bromelain and Ficin are also used because are similar to papain but from other plants and with pH-range and temperature- range different. Pepsin, that acts on the links adjacent to phenylalanine or leucine. Pancreatin (a variable mixture of trypsin and chimo- trypsin) that acts on the links adjacent to arginine, lysine, tyrosine, tryptophane, phenylalanine and leucine. Microbial Proteases are obtained from microbial cultures as extracellular enzymes and used for the peptones production. Acid and Neutral microbial proteases works in similar way as papain and alkaline microbial proteases as pancreatin. The papain is obtained from plant material and pepsin and pancreatin, that are animal enzymes, are of swine origin.

179 Culture Media Ingredients

Warranty of health and origin Series in Nutrition and Food. Section G. Vol III. CRC All the animal tissue raw materials used in the elabora- Press. Cleveland tion of the Scharlau Microbiology Peptones come from SYKES, J. (1956) Constituents of Bacteriologic Culture approved slaughterhouses and are covered by certifi- Media. Cambridge University Press. Cambridge. cates obtained from the veterinary authorities. The country of origin of bovine animal tissues and casein used in the manufacture of each batch of peptone is specified in the health certificate. These documents certify that animals from which tissues have been taken were in good health and fit for human consumption. They are used by Scharlau Microbiology Quality Control Department to edit a health certificate for each batch of product manufactured: a copy of this certificate is submitted to our customers upon each delivery.

Analytical data and control methods All data ﬁguring in the following documentation result from the analysis of a signiﬁcant number of batches of every product. These data may be: Typical data, which are in fact average values (comparative tables and technical data sheets). Norms of acceptability (Technical data sheets).

Bacteriological controls as well as most general physico- chemical controls are carried out each batch system- atically. The other characters are verified according to routine. For the control methodology, a norm is defined specifically for each character. When a pharmacopoeial monograph (Eur. Phar. 4th ed. 2002; USP 25th ed 2002) is available, it is adopted as routine method. If it is not possible an other documented methodology is accepted. The typical data are show in the following Tables 1 to 6 and in the specific description of any product. The information contained in this publication is based in own research and development work and is the best of our knowledge true and accurate. Users should however conduct their own tests to determine the suitability of our products for their own purpouses. Statements contained herein should not be considered as a warranty of any kind, expressed or implied, and no liability is accepted for the infringement of any patents.

References ATLAS, R.M. & L.C. PARKS ( 1993) Handbook of Micro- biological Media CRC Press London. BRYDSON, E.Y. (1978) Natural and Synthetic Culture Media for Bacteria. In M. Rechcigl Jr. (ed) Handbook

180 Culture Media Ingredients

181 Culture Media Ingredients

182 Culture Media Ingredients

183 Culture Media Ingredients

184 Culture Media Ingredients

185 Culture Media Ingredients

186 Agar-Agar

Ref. 07-490

Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as Agarophytes (several species and genera of the Class Rodophyceae). It consists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar-Agar is a solidifying agent with the same gelling strength as the Agar Technical but with an inferior grade of puriﬁcation, and shows a greater opacity and a higher salts content. Its use among the culture media is recommended only when the brightness and clarity is not a critical requirement.

The most important characteristics are shown in the following tables. Data are average values, which may vary from batch to batch.

Agar Bacteriological

Ref. 07-004

Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as Agarophyites (several species and genera of the Class Rodophyceae). It consists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar Bacteriological is a solidifying agent selected and prepared by mixing different agar from several zones of origin and is especially recommended for gelling the microbiological culture media where a great transparency and brightness is required.

The most important characteristics are shown in the following tables. Data are average values, which may vary from batch to batch.

187 Agar Technical

Ref. 07-521

Agar is the dried, hydrophilic, colloidal substance extracted from the algae known as agarophyites (several species and genera of the Class Rodophyceae). It consists of two polysaccharides, agarose and agaropectine, in a variable proportion depending on the geographical origin zone.

The Agar Technical is a solidifying agent with a gel strengh higher than Agar Bacteriological, especially suggested when the culture medium does not require a total brightness, since it shows a slight turbidity.

The most important characteristics are shown in the following tables. Data are average values, which may vary from batch to batch.

Beef Extract

Ref. 07-515 The totally desiccated version is easier to use than the For a long time beef extract has been the basic com- paste form, and require less quantity in order to obtain ponent of culture media, and initially it substituted meat the same effects. Beef extract solutions are clear, slightly infusions due to its easy usage. Now, there is a trend to coloured and with pH near to neutral. In culture media substitute it by peptones and different mixtures with a they are used in concentrations varying from 0,3-0,5%. more deﬁned composition, because they allow a greater reproducible result. The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg- Scharlau Microbiology Beef Extract is obtained from free gining of this chapter. Data are average values, which tendons and fat beef muscle, enzymatically predigested. may vary from batch to batch. Its production also includes the elimination of fermentable sugars.

Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the category 4 of the WHO classiﬁcation. The bovine tissues are sourced from New Zealand, and come from herds free from Bovine Spongiform Encephalopathy virus and foot-and-mouth disease after examination by the Veteri- nary Authorities. The enzymatic preparation is of porcine origin.

The product does not contain and is not derived from speciﬁed risk material as deﬁned in the European Com- mission Decision 97/534/EC. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

188 Beef Extract

07-515 Beef Extract 07-515 Beef Extract Molecular Weigth Distribution Amino Acids (Free/Total) x 100

Bile

Ref. 07-039 Dry powder has a colour varying form yellow-beige to yellow-green, and it allows transparent 5% solutions in Ox bile powder is obtained by spray drying the fresh bile water, that have a pH between 6 and 7,5. In alcoholic at high temperature, assuring the maintenance of the solutions (ethanol 84%) it provides less than 0,1% of most important characteristics or properties of fresh bile. insoluble substances. 1g of ox bile powder is corresponds to approx. 10 g of fresh bile. The most important characteristics are shown in the following tables and in Tables 1 to 6 at the beggining of this The bile used in its preparation is naturally of bovine chapter. Data are average values, which may vary from origin. It comes from animals which are raised and batch to batch. slaughtered in Holland, and are in good health and suitable for human comsumption on ante- and post- mortem examination by the Veterinary Authorities. These animals come from herds free from Bovine Spongiform Encepha- lopathy and Foot-and-Mouth disease.

The manufacturing process includes the rapid heating at 200ºC on spray drying.

In the culture media it is employed at concentrations varying from 1-2%. Ox bile act as a non enteric microbial ﬂora inhibitor, and it is used as selective agent in enterobacterial isolation media. Ox bile solutions at low concentrations are clear and have a slight colour, but at higher concentrations they are opalescent and have darker colours.

189 Bile Salts #3

Ref. 07-525 The Scharlau Bile Salts #3 is standardized batch by batch intending to supply a product as homogeneous as The bile salts for microbiological applications are ob- possible. tained from fresh animal (sheep, pig) bile by precipitation with chlorhidric acid, in a process that removes pigments Dry powder has a flowable blurred white aspect, with a and other toxic substances and concentrate the bile bitter in odour and taste, but provides clean, transparent salts. Nevertheless the standardization of the results is and pale yellow 2% solutions in water, which present very difficult because the composition of the final mixture an alkaline reaction (pH 8,0) and therefore its addition depends not only on the process but on the raw material to culture media may require pH adjustment. When this that is very variable in origin. product is used as culture media component it is advis- In the normal preparation of bile salts several compo- able do nod exceed never the 0,3 % (w/v) concentration. nents like gluconate, taurocholate, cholate and deoxycholate, and others, can be identified, and the inhibitory The most important characteristics are shown in the fol- character of the mixture depends on the relative rate lowing tables and in Tables 1 to 6 at the beggining of this between all these substances. Usually, in the micro- chapter. Data are average values, which may vary from biological culture media, the bile salts are dosed in a batch to batch. concentration of 0,5% (w/v) to inhibit the growth of grampositive bacteria.

There are other type of preparations of mixture o bile salts, with a higher level of puriﬁcation and concentration of actives substances. These ones are used, in microbiological applications, in lower concentration, that is, a third o the concentration of the normal preparations. Because its efﬁcacy, they are called Bile Salts #3 and its usual concentration in culture media is 0,15%.

Brain Extract

Ref. 07-076

For a long time animal brain infusions have been one of the basic components of some culture media for fastidious microorganisms, and nowadays, in most cases they are still necessary.

Scharlau Microbiology Brain Extract produces clear, clean and stable solutions at 121ºC, and provide the culture medium with very complex nutrients. It is obtained from brains of healthy pigs with ante- and post-mortem sanitary certiﬁcation.

Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the category 4 of the WHO classification. The product does not contain and is not derived from specified risk material as defined in the European Commission Decision 97/534/EC. All the constituents are of swine origin.

The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

The most important characteristics are shown in the following tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which may vary from batch to batch.

190 Casein Acid Hydrolysate

Ref. 07-151 The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg- Casein Acid hydrolysate is a protein hydrolysate ob- gining of this chapter. Data are average values, which tained by acid digestion, where all the casein com- may vary from batch to batch. pounds reduced to their aminoacids, except tryptophane which almost disappears. Vitamines also almost disappear due to the acid digestion process.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classification. The lactic casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot- and-mouth disease after examination by the Veterinary Authorities. The product does not contain and is not derived from specified risk material as defined in the European Com- mission Decision 97/534/EC. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

07-151 Casein Acid Hydrolisate 07-151 Casein Acid Hydrolisate Molecular Weigth Distribution Amino Acids (Free/Total) x 100

191 Casein Pancreatic Peptone

Ref. 07-154 The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at Casein Pancreatic Peptone is a protein hydrolysate, 200ºC on spray drying. obtained by digestion with pancreatic extracts. It differs from Tryptone (Ref. 07-119) only in the way it is ob- The most important characteristics are shown in the fol- tained, which produces a different aminoacid composi- lowing ﬁgures and tables and in Tables 1 to 6 at the beg- tion and a lesser molecular size. It is the most used type gining of this chapter. Data are average values, which of peptone in industrial fermentation operations. may vary from batch to batch.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classiﬁcation. The Milk casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veteri- nary Authorities.

The enzymatic preparation is of porcine origin. The product does not contain and is not derived from speciﬁed risk material as deﬁned in the European Com- mission Decision 97/534/EC.

07-154 Casein Pancreatic Peptone 07-154 Casein Pancreatic Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

192 Casein Trypsic Peptone (Tryptone)

Ref. 07-119 The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at The Casein Trypsic Peptone or Tryptone, is a protein 200ºC on spray drying. hydrolysate obtained by digestion of casein with an especially tripsin-enriched pancreatic enzymatic prepa- The most important characteristics are shown in the fol- ration. lowing ﬁgures and tables and in Tables 1 to 6 at the beg- Both, its nitrogen content and balanced amino-acid com- gining of this chapter. Data are average values, which position makes it more suitable for the manufacturing of may vary from batch to batch. culture media, producing exceptionally clear solutions.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classiﬁcation. The lactic casein from cow milk is sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities. The enzymatic preparation is of porcine origin.

The product does not contain and is not derived from speciﬁed risk material as deﬁned in the European Com- mission Decision 97/534/EC.

07-119 Casein Trypsic Peptone 07-119 Casein Trypsic Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

193 Gelatin Pancreatic Peptone

Ref. 07-153 gining of this chapter. Data are average values, which may vary from batch to batch. Gelatin peptone is a cream coloured powder, with characteristic odour, obtained by pancreatic digestion of gelatin.

The gelatin is of porcine origin. None of the raw materials and auxiliaries used in its preparation are of bovine origin. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

It has a low content of Tryptophan, and has no fermentable sugars. Their solutions, even at high concentrations (10%), take a light colours, without precipitate, due to their elaborated manufacturing process. It produces a slight acid reaction (after sterilization) and has no indole. Its nutritional capacity is low, but it may be used for non fastidious microorganisms, and complies with the peptone speciﬁcations for fermentation studies.

