Methods for Food- Borne Pathogens Detection and Enumeration Sabina Purkrtová Methods in Microbiology

Methods for food- borne pathogens detection and enumeration Sabina Purkrtová Methods in microbiology

Analyte – the measured component of a sample Analytes in microbiology

microorganisms as microbial cells

component of microorganisms – e.g. DNA, proteins

products of microorganisms – e.g. enterotoxins of S. aureus presence or absence of the amount of the analyte analyte measured either directly or in a certain amount of sample indirectly in a certain amount of sample qualitative method quantitative method

Amount: according to the legislative Amount: according to the legislative in EU usually 25 g (or occasionally 10 g) in EU usually 10 g Types of methods

1. International, regional or national standards

• contain sufficient and concise information on how to perform We're ISO, the the tests and/or calibrations International Organization for • do not need to be supplemented or rewritten as Standardization. We international procedures (if these standards are written in develop and publish International Standards. a way that they can be used as published by the operating staff in a lab)

• optional steps in the methods or additional details shall be documented

• ISO, International Dairy Foods Association (IDFA), FDA (Food and Drug Administration), AIJN (European Fruit Juice Association

2. Laboratory-developed methods

3. Non-standard methods Microbiological analysis procedures

CONVENTIONAL CELL CULTIVATION • relatively easy to use, but time (requires several days), labour (lots of procedural steps) and material consuming • many of them are recognised as approved for ISO and they are gold standard procedures • Colony count method (CCM) • pour plate techniques • spread plate techniques • Membrane filtration • Most Probable Number (MPN) RAPID METHODS • immunonological method (based on antigen/antibody-binding) • based on molecular biological method (based on PCR) • others (ATP Photometry, Direct Epifluorescent Filter Techniques (DEFT), Electrical impedance method, Flow cytometry, etc.) Phylogenetic tree of life

Γ-Proteobacteria: Enterobacteriaceae ε-Proteobacteria: Campylobacter sp.

http://www.sciencedirect.com/science/article/pii/S000527280600140X Cultivation methods Cultivation methods - classical microbiology methods Microorganisms are studied not as single cells, but like a mass population of daughter cells arisen from one single mother cell by multiplication

http://www.blackmould.me.uk/images/FungalLifeC ycle.gif Asexual life cycle of toxic black mould

Yeast: budding (most https://www.kullabs.com/uploads/Notes/PXlu4LSHCNgjaCpM.jpg yeasts), binary fission (Schizosaccharomyces sp.)

http://leavingbio.net/BACTERIA%20Page_files/image011.jpg 6 Bacteria: binary fission https://kollokvium.files.wordpress.com/2014/07/saccharo.png Cultivation methods Cultivation methods - classical microbiology methods Microorganisms are studied not as single cells, but like a mass population of daughter cells arisen from one single mother cell by multiplication

Example: Growth of bacteria in the logarithmic

phase (generation time: 0.5 h)

(number of cells) of (number

10 Log

Hours http://www.cs.montana.edu/webworks/projects/ stevesbook/artifacts/images/chapter_002/section 002/typical_growth_curve_002.jpg

7 Cultivation methods This population („mass of cells“) is visible on solid media as a colony Growth of a colony from one mother cell (teoretically)

Video: https://www.youtube.c om/watch?v=zrx7Xg0g kQ4 http://ibis.inrialpes.fr/rubrique51.html a well-isolated colony grown from only one mother cell ~ ideal state, but it can be theoretic (some cells in clumps etc.) the number of colonies = number of colony forming units (CFU) (not number of „cells“)

Those populations are visible to grow in Those populations are able to give the liquid media (broth) detectable signal in biochemical testing

8 http://accounts.smccd.edu/case/biol240/images/nutrientbroth2.jpg Cultivation methods The ability of microorganism to survive and to multiplicate („to grow“) under given envirommental conditions depends on the compatibility of these conditions and the physiological and biochemical featrues of the microorganism