The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg-

07-153 Gelatin Pancreatic Peptone 07-153 Gelatin Pancreatic Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

194 Heart Extract

Ref. 07-077 gining of this chapter. Data are average values, which may vary from batch to batch. Bovine heart extract has been widely used as an alternative to meat extract where very special nutrient requirements are needed. Scharlau Microbiology Heart Extract is obtained from the bovine cardiac muscle of healthy animals with explicit sanitary certiﬁcation.

Among the raw materials and auxiliaries used in its preparation, the bovine constituents belong to the category 4 of the WHO classiﬁcation. The bovine tissues are sourced from New Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot- and-mouth disease after examination by the Veterinary Authorities.

The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg-

07-077 Heart Extract 07-077 Heart Extract Molecular Weigth Distribution Amino Acids (Free/Total) x 100

195 Lecithin

Ref. 07-342

Phosphatidylcholine, a mixture of diglycerides of the stearic, palmitic and oleic acids, linked to the cholic ester of the phosphoric acid.

The SCHARLAU Lecithin is a clear brown powder, obtained from soy beans by extraction. It is especially treated to be included in culture media as an emulsiﬁer or as a nutrient factor for fastidious microorganisms.

The most important characteristics are shown in the following tables. Data are average values, which may vary from batch to batch.

Liver Peptone

Ref. 07-614 gining of this chapter. Data are average values, which may vary from batch to batch. Liver peptone is a proteic hydrolized by enzymatic digestion of fresh swine liver, followed by a careful desiccation process to maintain its fundamental characteristics.

Liver peptone is very employed in culture media for Tri- chomonas and other fastidious protozoa, and for some pathogen and saproﬁtic fungi, micoplasms and anaerobic bacteria.

Liver peptone provides clear solutions of a dark colour, and is perfectly compatible with other medium components. It accepts sterilization and does not loose its characteristics. Generally it uses to replace peptones, at the same weight. In culture media for protozoa, its concentration uses to be high (25-30 g/L), but for bacteria it uses to vary from 0,5 to 1 %, except for anaerobics enrichment media, where concentration may be higher.

The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg-

196 Liver Peptone

07-614 Liver Peptone 07-614 Liver Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

Malt Extract

Ref. 07-080

Malt extract is used in the culture media for fungi, as much as enrichment as a true nutritive base, because very often it substitutes the peptone. It is obtained by extraction of soluble fraction of malted barley, followed by a drying proccess at low tempertature so that there is only minimal alteration in its nitrogenated composition and high sugar content, especially maltose. All the raw materials and auxiliaries are of plant origin. It has no diastatic activity. Very hygroscopic product.

Malt extract solutions are usually opalascent or turbid. Should clear solutions are required, ﬁltration is necessary.

The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which may vary from batch to batch.

197 Meat Extract

Ref. 07-075 The totally desiccated (dried) version is easier to use and requires less quantity in order to obtain the same Meat extract has been the basic component of culture effects. Meat extract solutions are clear, slightly coloured media for a long time, and initially it substituted meat and with pH near to neutrality. In the culture media they infusions due to its easy usage. Now, there is a trend to are used in concentrations varying from 0,3-0,5%. substitute it by peptones and different mixtures with a more deﬁned composition, because they allow a greater The most important characteristics are shown in the fol- reproductivity result. lowing ﬁgures and tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which Meat extract is obtained from free tendons and fat may vary from batch to batch. tissues of the animals (sheep and pork), which are enzymatically predigested. Its production involves the elimination of fermentable sugars.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and foot- and-mouth disease after examination by the Veterinary Authorities. The product does not contain and is not derived from specified risk material as defined in the European Com- mission Decision 97/534/EC. The other constituents are of porcine origin. The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at 200ºC on spray drying.

07-075 Meat Extract 07-075 Meat Extract Molecular Weigth Distribution Amino Acids (Free/Total) x 100

198 Meat Peptone

Ref. 07-152 The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at Meat peptone is an hydrolysate obtained by a partial 200ºC on spray drying. digestion of meat by pepsine. It complies with the USP/ NF25 and Eur. Phar. 4th. Ed. specifications for the peptic The most important characteristics are shown in the fol- digestion of animal tissues. lowing figures and tables and in Tables 1 to 6 at the beggining of this chapter. Data are average values, which It is a fine powder, cream or brown coloured, that gives may vary from batch to batch. very clear and light solutions and is specially prepared for using in the culture media.

Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease after the examination by the Veterinary Authorities. The product does not contain and is not derived from specified risk material as defined in the European Com- mission Decision 97/534/EC. The other constituents are of porcine origin.

07-152 Meat Peptone 07-152 Meat Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

199 Peptone from Casein

Ref. 07-489 The digestion process by an exceptionally trysin-rich enzymatic preparation produces a high content of tryp- The Peptone from casein is a protein hydrolysate ob- tophane and assures the absence of fermentable sugars tained from trypsin-digested casein according to the USP and enzymatic activity. specifications. Both its nitrogen rate and its balanced amino-acid composition provide an adequate support for The most important characteristics are shown in the fol- the production of culture media, producing exceptionally lowing figures and tables and in Tables 1 to 6 at the beg- clear solutions. gining of this chapter. Data are average values, which may vary from batch to batch. Among the raw materials and auxiliaries used in its preparation the bovine constituents belong to the category 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from the herds free from Bovine Spongiform Encephalopathy and Foot-and-Mouth Disease after the examination by the Veterinary Authorities. The product does not contain and is not derived from specified risk material as defined in the European Com- mission Decision 97/534/EC.

07-489 Peptone from Casein 07-489 Peptone from Casein Molecular Weigth Distribution Amino Acids (Free/Total) x 100

200 Proteose Peptone

Ref. 07-213 The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at This peptone is obtained after a partial enzymatic (pep- 200ºC on spray drying. tic) digestion process of animal tissues. It is obtained in such a way that there is a high proportion of peptides The most important characteristics are shown in the fol- of low molecular weight, with free amino acids and lowing figures and tables and in Tables 1 to 6 at the beg- other growth factors. Although all these things make its gining of this chapter. Data are average values, which definition very difficult, it has a high nutritive capacity may vary from batch to batch. that makes it suitable for obtaining toxins and as a basic growth support for very fastidious microorganisms.

Among the raw materials and auxiliaries used in its production, the bovine constituents belong to the category 4 of the WHO classification. The bovine tissues are sourced from New-Zealand, and come from herds of cattle which are free from Bovine Spongiform Encephalopa- thy and foot-and-mouth disease after examination by the Veterinary Authorities. The product does not contain and is not derived from specified risk material as defined in the European Com- mission Decision 97/534/EC. The other constituents are of porcine origin.

07-213 Proteose Peptone 07-213 Proteose Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

201 Soy Peptone

Ref. 07-155

Soy peptone is a proteic hydrolysate obtained by papaic digestion of soy ﬂour. It complies with the USP/NF25 and Eur. Phar. 4th. Ed. speciﬁcations for these type of products, and it is a useful compound in laboratory culture media. However, due to its high content of sugar it is not recommendable for fermentation assays.

07-155 Soy Peptone 07-155 Soy Peptone Molecular Weigth Distribution Amino Acids (Free/Total) x 100

202 Tryptose

Ref. 07-197 The manufacturing process includes boiling at 100ºC for a minimum of 5 minutes and instantaneous heating at Tryptose is a mixed peptone with high nutrient properties 200ºC on spray drying. that make it appropiate for the use in culture media for very fastidious microorganisms. The most important characteristics are shown in the following ﬁgures and tables and in Tables 1 to 6 at the beg- Among the raw materials and auxiliaries used in its gining of this chapter. Data are average values, which preparation the bovine constituents belong to the cat- may vary from batch to batch. egory 4 of the WHO classiﬁcation. The bovine tissues are sourced from New-Zealand, and come from herds free from Bovine Spongiform Encephalopathy and foot-and-mouth disease after examination by the Veterinary Authorities.

The product does not contain and is not derived from speciﬁed risk material as deﬁned in the European Com- mission Decision 97/534/EC. The other constituents are of porcine origin.

07-197 Tryptose 07-197 Tryptose Molecular Weigth Distribution Amino Acids (Free/Total) x 100

203 Yeast Extract

Ref. 07-079

A water soluble extract of fresh autolyzed yeast cells. Prepared and standardized for use in microbiological culture media.

It is commonly added to culture media in concentrations between 0.2% and 1%.

07-079 Yeast Extract 07-079 Yeast Extract Molecular Weigth Distribution Amino Acids (Free/Total) x 100

204

Additives Dextrose Powder (D(+)- Glucose Powder)

Ref. 06-048 Speciﬁcations

Specific rotation ([α]20ºC/D, c=10, H2O) ...... +52,6 - +53,2 º Presentation Molecular weight ...... 180,16 g/mol 500 g Flask Acidity/alkalinity ...... passes test Insoluble in water ...... max. 0,01 % Description Chlorides (Cl)...... max. 0,01 % Sulfates (SO4) ...... max. 0,02 % Purified and standardized carbohydrate for the use in Sulfite (as SO2) ...... max. 0,001 % microbiological cultrure media as energy source for Arsenic (As) ...... max. 0,0001 % bacteria. Carbohydrate are adapted to the suitable basic Calcium (Ca) ...... max. 0,02 % media. Heavy metals (as Pb) ...... max. 0,0005 % Scharlau carbohydrates are pure and without mixtures, Iron (Fe) ...... max. 0,0005 % and this important characterístic is assured in order to Lead (Pb) ...... max. 0,00005% get always right results. Sulfated ash ...... max. 0,1 % Water (K.F.) ...... max. 1 % Physical Data Foreign sugars,starchs,dextrines .....passes test 3 Bulk density...... ~ 630kg/m Residual solvents (Eur. Phar./ICH) ...... excluded Solubility in water (20ºC) ...... ~ 470g/L Melting point ...... ~ 146ºC Ignition temperature ...... ~ 500ºC pH (100 g/L H2O, 20ºC) ...... 6-7

Egg’s Yolk Sterile Emulsions

Egg’s Yolk Sterile Emulsion The Egg’s Yolk Sterile Emulsion has been widely used Ref. 06-016 as bactericide neutralizer on sampling liquids. Such an effect can be considerably intensiﬁed by adding a bit of Polysorbate with the yolk in the formulation. 1% (v/v) is Presentation usually enough. 100 mL Flask Egg’s Yolk Tellurite Sterile Description Sterile egg’s yolk emulsion stabilized for use in bacte- Emulsion riology, especially with Bacillus cereus Agar Base (Ref. Xn 01-262), Bacillus cereus Selective Agar Base (Ref. 01- Ref. 06-026 487) and Tryptose Sulﬁte Cycloserine Agar Base (Ref. R-22 S-46 01-278). Presentation 100 mL Flask It is also used for the detection of lecithinase in species such as Bacillus, Clostridium and Staphylococcus, Description and in all processes related with this enzyme which are This emulsion has been especially formulated for its ad- present in dairy microorganisms and most psychrotroph- dition to the Baird Parker Agar Base (Ref. 01-030) ics. Aseptically add 50 mL of Egg’s Yolk Tellurite Sterile Lecithinase Assay Emulsion to 1 litre of sterile Baird Parker Agar Base The medium is prepared by aseptically adding 0,5-1,0 (Ref. 01-030), melted and cooled down to approximately mL of yolk emulsion to 10 mL of sterile melted solid 55-60°C. Mix uniformly avoiding bubbles and foam, and medium, cooled to 55-60°C. Tryptic Soy Agar (Ref. 01- pour into Petri dishes. 200), Nutrient Agar (APHA) (Ref. 01-144) and Nutrient The presumptive Staphylococcus aureus colonies show Agar (B. Ph.) (Ref. 01-140) are very adequate for these the lecithinase activity by the halo digestion around the purposes. The solid medium is inoculated with the assay colonies and simultaneously a black center due to the strain and incubated at 35-37° C for 5 days. If there is tellurite reduction. lecithinasic activity the broths will turn opalescent and solid media will present an opaque zone of clearing around the colonies. Bacillus cereus, with a strong lecithinase, produces visible results in just a few hours.