Physical factors Chemical factors • temperature* • Nutrition requirements* • pH* • Source of carbon • osmotic pressure* • Source of nitrogen • atmosphere* • Source of energy • pressure • Other • UV radiation (UV, γ...) • Antimicrobial and (bactericidal/bacteriostatic) and other chemical compounds*

Biological factors Physiological state of cells („fitness“) • The influence of other • cultivable cells microorganisms (synergism -helping • cells in good state grow or survive , competition, • cells demaged or suppressed by the antagonism, symbiosis, food processing, conditions… mutualism…) • uncultivable cells

*...Factors widely used in food microbiology cultivation methods 9 Cultivation methods

http://image.slidesharecdn.com/microbiologyunit2-3-150710082502-lva1-app6892/95/microbiologyunit-23-bacteria-36-638.jpg?cb=1436518851 Cultivation methods

obligate anaerobes: oxygen is toxic for them

https://o.quizlet.com/CXi.qp97gMpOFIXSYF0skA.png Cultivation methods

Bacteria Tmin (°C) Topt (°C) Tmax (°C) Listeria monocytogenes 1 30-37 45 Vibrio marinus 4 15 30 Pseudomonas maltophilia 4 35 41 Thiobacillus novellus 5 25-30 42 Staphylococcus aureus 10 30-37 45 Escherichia coli 10 37 45 Cultivation temperature for yeasts and Clostridium kluyveri 19 35 37 moulds: 25 °C. Streptococcus pyogenes 20 37 40 Streptococcus 25 37 42 pneumoniae Bacillus flavothermus 30 60 72 Thermus aquaticus 40 70-72 79

Archaebacteria Tmin (°C) Topt (°C) Tmax (°C) Methanococcus 60 85 90 jannaschii Sulfolobus acidocaldarius 70 75-85 90 Pyrobacterium brockii 80 102-105 115 12 Cultivation methods

Teplotní rozsah růstu 27 kvasinek. Optimální růstová teplota je označena kolečkem. S., Saccharomyces Sc, S. cerevisiae; Sp, S. paradoxus; Smik, S. mikatae; Sarb, S.arboricolus; Scar, S.cariocanus; Su, S. bayanus var. uvarum; Sk, S. kudriavzevii; Km, Kluyveromyces marxianus; Pf, Pichia fermentans; Td,Torulaspora delbrueckii; Cs, Candida stellata; Hu, Hanseniaspora uvarum.

Salvadó Z et al. Appl. Environ. Microbiol. 2011;77:2292-2302 Cultivation methods

http://agrosum14.weebly.com/uploads/4/4/9/5/44958631/4965520_orig.jpg Cultivation methods

http://image.slidesharecdn.com/microbiologicalprinciples-110819155057-phpapp01/95/food- microbiological-principles-fst-241-32-728.jpg?cb=1313769603 Cultivation methods

http://image.slidesharecdn.com/factors affectingthegrowthandsurvivalofmicro- organismsinfoods-150306050511- conversion-gate01/95/factors-affecting- the-growth-and-survival-of-microorganisms-in-foods-30- 638.jpg?cb=1425640055

http://aqualab.decagon.com.br/assets/Article-Graphics/Key-concepts-in-water-activity-Table-1.JPG Nutrition requirement

http://www.easynotecards.com/uploads/929/65/_4a88b4c5_140c98ff4b1__8000_00008247.png

17 Nutrition requirement

http://www.oxfordscholarship.com/doc/10.1093/acprof:oso/978 0199586936.001.0001/acprof_9780199586936_graphic_012.gif Most bacteria important in clinical and food microbiology, all yeasts and moulds belong to chemoheteroorganotrophes: the source of carbon and energy and redox compound are organic compounds. 18 Culture media - purpose Liquid medium: the cell absorbs by the whole surface