207 Lactose Powder

Ref. 06-051 Speciﬁcations

Speciﬁc rotation ([α]20ºC/D, c=10, H2O) ...... +54,4 - +55,9 º Presentation Molecular weight ...... 360,32 g/mol 500 g Flask Acidity/alkalinity ...... passes test Appaerance of solution (10% water) ...... passes test Description Proteins ...... passes test Arsenic (As)...... max. 0,00005 % Puriﬁed and standardized carbohydrate for the use in Copper (Cu) ...... max. 0,0025 % microbiological cultrure media as energy source for Heavy metals (as Pb) ...... max. 0,0005 % bacteria. Carbohydrate are adapted to the suitable basic Lead (Pb)...... max. 0,00005 % media. Zinc (Zn) ...... max. 0,0025 % Scharlau carbohydrates are pure and without mixtures, Sulfated ash ...... max. 0,1 % and this important characterístic is assured in order to Water (K.F.)...... 4,5 - 5,5 % get always right results. Residual solvents (Eur. Phar./ICH)...... excluded Physical Data Bulk density...... ~ 500 kg/m3 Solubility in water (20ºC) ...... freely soluble Melting point ...... ~ 223 ºC pH (50 g/L H2O, 20ºC) ...... 4-6

Maltose Powder

Ref. 06-052 Physical Data Bulk density...... ~ 320 kg/m3 Presentation Solubility in water (20ºC) ...... freely soluble 500 g Flask Melting point ...... ~ 160 - 165 ºC

pH (50 g/L H2O, 20ºC) ...... 4,5-6,0 Description Purified and standardized carbohydrate for the use in Specifications ([ ]20ºC/D, c=10, H O) microbiological cultrure media as energy source for Specific rotation α 2 ...... +137 - +139 º bacteria. Carbohydrate are adapted to the suitable basic Molecular weight ...... 342,31 g/mol media. Acidity/alkalinity ...... passes test Scharlau carbohydrates are pure and without mixtures, Appaerance of solution (10% water) ...... passes test and this important characterístic is assured in order to Proteins ...... passes test get always right results. Arsenic (As)...... max. 0,00005 % Copper (Cu)...... max. 0,0005 % Heavy metals (as Pb)...... max. 0,002 % Barium (Ba)...... max. 0,0005 % Calcium (Ca)...... max. 0,005 % Sulfated ash...... max. 0,1 % Water (K.F.)...... 4,5 - 5,5 % Residual solvents (Eur. Phar./ICH) ...... excluded

208 Mannitol Powder

Ref. 06-050 Specifications Assay (iodometric) ...... min. 98 % Presentation Acidity/alkalinity ...... passes test 500 g Flask Appaerance of solution (20% water) ...... passes test Conductivity (20ºC, 20% water) ...... passes test Specific rotation ([α]20ºC/D, c=8, Na B O ) ...+23 - +25 º Description 2 4 7 Molecular weight ...... 182,17 g/mol Purified and standardized carbohydrate for the use in Chlorides (Cl)...... max. 0,005 % microbiological cultrure media as energy source for Sulfates (SO ) ...... max. 0,01 % bacteria. Carbohydrate are adapted to the suitable basic 4 Arsenic (As) ...... max. 0,0001 % media. Copper (Cu) ...... max. 0,001 % Scharlau carbohydrates are pure and without mixtures, Heavy metals (as Pb) ...... max. 0,0005 % and this important characterístic is assured in order to Lead (Pb)...... max. 0,00005 % get always right results. Nickel (Ni) ...... max. 0,0001 % Zinc (Zn) ...... max. 0,0025 % Physical Data Related substances (sorbitol) ...... max. 2 % 3 Bulk density...... ~ 400 - 500 kg/m Red. impurities (as glucose) ...... max. 0,05 % 3 Spec. density...... 1,49 g/m Sulfated ash ...... max. 0,1 % Solubility in water (25ºC)...... 213 g/L Loss on drying (105ºC, 4h) ...... max. 0,3 % Melting point ...... ~ 164 -169 ºC Residual solvents (Eur. Phar./ICH)...... excluded Boiling point (4 hPa) ...... ~ 290 - 295 ºC pH (100 g/L H2O, 20ºC) ...... 5-7

Polysorbate 80 (Polyoxyethylene sorbitan monooleate)

Ref. 06-088 The incorporation of Polysorbate 80 to culture media may slightly affect the final pH, if the SCHARLAU Presentation medium is not originally formulated to be composed by 100 mL Flask Polysorbate. 1 L Flask Although it may bear sterilization in the autoclave when it is more than 1% in the medium, it is usual to homog- Description enize the medium after the sterilization, since with the Under the name of Polysorbate 80 or polyoxyethylene autoclaving sometimes the polysorbate is separated sorbitanmonooleate are included a serie of derivates of from the medium. polyose-1,2-ethanodiol sorbitan-mono-9-octodecenoate. Polysorbate is a tensioactive agent that makes decrease The product supplied by SCHARLAU is verified to be a the superficial tension of the cell, modifying at the same nutrient in some cases and an emulsionant in others, but time the cellular exchange speed. The response use to it is always compatible with the rest of components of be a quicker growht or the increase of some bacterial the culture medium. activities. It is a very thick liquid, amber colour, and density 1,08 approx. It is very soluble in water, it has an average solubility in organic diluents but it is not soluble in mineral lipids.

209 Potassium Tellurite Solutions

Potassium Tellurite Solution 1% It is used in media such as Giolitti Cantoni Broth (Ref. 02-230), Vogel-Johnson Agar (Ref. 01-206) and other Xn Ref. 06-089 selective media for staphylococci. This solution is also R-22 contained in selective media for corynebacteria, strepto- S-46 cocci and vibrios. Presentation 100 mL Flask There is high relation between the ability to reduce potassium tellurite to tellure and the staphylococci’s patho- Potassium Tellurite genity. Therefore, the presence of potassium tellurite in Solution 3,5% a medium helps to determine staphylococci of clinical interest, together with other tests. Xn Ref. 06-011 The Potassium Tellurite Solution should be stored at R-22 S-46 room temperature, since low temperatures will cause Presentation the crystallization and later precipitation of the product. 100 mL Flask Should this occur, intense agitation will help redissolve the precipitate. Due it its thermolabile qualities, the po- Description tassium tellurite is supplied sterile ﬁltered. Aquouse solutions of potassium tellurite at 1% or 3,5%, sterilized by ﬁltration and suitable to be used as an inhibitor additive in culture media.

The Potassium Tellurite Solution is added to culture media as an inhibitor. Its purpose is to prevent the growth of most gram-negatives and of those gram-positives unable of reducing it.

Ringer Powder

Ref. 06-073 Description Ringer saline solution is an isotonic medium which is Presentation more balanced than the simple sodium chloride saline 500 g Flask solution, and its formulation permits the autoclaving 100 g Flask without producing any precipitation.

Speciﬁcation For the routine work with bacteria the solution is diluted one fourth (to the fourth part) (Ringer 1/4), and is em- Isotonic solution for the cellular suspensions. ployed to get cell suspensions or to prepare the dilution banks. Formula (in g/L) Nonetheless, to dilute the food samples or substances Sodium chloride ...... 2,250 that have undergone thermal treatment, it is more advis- Potassium chloride ...... 0,105 able to use Peptone Water (Ref. 3-156) for the dilutions, Calcium chloride ...... 0,120 since the Peptone Water acts as a revitalizer also. Sodium bicarbonate ...... 0,050 References Directions DAVIS, J.G. (1956) Laboratory Control of Dairy Plant. To obtain an isotonic solution for eukaryotic cells, dis- Dairy Industries Ltd. London. solve 10 g of powder in 1 L of distilled water. To obtain ANONYMOUS (1937) Bacterial Tests for Graded an isotonic solution for prokaryotic cells, dissolve 2,5 g of Milk. Memo 139-Food. Dept. of Health and Social powder in 1 L of distilled water. Security,London. Distribute into suitable containers and sterilize in the autoclave at 121°C for 15 minutes.

210 Skimmed Milk

Ref. 06-019 Skimmed milk may be use alone or as an additive to other culture media. It is a very suitable medium for the Presentation culture of acid lactic bacteria and for identification in 500 g Flask general, in base to its capacity to coagulate o peptonize milk. With the additon of suitable indicators as bromcresol purple at 0,004% the pH variations in the transfor- Description mation may be screened. It also accepts oxid-reduction Powder of skimmed milk for bacteriology is obtained indicators like methylen blue, resazurine or TTC (Ref. after a depurated atomization process that keep it free 6-023) to verify microorganisms development. from thermophil organismsm that use to interfere with its use. Skimmed milk may be added to media as Tryptone Soy Agar (Ref. 01-200) or Nutrient Agar (Ref. 01-144 and 01- 100 g of powder produce 1 L of skimmed milk. Water 140) to detect caseolytic activity. addition must be gradual, until getting an homogeneous paste. Then fullfil with water to the desired volume. Fat ...... 0,5 % Sterilization may be under fluent vapor for 30 minutes Protein ...... 33,0 % and three consecutive days or in the autoclave at 121°C Ash ...... 8,0 % for 15 minutes or at 114°C for 15-20 minutes (this last Moisture ...... 5,0 % way is the better). Anycase, do not overheat prepared Lactic acid ...... 1,5 % milk since natural sugars may become caramelizated Antibiotic test ...... Negative and produce toxic compounds.

Sodium Biselenite

Ref. 06-615 T N The intended use of this product is to complete the following culture media by adding the speciﬁed amounts: R-23/25-33-50/53 Presentation S-20/21-28-45-60-61 100 g Flask 02-602 Selenite Cystine Broth Base ...... 4 g/L 02-603 Selenite Brilliant Green Broth Base ...... 4 g/L 02-598 Selenite Broth Base ...... 4 g/L Description Chemical compound to be added to selenite based The use of this product is restricted to technically quali- culture media. ﬁed personnal. Keep attention to the job limitations in inexpert personnal. The toxic and potetially theratogenicity of this product recommend its exclusion from the dehydrated powder mixture of culture media, to minimize the hazard of acci- dental inhalation or contact. The supply of this product in a separate form from the base medium, with all the risk considerations, enhances its safe and responsible use.

211 Sucrose Powder (D(+) Saccharose Powder)

Ref. 06-049 Speciﬁcations

Specific rotation ([α]20ºC/D, c=26, H2O) ...... +66,3 - +67,0 º Presentation Molecular weight ...... 342,30 g/mol 500 g Flask Acidity/alkalinity ...... passes test Appaerance of solution (50% water) ...... passes test Description Conductivity ...... max. 35 μS/cm Chlorides (Cl)...... max. 0,0035 % Purified and standardized carbohydrate for the use in Sulfites (as SO ) ...... max. 0,0015 % microbiological cultrure media as energy source for 3 Sulfates (SO ) ...... max. 0,005 % bacteria. Carbohydrate are adapted to the suitable basic 4 Dextrines ...... passes test media. Glucose and invert sugar ...... passes test Scharlau carbohydrates are pure and without mixtures, Calcium (Ca) ...... max. 0,001 % and this important characterístic is assured in order to Heavy metals (as Pb) ...... max. 0,0005 % get always right results. Lead (Pb) ...... max. 0,00005 % Physical Data Sulfated ash ...... max. 0,01 % 3 Bulk density...... ~ 800 - 950 kg/m Organic volatile impurities (NF) ...... passes test Solubility in water (25ºC) ...... freely soluble Residual solvents (Eur. Phar./NF) ...... max. 0,5 % Melting point ...... ~ 169 -170 ºC pH (100 g/L H2O, 20ºC) ...... ~ 7

212 TTC Sterile Solution 1%

Ref. 06-023 The general structure is the following:

Presentation 100 mL Flask

Description Sterile solution at 1% of 2-3-5-triphenyl-2H-tetrazolium chloride. It is used as an additive for culture media to show biological activity, since the colourless form gets hydrogenizated or reduced to a red insoluble pigment: triphenylformazan, which may be easily observed.