Semisolid: To see motility

Solid (can be converted to liquid): with agar, single colonies

Solid (cannot be liquified): carrot, potato etc. – good for moulds

http://intranet.tdmu.edu.ua/data/kafedra/internal/micbio/classes_stud/en/stomat/ptn/Microbiology,%2 0virology%20and%20immunology/2/Lesson_03_The%20main%20methods%20and%20principles%20of% 19 20pure%20cultures%20isolation.files/image052.jpg Culture media - composition Chemically defined media the exact chemical composition is known prepared from pure chemicals to contain all the growth requirement of the microorganism as source of carbon, energy, nitrogen, osmotic compounds, for auxotrophes vitamins and trace compounds etc. (they are not able to synthesize certain substances required for its growth and metabolism, it can be expansive)

http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T03_MediumFastid.jpg http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T02_MediumChemo.jpg Carbon and energy source (equal): glucose Fastidious bacterium: demanding for Nitrogen source: ammonium phosphate nutrition E. coli – easy to be cultivated 20 Culture media - composition Complex media Composed mainly from different natural materials and some simple pure chemicals Due to natural materials the exact chemical composition is not known and varies slightly from batch to batch of used natural materials and its preparation Products of hydrolysis of different source proteins (meat, soya, gelatine, caseine, vegetable tissue...) by proteolytic enzymes (as pepsin, trypsin, papain, pankreatin) = peptones/tryptones from...., protein hydrolysates = main source of nitrogen (also of carbon and energy, but this utilisation runs slower, some tryptones also source of vitamins) Simple saccharides (as glucose, lactose, maltose...) – source of energy and and carbon Other growth factors: sheep/horse blood,

http://images.slideplayer.com/20/5940717/slides/slide_8.jpg NADH+ Mineral salts: Meat extract, yeast extract: NaCl – osmotic pressure ( 0,85-0,90 % NaCl Source of vitamins (yeast extract saline solution), phosphates, sulphates, Ca2+, particularly rich in B vitamins and other Mg2+, Fe2+,3+, Mn2+ and traces compound – organic growth factors improving MO growth in some level (source of energy, carbon, minor source of Buffering salts nitrogen) for maintaing the stable pH during the cultivation (soluble phosphates, acetates....)21 Culture media - consistency Solidified by agar or gelatine • By agar in 1-3 % (polymerisation at 37-45 °C) - solid media – enabling to grow in single well-isolated colonies • By agar 0.2-0.5 % - semisolid media – detection of motility (bacteria can move) • By gelatine – biochemical testing for liquidifying of gelatine in some bacterial species (e.g. Clostridium perfringens) agar (agar-agar) – complex polysaccharides (agarose – 70 % + agaropektin – 30 %), derived from the cell wall of a marine alga (Floridae a Gelidium), only few microorganism can degrade, so it remains solid, in water it liquifies around at 95 °C, by cooling (~40 °C) solidifies and polymerises gelatine – produced by hydrolysis of colagen tissue (from gristle, chord, mixture of partially degradated peptides and proteins)

STRUCTURE In the natural state, agar occurs as structural carbohydrate in the cell walls of agarophytes algae, probably existing in the form of its calcium salt or a mixture of calcium and magnesium salts. It is a complex mixture of polysaccharides composed of two major fractions - agarose, a neutral polymer, and agaropectin, a charged, sulfated polymer. Agarose, the gelling fraction, is a neutral linear molecule essentially free of sulfates, consisting of chains of repeating alternate units of β-1,3-linked- D-galactose and α- 1,4-linked 3,6-anhydro-L-galactose. Agaropectin, the non gelling fraction, is a sulfated polysaccharide (3% to 10% sulfate), composed of agarose and varying percentages of ester sulfate, D-glucuronic acid, and small amounts of pyruvic acid. The proportion of these two polymers varies according to the species of seaweed. Agarose normally represents at least two-thirds of the natural agar-agar. http://www.agargel.com.br/agar-tec-en.html http://www1.lsbu.ac.uk/water/images/gelatin.gif 22 Culture media

Ready-to-use Dehydrated mixture prepared by the producer

Mixed from single parts 23 Culture media - purpose

http://classes.midlandstech.edu/carterp/courses/bio225/chap06/06-T05_CultureMedia.jpg

24 Culture media – general purpose General (universal) culture media • applicable for the growth of all those microorganisms, for which the nutrition composition is suitable (no selective additive) • the restriction of well-grown microorganisms may be partly done by the cultivation temperature E.g. Plate Count Agar, Nutrient agar, Tryptic soy agar Brain heart infusion broth, Tryptic soy broth, Buffered peptone water Enriched media • More rich in nutrient to support the growth of a wide variety of organisms, including some of the more fastidious ones E.g. Columbia agar with 5 % of sheep blood – very nutrient, also for more fastidious bacteria Blood agar is an enriched medium in which nutritionally rich whole blood supplements the basic nutrients. Chocolate agar is enriched with heat-treated blood (40–45 °C), which turns brown and gives the medium the color for which it is named. 25 Nutrient Agar Universal culture media for cultivating less fastidious microorganisms. Typical Composition (g/litre) Peptone from meat 5.0; meat extract 3.0; agar- agar 12.0. pH: 7.0 ± 0.2 at 25°C.

26 Plate Count Agar (Casein-peptone Dextrose Yeast Agar) This medium does not contain any inhibitors or indicators; it is mainly used to determine the total microbial content in milk, dairy products, water and other materials. Typical Composition (g/litre) Peptone from casein 5.0; yeast extract 2.5; D(+)glucose 1.0; agar-agar 14.0. pH: 7.0 ± 0.2 at 25°C.

27 Tryptic Soy Agar (TSA) Universal culture media free from inhibitors and indicators for a wide spectrum of applications. Typical Composition (g/litre) Peptone from casein 15.0; peptone from soymeal 5.0; sodium chloride 5.0; agar- agar 15.0. pH: 7.3 ± 0.2 at 25°C. Tryptic Soy Broth (TSB) CASO Broth (Casein- peptone Soymeal-peptone Broth) Universal culture media free from inhibitors and indicators for a wide spectrum of applications Typical Composition (g/litre) Peptone from casein 17.0; peptone from soymeal 3.0; D(+)glucose monohydrate 2.5; sodium chloride 5.0; di-potassium hydrogen phosphate 2.5 pH: 7.3 ± 0.2 at 25 °C. Buffered Peptone Water (BPW) For the preliminary, non-selective enrichment of bacteria, particularly pathogenic Enterobacteriaceae, from foodstuffs and other materials. Typical Composition (g/litre) Peptone from casein 10.0; sodium chloride 5.0; disodium hydrogen phosphate dodecahydrate 9.0; potassium dihydrogen phosphate 1.5.

pH: 7.0 ± 0.2 at 25 °C. Brain Heart Infusion Broth For the cultivation of various fastidious pathogenic microorganisms. Typical Composition (g/litre) Nutrient substrate (brain extract, heart extract and peptones) 27.5; D(+)glucose 2.0; sodium chloride 5.0; disodium hydrogen phosphate 2.5. pH: 7.4 ± 0.2 at 25 °C Columbia Agar This superior, complete medium proposed by ELLNER et al. (1966) can be used for the cultivation of even fastidious microorganisms and also as a base for the preparation of various special culture media. (universal + differential when used )

Typical Composition (g/litre) Peptrone from casein 10.0; peptone from meat 5.0; heart extract 3.0; extract from yeast 5.0; starch 1.0; sodium chloride 5.0; agar-agar 13.0. pH: 7.3 ± 0.2 at 25°C. Preparation of blood agar: Mix 5 ml blood homogeneously with 95 ml sterile culture medium base. Pour plates. (usually Columbia 5 % sheep blood agar) (universal + differential when used )

Preparation of gentamicin blood agar: Mix 100 ml defibrinated sheep blood and 0.11 ml gentamicin solution homogeneously with 900 ml sterile culture medium base. Pour plates. Preparation of boiled agar: Add 10 ml blood to 90 ml sterile culture medium base. Heat the mixture in a water bath for about 10 minutes to 80°C swirling all the time until the medium becomes chocolate brown in colour, pour plates. Preparation of lactose milk egg-yolk agar: Dissolve 42 g dehydrated culture medium, 12 g lactose, 1 g agar-agar in 1 litre demineralized water. Mix in 33 ml/litre of a 0.1 % aqueous solution of neutral red, adjust the pH to 7.0 and autoclave (15 min at 121°C). Cool to 45-50°C, add approximately 35 ml egg-yolk emulsion/litre and 10 g dried milk/litre and mix homogeneously. Pour plates. http://image.slidesharecdn.co m/copyofrevision2014- 140501141600- phpapp02/95/revision-2014- 66-638.jpg?cb=1398954082