Despite of TTC decomposes at 243°C, it is not advisable to incorporate it to culture media before sterilization, because it lose efﬁcacy. Very good results may be achieved when the addition is carried out asseptically with cold medium at 60°C maximum. TTC is photolabile and becomes yellow by the effect of light, therefore keep This product is especially produced to be added to it in the refrigerator and avoid direct light. the following media: Chapman TTC Agar Base (Ref. 01-053), KF Media (Ref. 01-294 and Ref. 02-294) and Concentration of use vary depending on the medium, but Slanetz Bartley Agar Base (Ref. 01-579). generally it goes between 0,3 and 1% (v/v).

Urea Sterile Solution 40%

Ref. 06-083 It is supplied to be used with the dehydrated media Urea Agar acc. to Christensen (Ref. 01-261) and Urea Broth Presentation (Ref. 02-202). It must be added to these media after the 100 mL Flask sterilization and with the media cooled below 55°C. Once it is added, do not reheat the media. Description Aquose urea solution 40%, sterilized by ﬁltration and suitable to be used as an additive in culture media.

Vaseline Sterile

Ref. 06-077 incorporation of air to the medium. To seed the tubes, use a capilar pipette or inoculate them previously to the Presentation vaseline addition. 100 mL Flask Vaspar is another method used to achieve an hermetic lock. It is prepared melting vaseline and solid paraﬁne Description toghether in equal parts. Liquid media may be kept in anaerobic conditions if Vaseline sterile is especially suggested for the addition sterile compounds, as vaseline, are used to grant an to O/F Medium (Ref. 03-037). For solid media is more hermetic lock. suitable to use the Sealing Agar (Ref. 01-174). To achieve an hermetic lock in tubes with liquid medium, heat them up in boiling bath for 10 minutes to remove the oxygen and add after the vaseline to avoid the

213

Supplements Improved NEW presentation

PRESENTATION: 10 VIALS INSIDE A RESISTANT CASE

REFERENCE DESCRIPTION REFERENCE DESCRIPTION 06-012CASE SC Selective Supplement for Clostridium 06-111CASE Listeria Selective Supplement for Secondary Enrichment VIAL CONTENTS: 120 mg of Sodium azide, 90 mg of Neomycin (UVM II/Fraser) sulphate and solvent. VIAL CONTENTS: 10 mg of Nalidixic acid, 12,5 mg of Acriflavine Each vial is sufficient to supplement 500 ml of Blood and solvent. Columbia Agar Base (Ref. 01-034) or Blood Agar Base Each vial is sufficient to supplement 500 ml of Listeria (Ref. 01-352) in order to prepare Clostridium spp. Selective Agar. Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare 06-013CASE CP Gram-positive cocci in Blood Agar Selective Supplement 500 ml of Listeria Secondary Enrichment Medium (UVM II VIAL CONTENTS: 5 mg of Colistin sulphate, 7,5 mg of Nalidixic acid formulation); or to supplement 500 ml of Listeria Enrichment and solvent. Broth acc. Fraser (Ref. 02-496) in order to prepare 500 ml Each vial is sufficient to supplement 500 ml of Blood Columbia of Fraser Broth. Agar Base (Ref. 01-034) or Blood Agar Base (Ref. 01-352) and 06-112CASE Ferric Ammonium Citrate for Bacteriology obtain Staphylococcus and Streptococcus Selective Agar. VIAL CONTENTS: 250 mg of Ferric ammonium citrate and solvent. 06-017CASE Brilliant Green + Novobiocin Selective Supplement Each vial is sufficient to supplement 500 ml of Listeria VIAL CONTENTS: 5 mg of Brilliant green, 20 mg of Novobiocin Enrichment Broth Base acc. Fraser (Ref. 02-496) and solvent. 06-113CASE Ferric Ammonium Citrate for bacteriology Each vial is sufficient to supplement 500 ml of Tetrationate VIAL CONTENTS: 312 mg of Ferric ammonium citrate and solvent. Base Broth (Ref. 02-033) and Muller-Kauffmann Medium Each vial is sufficient to supplement 500 ml of Lactose Sulphite (Ref. 02-335). Broth Base (Ref. 02-519). 06-021CASE Polymyxin B Sulphate Selective Supplement 06-114CASE Disodium disulphite Selective Supplement for bacteriology VIAL CONTENTS: 50 mg Polymyxin B sulphate and solvent. VIAL CONTENTS: 375 mg of Disodium disulphite and solvent. Each vial is sufficient to supplement 500 ml of Bacillus cereus Each vial is sufficient to supplement 500 ml of Lactose Sulphite Agar Base (Ref. 01-262). Broth Base (Ref. 02-519). 06-022CASE Cycloheximide Selective Supplement 06-115CASE Oxytetracycline Selective Supplement VIAL CONTENTS: 2 mg of Cycloheximide and solvent. VIAL CONTENTS: 50 mg of Oxytetracycline and solvent. Each vial is sufficient to supplement 500 ml of WL Nutrient Each vial is sufficient to supplement 500 ml of Sabouraud with Agar (Ref. 01-210) or WL Nutrient Broth (Ref. 02-210) Oxytetracycline Agar Base (OGYEA) (Ref. 01-275). and converted in WL Differential Agar or Broth. 06-116CASE Cycloserine Selective Supplement 06-025CASE Brucella Selective Supplement VIAL CONTENTS: 100 mg of Cycloserine and solvent. VIAL CONTENTS: 50 mg of Cycloheximide, 3000 u.i. of Each vial is sufficient to supplement 250 ml of Tryptose Sulphite Polymyxin B Sulphate, 12500 u.i. of Bacitracin sulphate Cycloserine (TSC) agar Base for Clostridium perfringens and solvent. Ref. 1-278). Each vial is sufficient to supplement 500 ml of Brucella Selective 06-118CASE Chloramphenicol Selective supplement Agar (Ref. 01-042) and Brucella Selective Broth (Ref. 02-042). VIAL CONTENTS: 25 mg of Chloramphenicol and solvent. 06-085CASE Rosolic Acid Selective Supplement Each vial is sufficient to supplement 500 ml of Sabouraud VIAL CONTENTS: 50 mg of Rosolic acid and solvent. Dextrose Agar (Eur. Phar. Agar Medium C), (Ref. 01-165). Each vial is sufficient to supplement 500 ml of Faecal Coliforms 06-124CASE Nalidixic Acid Selective Supplement Agar (m-FC Agar) (Ref. 01-287) or Faecal Coliforms Broth VIAL CONTENTS: 7,5 mg of Nalidixic acid and solvent. (m-FC Broth) (Ref. 02-287). Each vial is sufficient to supplement 500 ml of CN Selective 06-091CASE CPB Selective Supplement for Campylobacter Agar Base for Pseudomonas (Ref. 01-609). VIAL CONTENTS: 2,5 mg of Trimethoprim, 5 mg of Vancomycin, 06-125CASE m-CP Selective Supplement 7,5 mg of Cephalotin, 1250 u.i. of Polymyxin B sulphate, VIAL CONTENTS: 200 mg of D-Cycloserine, 12,5 mg of Polymyxin 1 mg of Amphotericin B and solvent. B sulphate, 30 mg of 3-indoxyl-ß-D-glucopyranoside, 50 mg Each vial is sufficient to supplement 500 ml of Blood Columbia of Phenolphthalein diphosphate, 45 mg of Iron III Chloride, Agar Base (Ref. 01-034) and obtain 500 ml of Campylobacter and solvent. Selective Agar acc. Blaser-Wang. Each vial is sufficient to supplement 500 ml of m-CP Agar Base 06-102CASE MUG Supplement. Fluorescent Agent for Escherichia coli (Clostridium perfringens Agar) (Ref. 01-513). VIAL CONTENTS: 50 mg of MUG (4-methilumbeliferil-ß-D glucuronide) and solvent. Each vial is sufficient to supplement 500 ml of Coliforms Agar Storage conditions: or Broth. Should be stored at 2-8ºC in the dark. 06-106CASE Listeria Selective Supplement for Primary Enrichment (UVM I) Shelf life: VIAL CONTENTS: 10 mg of Nalidixic acid, 6 mg of Acriflavine and 18-24 months depending the reference. Larger vial-larger variety of supple- solvent. ments. Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base (UVM) (Ref. 02-472) in order to prepare 500 ml of Listeria Primary Enrichment Medium (UVM I formulation). 06-107CASE Listeria Selective Supplement for Enrichment acc. FDA and IDF/FIL VIAL CONTENTS: 20 mg of Nalidixic acid, 25 mg of Cycloheximide, MORE INFORMATION 7,5 mg of Acriflavine and solvent. AVAILABLE IN OUR WEB Each vial is sufficient to supplement 500 ml of Listeria Enrichment Broth Base acc. Lovett (Ref. 02-498). AND CD-ROM CATALOGUE 06-109CASE Oxford Agar Selective Supplement for Listeria VIAL CONTENTS: 5 mg of Phosphomycin, 1 mg of Sodium Cephotaxim, 10 mg of Colistin, 200 mg of Cycloheximide, 2,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Oxford Agar Base (Ref. 01-471) in order to prepare Listeria Selective Agar (Oxford formulation). 06-110CASE Palcam Agar Selective Supplement for Listeria VIAL CONTENTS: 5 mg of Polymyxin B sulphate, 10 mg of Sodium ceftazidime, 2,5 mg of Acriflavine and solvent. Each vial is sufficient to supplement 500 ml of Palcam Agar Base (Ref. 01-470) in order to prepare Listeria Selective Agar (Palcam formulation).

216 Selective Supplements

Culture media supplements in a practical presentation: an extemporaneous solution. Main advantages:

• fast • simple • easy • safe • ready to add • easy storage • longer shelf life • less risk of contamination

With this 10 vial-case format you no longer have to worry about things like sterile solvents, sterile syringes, sterilis- ing the supplements that must be added to the medium by ﬁltration ... With one simple pressure on the lid you obtain the sterile supplement solution, ready to add to the medium base. Selective supplements should be stored at 2-8°C in the dark. When stored as directed the reagents remain stable until the expiry date shown on the label.

Method of use: 4 simple steps 1 2 Press on the cap brea- The solid then falls king the container that into the solvent. Shake holds vigorously the solid. for total dissolution.

3 4 The supplement is now Homogenize and ready. Open the vial distribute into the suitable aseptically, close to a ﬂa- container: ﬂasks, tubes or me or in a safety cabinet. plates. Pour over the medium base, which has been cooled down to 45-50°C.

217 Basic Fuchsin 200 Selective Supplement

Ref. 06-617CASE F Precautions

R-11 • This product should be for laboratory use only. Contents S-7-16 • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 250 mL of Endo LES (Ref. 01-604). Applicable media Ref. 01-604 Endo LES Agar Base Vial contents Necessary amount for 250 mL of medium. Basic Fuchsin ...... 200 mg Ethanol ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 250 mL of Endo LES Agar Base. Note: Don’t heat the media once the supplement has been added.

Basic Fuchsin 250 Selective Supplement

Ref. 06-607CASE F

R-11 Precautions Contents S-7-16 • This product should be for laboratory use only. The box contains 10 vials. Each vial is sufﬁcient to sup- • Do not use beyond stated expiry date. plement 500 mL of Endo Agar Base (Ref. 01-589), Endo DEV Agar Base (Ref. 01-606) and only 250 mL of Endo Applicable media Base Broth (Ref. 02-605). Ref. 01-589 Endo Agar Base Ref. 01-606 Endo DEV Agar Base Vial contents Necessary amount for 500 mL of solid medium or 250 mL of liquid broth. Basic Fuchsin ...... 250 mg Ethanol ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to the boiled medium. Homogenize and use as per each monography medium. Do not remelt the solid media.

218 Brilliant Green + Novobiocin Selective Supplement

Ref. 06-017CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial contains inhibitors sufﬁcient to add to 500 mL of Tetrathionate Broth Base Applicable media Ref. 02-033 or 02-335 Muller Kauffmann Medium. Ref. 02-033 Tetrathionate Base Broth Ref. 02-335 Muller-Kauffmann Medium Vial contents Necessary amount for 500 mL of medium. Brilliant Green ...... 5 mg Novobiocin, sodium salt ...... 20 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the vial contents to 500 mL of boiled broth base cooled to 50°C. Homogenize and use as per each monography medium. Note: Don’t heat the media once the supplements have been added.

219 Chloramphenicol Selective Supplement

Ref. 06-118CASE T Precautions R-45 • This product should be for laboratory use only. Contents S-53-45 • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Sabouraud Dextrose Agar (Ref. Applicable media 01-165). Ref. 01-165 Sabouraud Dextrose Agar (Eur. Phar. Agar Medium C) Vial contents Necessary amount for 500 mL of medium. Chloramphenicol ...... 25 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C. Ho- mogenize and distribute the complete medium into the containers. Note: Don’t heat the media once the supplement has been added.

m-CP Selective Supplement

Ref. 06-125CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of m-CP Agar Base, Ref. 01-513, Applicable media in order to prepare 500 mL of m-CP (Clostridium perfrin- Ref. 01-513 m-CP Agar Base gens) Agar.

Vial Contents Necessary amount for 500 mL of complete medium. D-Cycloserine ...... 200,0 mg Polymixin B sulfate ...... 12,5 mg 3-Indoxyl-ß-D-Glucopyranoside ...... 30,0 mg Pehnolphthalein di-phosphate ...... 50,0 mg Iron III Cloride ...... 45,0 mg Distilled water ...... 5,0 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50ºC. Note: Don’t heat the media once the supplement has been added.

220 CP Gram-positive cocci in Blood Agar Selective Supplement

Ref. 06-013CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Blood Columbia Agar Base Ref. 01- 034 or Blood Agar Base Ref. 01-352 in order to prepare Applicable media 500 mL of Staphylococcus and Streptococcus selective Ref. 01-034 Blood Agar Base (Columbia) blood agar. Ref. 01-352 Blood Agar Base

Vial contents Necessary amount for 500 mL of complete medium. Colistin sulfate ...... 5,00 mg Nalidixic Acid, sodium salt ...... 7,50 mg Distilled Water ...... 5,00 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500mL of blood agar. Homogenize and use as per each monography medium. Note: Don’t heat the media once the supplements have been added.

221 Cycloheximide Selective Supplement

Ref. 06-022CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of WL Nutrient Agar or Broth Ref. Applicable media 01-210 or 2-210 in order to prepare 500 mL of WL Dif- Ref. 01-210 WL Nutrient Agar ferential Agar or Broth. Ref. 02-210 WL Nutrient Broth

Vial contents Necessary amount for 500 mL of complete medium. Cycloheximide ...... 2 mg Sodium chloride ...... 8 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C. Note: Don’t heat the media once the supplement has been added.

Cycloserine Selective Supplement

Ref. 06-116CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufficient to supplement 250 mL of Tryptose Sulfite Cycloserine Agar Applicable media Base (TSC Agar) Ref. 01-278. Ref. 01-278 Tryptose Sulfite Cycloserine Agar (TSC Agar) Vial contents Necessary amount for 250 mL of medium. D-Cycloserine ...... 100 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 250 mL of sterile agar base cooled to 50°C. If it is desired, add 20 mL of Egg Yolk Sterile Emulsion (Ref. 06-016). Homogenize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplements has been added.

222 Ferric Ammonium Citrate for Bacteriology

Ref. 06-112CASE Ref. 06-113CASE

Contents Contents The box contains 10 vials. Each vial is sufficient to sup- The box contains 10 vials. Each vial is sufficient to supple- plement 500 mL of Listeria Enrichment Broth acc. Fraser ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in Ref. 02-496 in order to prepare 500 mL of Fraser broth. order to prepare 500 ml of Lactose Sulfite Broth.

Vial contents Vial contents Necessary amount for 500 mL of complete medium. Necessary amount for 500 ml of complete medium. Ferric ammonium citrate ...... 250 mg Ferric ammonium citrate ...... 312 mg Distilled water ...... 5 mL Distilled water ...... 5 mL

Directions Directions Mix the liquid with the powder by pressing down on the Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution cap. Shake to dissolve and aseptically add the solution to to 500 mL of sterile broth base cooled to 50°C. 500 ml of sterile broth base cooled to 50°C. Note: Don’t heat the media once the supplement has Note: Don’t heat the media once the supplement has been been added. added.

Precautions Precautions • This product should be for laboratory use only. • This product should be for laboratory use only. • Do not use beyond stated expiry date. • Do not use beyond stated expiry date.

Applicable media Applicable media Ref. 02-496 Listeria Enrichment Broth Base Ref. 02-519 Lactose Sulﬁte Broth Base acc. to Fraser

Listeria Selective Supplement for Enrichment acc. FDA/IDF

T Directions Mix the liquid with the powder by pressing down on the Ref. 06-107CASE R-61-25-68 S-36/37-45-53 cap. Shake to dissolve and aseptically add the solution Contents to 500 mL of sterile broth base cooled to 50°C. Note: Don’t heat the media once the supplement has The box contains 10 vials. Each vial is sufﬁcient to sup- been added. plement 500 mL of Listeria Enrichment Broth Base acc. Lovett Ref. 02-498 in order to prepare 500 mL of Listeria enrichment broth according FDA and IDF/FIL. Precautions • This product should be for laboratory use only. Vial contents • Do not use beyond stated expiry date. Necessary amount for 500 mL of complete medium. Nalidixic acid, sodium salt ...... 20,0 mg Applicable media Cycloheximide ...... 25,0 mg Ref. 02-498 Listeria Enrichment Broth Base acc. Lovett. Acriﬂavine ...... 7,5 mg Distilled water ...... 5,0 mL

223 Listeria Selective Supplement for Primary Enrichment (UVM I)

T Precautions Ref. 06-106CASE • This product should be for laboratory use only. R-61-25-68 • Do not use beyond stated expiry date. S-36/37-45-53 Contents The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Listeria Enrichment Broth Base Applicable media (UVM) Ref. 02-472 in order to prepare 500 mL of Liste- Ref. 02-472 Listeria Enrichment Broth Base (UVM) ria primary enrichment medium.

Vial contents Necessary amount for 500 mL of complete medium. Nalidixic acid, sodium salt ...... 10 mg Acriﬂavine ...... 6 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile broth base cooled to 50°C. Note: Don’t heat the media once the supplement has been added.

Listeria Selective Supplement for Secondary Enrichment (UVM II / Fraser)

Ref. 06-111CASE T Precautions

R-61-25-68 • This product should be for laboratory use only. Contents S-36/37-45-53 • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Listeria Enrichment Broth Base (UVM) Ref. 02-472 in order to prepare 500 mL of Liste- Applicable media ria secondary enrichment medium (UVM II formulation); Ref. 02-472 Listeria Enrichment Broth Base (UVM) or to supplement 500 mL of Listeria Enrichment Broth Ref. 02-496 Listeria Enrichment Broth Base acc. to acc. Fraser Ref. 02-496 in order to prepare 500 mL of Fraser Fraser Broth.

Vial contents Necessary amount for 500 mL of complete medium. Nalidixic acid, sodium salt ...... 10,0 mg Acriﬂavine ...... 12,5 mg Distilled water ...... 5,0 mL

224 MUG Supplement

Ref. 06-102CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufficient to supplement 500 mL of Coliforms Agar or Broth. Applicable media Ref. 01-047 CLED Agar (Brolacin Agar) Vial contents Ref. 01-118 MacConkey Agar (Eur. Phar. Medium H) Necessary amount for 500 mL of complete medium. Ref. 01-164 Violet Red Bile Agar (VRB Agar) MUG (4-methilumbeliferil-ß-D-glucuronide) .....50 mg Ref. 01-220 Violet Red Bile Dextrose Lactose Agar Distilled water ...... 5 mL (VRBDL Agar) Ref. 01-484 E.coli Direct Agar (ECD Agar) Ref. 02-041 Brilliant Green Bile 2% Broth Directions Ref. 02-060 EC Broth Mix the liquid with the powder by pressing down on the Ref. 02-105 Lactose Broth (Eur. Phar. Broth Medium D) cap. Shake to dissolve and aseptically add the solution Ref. 02-108 Tryptose Lauryl Sulfate Broth to 500 mL of agar or broth cooled to 50°C. Ref. 02-118 MacConkey Broth MUG supplement may be added to almost all the media Ref. 02-120 MacConkey Modified Broth that allow the growth of Escherichia coli for its identifica- Ref. 02-611 MacConkey G Broth (Eur. Phar. Medium G) tion. However, results will be more reliable in all those media that are selective for coliforms. Attached is a list of the most currently used. In our Culture Media Handbook you will find a table indicating the aspect of Escherichia coli in each medium. Note: Don’t heat the media once the supplement has been added.

Nalidixic Acid Selective Supplement

Ref. 06-124CASE Directions Mix the liquid with the powder by pressing downd on the Contents cap. Shake to dissolve and asseptically add the solution The box contains 10 vials. Each vial is sufﬁcient to sup- to 500 mL of sterile agar base cooled to 50ºC. plement 500 mL of CN Selective Agar for Pseudomonas Note: Don’t heat the media once the supplement has (Ref. 01-609). been added.

Vial Contents Precautions Necessary amount for 500 mL of complete medium: • This product should be for laboratory use only. Nalidixic acid sodium salt ...... 7,5 mg • Do not use beyond stated expiry date. Distilled water ...... 5,0 mL Applicable media Ref. 01-609 CN Selective Agar Base for Pseudomonas.

225 Oxford Agar Selective Supplement

Ref. 06-109CASE R-25-52/53-61/68 S-36/37-45-53-61 Precautions Contents • This product should be for laboratory use only. The box contains 10 vials. Each vial is sufﬁcient to sup- •Do not use beyond stated expiry date. plement 500 mL of Oxford Agar Base Ref. 01-471 in order to prepare 500 mL of Listeria selective agar (Oxford formulation). Applicable media Ref. 01-471 Oxford Agar Base Vial contents Necessary amount for 500 mL of complete medium. Acriﬂavine ...... 2,5 mg Fosfomicyn ...... 5,0 mg Sodium cefotaxim ...... 1,0 mg Colystin ...... 10,0 mg Cycloheximide ...... 200,0 mg Distilled water ...... 5,0 mL

Oxytetracycline Selective Supplement

Ref. 06-115CASE Precautions Contents • This product should be for laboratory use only. The box contains 10 vials. Each vial is sufﬁcient to sup- • Do not use beyond stated expiry date. plement 500 mL of Sabouraud with Oxytetracycline Agar Base Ref. 01-275. Applicable media Ref. 01-275 Saboraud with Oxytetracycline Agar (OG- Vial contents YEA) Necessary amount for 500 mL of medium. Oxytetracycline HCl ...... 50 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 mL of sterile agar base cooled to 50°C. Homoge- nize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplement has been added.

226 Palcam Agar Selective Supplement

Ref. 06-110CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Palcam Agar Base Ref. 01-470 in order to prepare 500 mL of Listeria Selective Agar Applicable media (Palcam formulation). Ref. 01-470 Palcam Agar Base

Vial contents Necessary amount for 500 mL of complete medium. Acriﬂavine ...... 2,5 mg Polymixin B sulphate ...... 5,0 mg Sodium ceftazidime ...... 10,0 mg Distilled water ...... 5,0 mL

Polymixin B Sulfate Selective Supplement

Ref. 06-021CASE Precautions • This product should be for laboratory use only. Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Bacillus cereus Agar Base Ref. Applicable media 01-262. Ref. 01-262 Bacillus cereus Agar Ref. 01-487 Bacillus cereus Selective Agar Vial contents Necessary amount for 500 mL of medium. Polymixin B sulphate ...... 50 mg Distilled water ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap Shake to dissolve and aseptically add the solution to 450 mL of sterile agar base cooled to 50°C. Add also 50 mL of sterile Egg Yolk Emulsion. Homoge- nize and distribute the complete medium into the plates. Note: Don’t heat the media once the supplements have been added.

227 Rosolic Acid Selective Supplement

F Ref. 06-085CASE Precautions R-11 • This product should be for laboratory use only. S-7-16 Contents • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Fecal Coliforms Agar or Broth (m-FC) Ref.1-287 or 2-287 in order to prepare 500 mL of m-FC Applicable media complete medium. Ref. 01-287 Fecal Coliforms Agar (FC Agar) Ref. 02-287 Fecal Coliforms Broth (FC Broth) Vial contents Necessary amount for 500 mL of complete medium. Rosolic Acid ...... 50 mg Ethanol ...... 5 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake till total dissolution and aseptically add the solution to 500 mL of agar or broth cooled to 50°C. Use medium newly made. Note: Don’t heat the media once the supplement has been added.

228 SC Selective Supplement

Ref. 06-012CASE T N Precautions

R-25-32-42/43-51/53 • This product should be for laboratory use only. Contents S-7-22-24-37-45-61 • Do not use beyond stated expiry date. The box contains 10 vials. Each vial is sufﬁcient to supplement 500 mL of Blood Columbia Agar Base Ref. 01- Applicable Media 034 or Blood Agar Base Ref. 01-352 in order to prepare Ref. 01-034 Blood Columbia Agar Base 500 mL of Clostridium ssp. selective agar. Ref. 01-352 Blood Agar Base

Vial contents Necessary amount for 500 mL of complete medium. Sodium Azide ...... 120,0 mg Neomycine sulfate ...... 90,0 mg Distilled water ...... 5,0 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 475 mL of sterile agar base cooled to 50°C. Add 25 mL of deﬁbrinated blood . Homogenize and distribute into the plates. Note: Don’t heat the media once the supplements have been added.

Disodium disulﬁte (meta-bisulﬁte) for Bacteriology

Ref. 06-114CASE

Contents Precautions The box contains 10 vials. Each vial is sufficient to supple- • Reagent for laboratory use only. ment 500 ml of Lactose Sulfite Broth Base Ref. 02-519 in • Do not use beyond stated expiry date. order to prepare 500 ml of Lactose Sulfite Broth. Applicable Media Ref. 02-519 Supplement for Lactose Sulfite Broth Vial contents Necessary amount for 500 mL of complete medium. Disodium sulfite ...... 375,0 mg Distilled water ...... 5,0 mL

Directions Mix the liquid with the powder by pressing down on the cap. Shake to dissolve and aseptically add the solution to 500 ml of sterile broth base cooled to 50°C. Note: Don’t heat the media once the supplement has been added.

229 Reagents Barrit’s Reagent

Ref. 06-027 F Technique

R-11-36 Microorganism to be assayed is inoculated in MRVP Presentation S-7-16-26 Broth (Ref. 02-207) and is incubated at 30°C for a period 100 mL dropper flask between 3 and 5 days maximum. Just before read, add Barrit’s Reagent (Ref. 06-027) Specification until all the medium gets a milky look. Following, add O’Meara’s Reagent (Ref. 06-006) until the milky look Reagent for the Voges-Proskauer test in enterobacte- disappears and then shake chiefly. Relative volumes of riaceae. each reagent depend on the initial volume of inoculated medium. Description When test is positive a violet pinked colour appears be- All the enterobacteria ferment dextrose, but some spe- fore 5 minutes, starting from top. When test is negative cies like Klebsiella, Enterobacter, etc..., do it following there is no change ol colour. the 2-3-butanediol path and other species like E.coli, There is a quicker way to perform the Voges-Proskauer Salmonella, etc..., do it by the mix acid path. test, with very little volumes of medium and massive Voges Proskauer test shows the production of 2-3- inocules. This way allows very short incubations (18-20 butanediol and acetoine, that are only produced in big hours) and the read may be accelerated by heating up amounts in the 2-3-butanediol path. The basis of the test the culture almost to boiling after adding the reagents. is that these compounds, in alkaline medium and with However, this method increases the possibility of getting air, bear an oxidation and become diacethyl, which at the wrong results. same time reacts with guanidine producing very visible coloured compounds. O’Meara, in 1931, observed that adding creatine to the References BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids for the Voges Proskauer Test. Appl. Microbiol. 15:1138- diacethyl reaction with guanidine, and then it was easier 1141. to detect the red coloured compounds. BARRIT, M.M. (1936) The intensification of The Voges Later, in 1936, Barrit demonstrated that the addition Proskauer Reaction by the Addition of alpha-Naphtol. J. of an alcoholic solution of alpha-naphtol 5% (Barrit’s Pathol. Bacteriol. 42:441-453. Reagent, Ref. 06-027) increased very much sensibility, BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles and it was possible to obtain positive reaction even when of Biochemical Tests in Diagnostics Microbiology. John the final concentration of diacethyl was very low. It is Wiley Sons. N.Y. important to add the Barrit’s Reagent before the alkaline O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid solution. Method of Detecting the Formation of Acetylmethyl-carbinol by Bacteria Fermenting Carbohydrate. J. Pathol. Bacteriol. 34:401-406. McFADDIN, J.E. (2000) Biochemical tests for identification of medical bacteria. 3rd. Ed. Cippincott William & Wilkins. Philadelphia.

233 Crystal Violet Dye Solution for Gram Staining

Ref. 06-029 Xn Cover the preparation with Safranine Dye Solution (Ref. 06-032) and let it act for 1 minute. R-40-52/53 Presentation S-36/37-61 Wash gently to remove the excess of colouriser, putting 100 mL dropper flask the preparation in fluent water for 1-2 seconds. 1 L dropper flask Dry and observe under microscope in homogeneous inmersion. Description This solution has been prepared according to the speci- Microorganisms that get coloured by the first colouriser, fications by Hucker for the Gram staining, and it is very Crystal Violet, become dark blue coloured and it is said stable though, when it is too old, it may require filtration that they take the gram, and they are call grampositive immediately before the use. Elderliness does not affect (G+). Those microorganisms that just get coloured by the staining properties but may force to make decoulor- the contrast colouriser become red and they are called ing times longer. gramnegative (G-).

Technique Most of eukariote cells, except yeasts, are coloured as Fix the smear following the habitual method and let it gramnegative and thus the staining is not very significa- cool or dry. tive. In spite of, it is one of the first levels in the system- Cover the extension with Crystal Violet Dye Solution atic identification of prokariote: between the bacteria, all (Ref. 06-029) and let it act for 1 minute the coci, except Neisseria and Veillonella, are gramposi- Wash the exceed of colouriser. The best way is to put tive, and all the sporogen bacilli and some part of the the preparation in a precipitate glass with fluent water. other bacilli are grampositive too. Spiriles, vibria, rikett- Do not wash excessively. This step may be critical for sia, clamidia and most bacilli are gramnegative. the rest of the test. Should actinomycete presence is suspected or microor- Cover the preparation with Lugol Solution (Ref. 06-030) ganisms are not well coloured as grampositives, it is ad- and drain immediately. Cover again with new solution visable to use Crystal Violet with Anilin for Actinomycete. and let it act for 1 minute. Wash softly again with water. To put the preparation in water fluent for 5 seconds will be enough. References BARTHOLOMEW, J.W. (1962) Variables Influencing Re- Decolourate, pouring the Gram Decoluriser (Ref. 06- sults, and the Precise Definition of Steps in Gram Stain- 031), drop to drop, over the slanted microscopical slide ing as a Means of Standardizing the Results Obtained. until total decolourising. Anycase, this step may not be Stain Technol. 37:139-155. longer than 60 seconds. PAIK, G. (1980) Reagents, Stain and Miscellaneous Wash with water to stop the decolouring action. Procedures, in Manual of Clinical Microbiolgy by Lenette, Balows, Hausler and Truant (eds.). ASM, Washington. CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William & Wilkins. Baltimore.

234 Decolouriser for Gram Staining

Ref. 06-031 F Xi Dry and observe under microscope in homogeneous inmersion. R-11-36-66-67 Presentation S-7-9-16-26 100 mL dropper flask Microorganisms that get coloured by the first colouriser, 1 L dropper flask Crystal Violet, become dark blue coloured and it is said that they take the gram, and they are call grampositive (G+). Those microorganisms that just get coloured by Description the contrast colouriser become red and they are called Gram’s decolouriser is a mixture of alcohol and acetone gramnegative (G-). especially adapted to act softly and quickly over base colourings. It use to be enough 15 or 20 drops to achive Most of eukariote cells, except yeasts, are coloured as an total decolourising of a correctly coloured smear. gramnegative and thus the staining is not very significa- tive. In spite of, it is one of the first levels in the system- Technique atic identification of prokariote: between the bacteria, all Fix the smear following the habitual method and let it the coci, except Neisseria and Veillonella, are gramposi- cool. tive, and all the sporogen bacilli and some part of the Cover the extension with Crystal Violet Dye Solution other bacilli are grampositive too. Spiriles, vibria, rikett- (Ref. 06-029) and let it act for 1 minute sia, clamidia and most bacilli are gramnegative. Wash the exceed of colouriser. The best way is to put the preparation in a precipitate glass with fluent water. Should actinomycete presence is suspected or microor- Do not wash excessively. This step may be critical for ganisms are not well coloured as grampositives, it is ad- the rest of the test. visable to use Crystal Violet with Anilin for Actinomycete.

Cover the preparation with Lugol Solution (Ref. 06-030) and drain immediately. Cover again with new solution References and let it act for 1 minute. BARTHOLOMEW, J.W. (1962) Variables Influencing Re- Wash softly again with water. To put the preparation in sults, and the Precise Definition of Steps in Gram Stain- water fluent for 5 seconds will be enough. ing as a Means of Standardizing the Results Obtained. Stain Technol. 37:139-155. Decolourate, pouring the Gram Decoluriser (Ref. 06- PAIK, G. (1980) Reagents, Stain and Miscellaneous 031), drop to drop, over the slanted microscopical slide Procedures, in Manual of Clinical Microbiolgy by Lenette, until total decolourising. Anycase, this step may not be Balows, Hausler and Truant (eds.). ASM, Washington. longer than 60 seconds. CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. Wash with water to stop the decolouring action. William & Wilkins. Baltimore. Cover the preparation with Safranine Dye Solution (Ref. 06-032) and let it act for 1 minute. Wash gently to remove the excess of colouriser, putting the preparation in fluent water for 1-2 seconds.

235 Kovac’s Reagent

Ref. 06-018 C Since indole is one of the most volatile compounds of the group, some authors choose to conduct the test R-10-20-34 Presentation S-26-36/37/39-45 using strips impregnated in reagent. Others suggest 100 mL flask placing the reagent directly on the cap’s swabs, avoiding 1L in flask all contact with the culture medium so that the reaction will occur with just the indole’s steam. Isenberg and Suddenheim demonstrated that if a previous extraction Description with Toluene was performed, only indole and Alpha- Many microorganisms can produce indole (=benzopyr- Methylindole (=indoleacetic acid) were detected. Such role) from Tryptophane thanks to a Tryptophanase, in is the most common practice nowadays when using the a process favored by oxygen and inhibited by glucose. Ehrlich-Böhme reagent. We therefore recommend that media used in this test contain no glucose, present a high Tryptophane content and are incubated aerobically. Technique The indole production property constitutes a classical When conducting the indole production test on vari- test for the differentiation of Escherichia and Entero- ous groups of bacteria, an appropriate reagent for each bacter, integrated in the IMViC. It is also widely used for group must be considered. Kovacs’s reagent (Ref. the differentiation of other non enteric microorganisms. 06-018) is recommended for enterobacteria while the The indole test can be conducted by various means, still Ehrlich reagent is for non-fermentating and anaerobes in the biochemical basis of the reaction remain the same. general. When a Pyrrol is mixed with a heated alcoholic p-dimeth- The directions to follow during the assay are: ylaminobenzaldehyde solution, a peculiar cherry-red Inoculate the pure culture to be verified in a high Tryp- coloring develops (Rosindole). If the reagent’s solution tophane content medium, as for example the Indole-Ni- is prepared with concentrated hydrochloric acid it will not trite Fluid Medium (Ref. 03-101), the SIM Medium (Ref. be necessary for it to progress in hot, as is the case with 03-176) or a non-glucose tryptone broth. Incubate at our reagents. 35°C for 48 hours. Incubation time can be reduced to 4 The reagent which was initially used was that of Ehrlich- hours if a massive inoculum in solid medium is done fol- Böhme (Ref. 06-024) with a previous extraction and con- lowed by seeding of a small volume (0,5 mL) of culture centration in Xylene, Ether, Chloroform or Toluol. Later medium. on Kovacs modified the original reagent by replacing the ethanol with amyl-alcohol, so that a previous extrac- In both cases, examination after incubation in the follow- tion was no longer necessary. In 1956 Gadenbusch and ing way: Gabriel proved that the Kovacs’s reagent was much a) Kovacs’s Reagent (Ref. 06-018, for enterobacteria) stable if instead of Amyl alcohol, Butilic or Isoamyl were Add 0,5 mL of reagent to the broth’s surface, shaking used. lightly to help extraction. If a cherry-red color develops in Nevertheless, the indole test reaction with paradiami- less than a minute it will be considered a POSITIVE RE- nobenzaldehyde is not very specific since at least 17 ACTION. No change in the original coloring constitutes a compounds close to Indole are known to react similarly. negative reaction. Although other reagents such as Oxalic acid and Hy- droxilamine HCI have been proposed, their use has not b) Ehrlich-Böhme Test been widespread. Add 1 mL of Xylene or Toluene to the broth and shake energically to help extraction. Allow to stand for 2 minutes until both layers separate. Then slide 0.5 mL of the reagent carefully down the sides of the tube, making sure there is no agitation. Should a dark red colored ring appear in the interface, it will be considered a POSITIVE REACTION.

Storage Reagents must be stored refrigerated and avoiding direct light.

References BÖHME, A. (1905) Die Anwendung der Ehrlichschen In- dolreaktion für bakteriologische zwecke. Zentralbl. Bakt. Parasit. Abt 1, Jena 40:129-133

236 Kovac’s Reagent

KOVACS, N. (1928) Eine vereinfachte Methode zum Nachweis der Indolbihdung duch bakterien. Z. Immuni- Kovac’s reaction on: Ref. 02-277 Tryptone Phosphate tats. Forsch. Exp. Ther. 55:311-315 Water, Ref. 02-460 Tryptose Lauryl Sulfate Mannitol GADEBUSCH, H.H. and GABRIELS, S. (1956) Modified Tryptophan Broth, Ref. 02-494 Buffered Peptone Water Stable Kovacs’s Reagent for the detection of Indol. Am. Eur. Phar., Ref. 03-156 Tryptone Water. J. Clin. Pathol. 26:1373-1375 ISENGERG, H.D. and SUNDHEIM, L.H. (1958) «Indole Reactions in Bacteria» J. Bact. 75:682-690 CENTER FOR DISEASE CONTROL (1968)»Identifica- tion of Unusual Pathogenic Bacteria» Atlanta G. VIRGINIA POLYTECHNICAL INSTITUTE (1972) Anaer- obe Laboratory Manual Blaksburg,Va. EDWARDS, P.R. and EWING, W.H. (1972) «Identifica- tion of Enterobacteriaceae» 3rd Ed. Burgess Pub. Co. Minneapolis McFADDIN, J.F. (2000) Biochemical tests for identification of medical bacteria. 3rd. Ed. Lippincott William & Wilkins. Philadelphia. ISO 9308-2 Standard (1990) Water Quality - Detection of coliforms thermotolerant coliforms and presumptive E.coli - MPN method.

Left: control; center: Salmonella typhimurium ATCC 14028 (negative reaction); right: Escherichia coli ATCC 25922 (positive reaction).

Lactophenol Blue

Ref. 06-037 C efficacy in the staining of moulds and plants material has been demonstrated. R-21/22-34-41 Presentation S-26-36/37/39-45 100 mL flask Technique 1 L flask Put a bit of mould to be assayed in a clean microscopical slide and pour one or two drops of Lactophenol Blue. Specification Disperse the material with two needles, mixing it with the Reagent for staining of fungi in fresh and fixed prepara- colouriser. tions. Add a couple of drops of water and homogenize all before putting the overglass. Heat slightly the preparation over a flave until it will be Description almost boiling. In that precise moment, press to remove Lactophenol Blue is an excellent colouriser for fresh all the excess of liquid and seal the borders with vaspar preparation of fungi, since it has, in a single solution, the or nails varnish. propierties of a mordant, a fixer and a colouriser. Preparation is ready for the microscopical examination. In fungi preparations for microscopical examination is not usual to use water neither aquose colouriser solutions, since most of the moulds expel water and remain References trapped in air microbubbles. This reason makes Lac- HARRIGAN, W.F. and McCANCE, M.E. (1976) Labora- tophenol Blue an idoneous medium for the examination, tory Methods in Food and Dairy Microbiology. Academic becase it moistens the structures at the same time that it Press. London. acts like a fixer and soft mordant. CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. In other hand, its nature makes the preparations useful William and Wilkins. Baltimore longer because evaporation is smaller. This effect can be LARONE, D.H. (2002) Medically important fungi. ASM enhanced if the preparations are sealed with vaspar or Press. Washington, DC. nails varnish. The Cotton Blue, China Blue or Soluble Anilin Blue is probably, an impurified and complex colouriser but, its

237 Lugol Solution for Gram Staining

Ref. 06-030 Dry and observe under microscope in homogeneous Presentation inmersion. 100 mL dropper flask 1 L dropper flask Microorganisms that get coloured by the first colouriser, Crystal Violet, become dark blue coloured and it is said that they take the gram, and they are call grampositive Description (G+). Those microorganisms that just get coloured by Iodine solution has been prepared according to the the contrast colouriser become red and they are called specifications by Burke, therefore it is more stable than gramnegative (G-). the classical Lugol formulation, and it does not affect the colouring. The solution may be stored for months at Most of eukariote cells, except yeasts, are coloured as room temperature, but if a characteristic amber colour is gramnegative and thus the staining is not very significa- observed, it must be discarded. tive. In spite of, it is one of the first levels in the systematic identification of prokariote: between the bacteria, all Technique the coci, except Neisseria and Veillonella, are gramposi- Fix the smear following the habitual method and let it tive, and all the sporogen bacilli and some part of the cool. other bacilli are grampositive too. Spiriles, vibria, rikett- Cover the extension with Crystal Violet Dye Solution sia, clamidia and most bacilli are gramnegative. (Ref. 06-029) and let it act for 1 minute Wash the exceed of colouriser. The best way is to put Should actinomycete presence is suspected or microor- the preparation in a precipitate glass with fluent water. ganisms are not well coloured as grampositives, it is ad- Do not wash excessively. This step may be critical for visable to use Crystal Violet with Anilin for Actinomycete. the rest of the test.

Cover the preparation with Lugol Solution (Ref. 06-030) References and drain immediately. Cover again with new solution BARTHOLOMEW, J.W. (1962) Variables Influencing Re- and let it act for 1 minute. sults, and the Precise Definition of Steps in Gram Stain- Wash softly again with water. To put the preparation in ing as a Means of Standardizing the Results Obtained. water fluent for 5 seconds will be enough. Stain Technol. 37:139-155. PAIK, G. (1980) Reagents, Stain and Miscellaneous Decolourate, pouring the Gram Decoluriser (Ref. 06- Procedures, in Manual of Clinical Microbiolgy by Lenette, 031), drop to drop, over the slanted microscopical slide Balows, Hausler and Truant (eds.). ASM, Washington. until total decolourising. Anycase, this step may not be CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. longer than 60 seconds. William and Wilkins. Baltimore. Wash with water to stop the decolouring action.

Cover the preparation with Safranine Dye Solution (Ref. 06-032) and let it act for 1 minute. Wash gently to remove the excess of colouriser, putting the preparation in ﬂuent water for 1-2 seconds.

238 Malachite Green

Ref. 06-038 Technique Prepare a smear of microorganism, in the habitual way, Presentation and let it dry. Fix strongly by passing the microscopical 100 mL flask slide over a flame about 20 times. 1 L flask Before doing the staining, let cool the microscopical slide. Cover all the smear with Malachite Green for Description Spores Stain and let it act for 10 minutes. Wash with water to remove the excess of colouriser. Malachite Green for Spores has been prepared accord- Contrast by covering the smear with Safranine Dye Solu- ing to the formulation by Barholomew and Mittwer, in tion (Ref. 06-032) and letting it act for 15-30 seconds. 1950, which was a modification over the procedure by Wash again, dry and perform the microscopical exami- Schaeffer and Fulton, in 1933, that was based in the nation in homogeneous inmersion. original one by Wirtz. Cellular bodies appear red or pinked coloured, whereas spores are green. Essencially, the modification is the supression of the step Should the Schaeffer’s technique is wanted to be used where the preparation was heated up. To achieve this diluting the colouriser, once the smear is covered with without affecting the test, they prepared a more concen- Malachite Green, do not let it act for 10 minutes and trated colouriser and let it act longer, and afterwards they instead of this, bring it to the boiling 3 consecutive times. did the contrast with a softer colouriser, Safranine Dye Then follow the described technique. Solution (Ref. 06-032).

This stain is a saturated and stabilized aquose solution References of malachite green. To use it with the Schaeffer tech- BARTHOLOMEW, J.W., MITTWER, T. (1950) A Simpli- nique it has to be diluted at 50% to avoid the formation ﬁed Bacterial Spore Stain. Stain Technol. 24:153-156. of precipitates. SCHAEFFER, A.B., M. FULTON (1933) A Simpliﬁed Method of Staining Endospores. Science, 77, 194. CLARK, G. (Ed.) (1981) Staining Procedures. 4th. Ed. William and Wilkins. Baltimore.

239 Methyl Red

Ref. 06-007 F Technique

R-11 Inoculate a tube of MRVP Broth (Ref. 02-207) and Presentation S-7-16 incubate at 30°C for 3-5 days. Take 5 mL of culture and 100 mL flask transfer them to a clean tube and then add 5 or 6 drops 1 L flask of indicator. Positive reaction is shown by the presence of a red Specification colour, whereas the negative reaction produce a yellow or orange colour. Indicator solution for the fermentation test in enterobac- Most cases, a 48 hours incubation at 37°C is enough, teria. but if the results are doubtous, the assay must be repeated incubating at 30°C for 5 days. Description There is a quicker way to perform the test: suspend a Clark and Lubs, in 1915, described the Methyl Red test loop of bacterial growth from a solid medium in 0,5 mL to distinguish between the E. coli group and Entero- of Azide Dextrose Broth acc. to Rothe (Ref. 02-027) and bacter. incubate at 37°C for 18 hours. Add a couple of drops of All the enteric bacteria ferment dextrose, some do it indicator and read the results as in the last case. following the 2-3-butanediol path, like Klebsiella, En- terobacter, etc..., and other follow the mix acid path, like E.coli, Salmonella, etc...In this last case, there is an References BARRY, A.L., K.L. BERNSOHN, A.P. ADAMS, L.D. accumulation of acid products produced by the sugar THRUPP (1970) Improved 18-hour methyl red test. Appl. unfoldment. This causes a decrease of pH, reaching a Microbiol. 20:886-870. value up to 4 after the incubation. Adding the methyl red CLARK, W.M. and LUBBS, H.A. (1915) The Differentia- alcoholic solution the change of the indicator may be de- tion of Bacteria of the Colon-Aerogenes Family by the tected: it remains yellow over pH 5,1 and red below pH use of indicators. J. Infect. Dis. 17:161-173. 4,4. If the change is positive it means that the dextrose EDWARDS, P.R., W.H. EWING (1972) Identification of fermentation followed the mix acid path, since in the 2- Enterobacteriaceae. 3rd. Ed. Burgess Pub Co. Minne- 3-butanediol path, after 3 days of incubation, predomi- apolis. nate the neutral or alkaline products. BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles Too early reads may produce false positive results. of Biochemical Tests in Diagnostics Microbiology. John Wiley Sons. N.Y. McFADDIN, J.F. (2000) Biochemical tests for identification of medical bacteria. 3rd. Ed. Lippincott William and Wilkins. Philadelphia.

240 Nitrates Reduction Reagents

Nitrates A Solution The scheme for the global process is the following:

C Ref. 06-003 R-10-35 S-23.2-51-26-36/37/39-45 Presentation 100 mL dropper ﬂask

Nitrates B Solution

C Ref. 06-004 R-10-35 Generally, the Griess-Ilosvay’s reagents detect the S-23.2-51-26-36/37/39-45 Presentation presence of nitrites with bacterial origin in a medium that 100 mL dropper flask initially has no nitrites (Indole Nitrite Fluid Medium, Ref. 03-101 and Nitrate Broth, Ref. 02-138). The scheme for the complete reaction is the following: Specification Griess-Ilosvay’s Reagents for the verification of the nitrates reduction through nitrites detection.

Description To use them, mix equal parts of the solutions A and B. Once they are mixed, the reagents are stable just for a few hours. Alone they may be stored for several months at room temperature. Nitrates B solution may produce a slight cristalization that does not affect its efﬁcacy. This process is accelerated with refrigeration, therefore it is recommended not to store them in the refrigerator.

Nitrates reduction in bacteria is performed through sev- References eral ways and it obeys different procedures. WALLACE, G.I., S.L. NEAVE (1927) The nitrite test as applied to bacterial cultures. J.Bact. 14:377-384. Nitrates Assimilation involves a reduction to ammonia BLAZEVIC, D.J., G.M. EDERER (1975) Principles of in several steps where nitrite may be detected. The am- Biochemical Tests in Diagnostics Microbiology. John monia that is produced is finally incorporated to the cel- Wiley Sons. NY. lular material. However, in the Deassimilation process, FORBES, B.A., D.F. SAMM, A.S. WEISSFELD (1998) nitrite is used as the final receiver of electrons, and thus, Bailey & Scott’s Diagnostics Microbiology. 10th. Ed. more than an assimilation process it is an energetic re- Mosby. St. Louis. action of respiration without oxygen, and this fact allows GRIESS, P. (1879) Liebereinige Azoverbindungen. Ber. the facultative growth of many aerobic in anaerobiosis. Deutsch. Chem. Geselkch. 12:426-427. In this case, it is usual the presence of nitrite acumula- McFADDIN, J.F. (2000) Biochemical Tests for identifica- tions, which may be toxical for the microorganism. In tion of medical bacteria. 3rd. Ed. Lippincott William and other cases, nitrate may reduce itself to gas states and it Wilkins. Philadelphia. is expeled as free nitrogen bubles. This process is called

Denitriﬁcation, since it makes the active ion (NO3) an inert gas (N2).

241 O’Meara’s Reagent

Ref. 06-006 C Technique Microorganism to be assayed is inoculated in MRVP R-22-35 Presentation S-26-36/37/39-45 Broth (Ref. 02-207) and is incubated at 30°C for a period 100 mL dropper flask between 3 and 5 days maximum. Just before read, add Barrit’s Reagent (Ref. 06-027) Specification until all the medium gets a milky look. Following, add O’Meara’s Reagent (Ref. 06-006) until the milky look Reagent for the Voges-Proskauer test in enterobacte- disappears and then shake chiefly. Relative volumes of riaceae. each reagent depend on the initial volume of inoculated medium. Description When test is positive a violet pinked colour appears be- All the enterobacteria ferment dextrose, but some spe- fore 5 minutes, starting from top. When test is negative cies like Klebsiella, Enterobacter, etc..., do it following there is no change ol colour. the 2-3-butanediol path and other species like E.coli, There is a quicker way to perform the Voges-Proskauer Salmonella..., do it by the mix acid path. test, with very little volumes of medium and massive Voges Proskauer test shows the production of 2-3- inocules. This way allows very short incubations (18-20 butanediol and acetoine, that are only produced in big hours) and the read may be accelerated by heating up amounts in the 2-3-butanediol path. The basis of the test the culture almost to boiling after adding the reagents. is that these compounds, in alkaline medium and with However, this method increases the possibility of getting air, bear an oxidation and become diacethyl, which at the wrong results. same time reacts with guanidine producing very visible coloured compounds. O’Meara, in 1931, observed that adding creatine to the References BARRY, A.L., K.L. FEENEY (1967) Two Quick Methods alkaline solution (O’Meara’s Reagent, Ref. 06-006) aids for the Voges Proskauer Test. Appl. Microbiol. 15:1138- diacethyl reaction with guanidine, and then it was easier 1141. to detect the red coloured compounds. BARRIT, M.M. (1936) The intensification of The Voges Later, in 1936, Barrit demonstrated that the addition Proskauer Reaction by the Addition of alpha-Naphtol. J. of an alcoholic solution of alpha-naphtol 5% (Barrit’s Path. Bact. 42:441-453. Reagent, Ref. 06-027) increased very much sensibility, BLAZEVIC, D.J. and EDERER, G.M. (1975) Principles and it was possible to obtain positive reaction even when of Biochemical Tests in Diagnostics Microbiology. John the final concentration of diacethyl was very low. It is Wiley Sons. N.Y. important to add the Barrit’s Reagent before the alkaline O’MEARA, R.A.Q. (1931) A simple, Delicated and Rapid solution. Method of Detecting the Formation of Acetylmethyl-carbinol by Bacteria Fermenting Carbohydrate. J. Pathol. Bact. 34:401-406. McFADDIN, J.F. (2000) Biochemical tests for identification of medical bacteria. 3rd. Ed. Lippincott William and Wilkins. Philadelphia.

242 Oxidase Reagent

Ref. 06-057 The basis of the test is the following: Citochromes are hemoproteins that act as oxidant enz- Presentation imes in the transportant chains of oxidative phosphorila- 5 g flask tion electrons. Generally, citochromes are found just in the aerobic bacteria, whereas the strict anaerobic does not present them. Specification Citochrome-oxidase found even in the enterobacte- Reagent for the detection and verification of bacterial riaceae is composed by a citochrome. However, the citochrome-oxidase. 3 substance that the Oxidase Reagent is able to reduce is c citochrome, thus the test just determinates the pres- Description ence of c citochrome and therefore test is positive only The chemical formulation for the Oxidase Reagent is for the bacteria that have c citochrome in their respira- N,N-dimethyl-P-phenyldiamine-2-HCl, and it is also tory chains. designated as 4-amino-N,N-dimethylaniline-2-HCl. It is advisable to store it in powder at 4°C since it has a very Reagent is very unstable to oxygen, and light acts as a short life time when is dissolved. catalyst in the self oxidation and so the reagent may be stored in amber flask. When the reaction in the colony is Directions very slow, it has to be considered negative or very weak, Prepare an aquose solution of Oxidase Reagent 1% im- since the lately appaerance of colour is more attributable mediately before the use. It is recommended to prepare to the spontaneous oxidation than to the truly presence just the amount that is going to be used, since once of c citochrome. diluted it will work just for a week, even if it is kept at Generally, except a few cases, oxidase production is 4°C and avoiding direct light. Althought self oxidation is linked to flagelation in the following way: Polar flagela- restrained with the addition of ascorbic acid 0,01%, if the tion is related with oxidase positive; peritrichous flagela- liquid is dark it must not be used. The normal colour for tion is related with oxidase negative. the solution is transparent or slightly pinked. The oxidation of c citochrome for the positive reaction of oxidase is the following and it may be observed that Technique molecullar oxygen is absolutely necessary: There are several techniques to determinate citrocrome- oxidase in the different genus. The more standardized are the following: a) Soak a filtration paper disc with the reagent and put it over a clean Petri plate. Take a colony and spread it over the paper. This step must be performed with a Platinum-Iridium loop (Ref. 5-006) or a Pasteur pi- References pette. The use of metallic objects (nicrom loop, etc...) GABY, W.L., C. MARTLEY (1957) Practical laboratory may produce wrong positive results. test for the identification of Pseudomonas aeruginosa. J. b) Flood the colony with reagent directly in the plate. Fol- Bact. 74:356-358. lowing this way, colonies are not able to subculture, BLAZEVIC, D.J., G.M. EDERER (1975) Principles of but the test does not interfere with the Gram staining biochemical test in diagnostic Microbiology. John Wiley and the colony may be observed at the microscope. Sons. NY. Positive reaction is shown by the presence of a pink FORBES, B.A., D.F. SAHM, A.S. WEISSFELD (1998) colouring, that becomes dark red and finally black after Bailey & Scott’s Diagnostic Microbiology. 10th. Ed. 10 minutes. Mosby. St. Louis. ISO 9308-2 Standard (1990) Water Quality - Detection of coliforms, thermotolerant coliforms and presumptive E.coli - MPN method.

243 Safranin Dye Solution for Gram Staining

Ref. 06-032 Dry and observe under microscope in homogeneous inmersion. Presentation 100 mL dropper flask Microorganisms that get coloured by the first colouriser, 1 L dropper flask Crystal Violet, become dark blue coloured and it is said that they take the gram, and they are call grampositive (G+). Those microorganisms that just get coloured by Description the contrast colouriser become red and they are called The contrast colouriser is composed by the classic gramnegative (G-). Safranine solution 0,25%. It is demonstrated that this solution is more effective than the fuchsine. This con- Most of eukariote cells, except yeasts, are coloured as trast colouriser is employed also in many other staining gramnegative and thus the staining is not very significa- methods. tive. In spite of, it is one of the first levels in the systematic identification of prokariote: between the bacteria, all Technique the coci, except Neisseria and Veillonella, are gramposi- Fix the smear following the habitual method and let it tive, and all the sporogen bacilli and some part of the cool. other bacilli are grampositive too. Spiriles, vibria, rikett- Cover the extension with Crystal Violet Dye Solution sia, clamidia and most bacilli are gramnegative. (Ref. 06-029) and let it act for 1 minute Wash the exceed of colouriser. The best way is to put Should actinomycete presence is suspected or microor- the preparation in a precipitate glass with fluent water. ganisms are not well coloured as grampositives, it is ad- Do not wash excessively. This step may be critical for visable to use Crystal Violet with Anilin for Actinomycete. the rest of the test.

Cover the preparation with Safranine Dye Solution (Ref. 06-032) and let it act for 1 minute. Wash gently to remove the excess of colouriser, putting the preparation in ﬂuent water for 1-2 seconds.

244