RBC – red blood cell http://image.slidesharecdn.com/strpyosequelae-100123053606-phpapp02/95/str-pyo-sequelae-6-728.jpg?cb=1435204288 Columbia Agar with 5 % sheep blood

Escherichia coli

http://www.eolabs.com/columbia-agar-sheep-blood.html Streptococcus pyogenes Culture media – selective/differential Selective culture media • Suppression of unwanted microorganisms/Encouraging target (desired) microorganisms • Selective compounds: • Surface active compounds: bile, bile salts, sodium laureth sulfate, tergitol • Antibiotics: by choice to select the target microorganism • Inorganic salts: lithium chloride (against G- and enterococci), tetrathionate (against G+ and coliform bacteria), sodium azide (against G-) • Dyes: acridine, triphenyl dyes, crystal violet, briliant green, malachite green • Enrichment: Similar to selective media but designed to increase numbers of desired microbes to detectable levels Differential culture media • Differentiation of colonies of target (desired) microorgnisms from others based on some biochemical reaction • E.g. fermentation of glucose, lactose / indicated by acidobasic indicators; • E. g. degradation of chromogenic compound…. Selective-differential culture media • Suppression of unwanted microorganisms • Encouraging target (desired) microorganisms and their differentiation 35 Acidobazic indicator

http://image.slidesharecdn.com/lab17-141201085153-conversion-gate01/95/-10- 638.jpg?cb=1417424219 Chemical compounds changing colour http://mslavenda.com/images/bromocresol_purple_chart.jpg according to the pH (by accepting or + http://www.dlt.ncssm.edu/tiger/diagrams/acid-base/WeakAcidTitration- releasing H ) General.gif

http://mslavenda.com/images/bromothymol_chart.jpg

http://2.bp.blogspot.com/-XaGKyJzCBPk/UgS8FU96L3I/AAAAAAAAAt8/7B6bSjxLjjI/s1600/lol.PNG Selective-differential culture media

Composition Ingredients Gms / Litre Peptones (meat and casein) 3.000 Pancreatic digest of gelatin 17.000 Sodium chloride 5.000 Bile salts 1.500 Crystal violet 0.001 Lactose monohydrate 10.000 Neutral red 0.030 Agar 13.500 pH after sterilization( at 25°C) 7.1±0.2 Essential nutrients, vitamins and nitrogenous factors: pancreatic digest of gelatin and peptones (meat and casein) Osmotic balance: sodium chloride Selective action MacConkey Agar crystal violet and bile salts – recommended for selective isolation of Escherichia coli. It inhibitory to most species of is also recommended for selective isolation and gram-positive bacteria (e.g. differentiation of lactose fermenting and lactose non S. aureus – no growth). fermenting enteric bacteria = Selective and differential Gramnegative, specially enteric medium for cultivation of coliform organisms bacteria, grow well. Selective-differential culture media Lactose fermenting strains MacConkey Agar (coliform bacteria) growth as red or pink and may be surrounded by a zone of acid precipitated bile. Lactose non-fermenting strains, (Shigella, Salmonella, Proteus...) are colourless and transparent and typically do not alter appearance of the medium. Yersinia enterocolitica may appear as small, non-lactose fermenting colonies after incubation at room temperature. Composition Ingredients Gms / Litre Differential action (by the ability to ferment lactose): Peptones (meat and casein) 3.000 Pancreatic digest of gelatin 17.000 Lactose monohydrate (fermentable source of carbohydrat Sodium chloride 5.000 + Neutral red – acidobasic indicator Bile salts 1.500 Fermentation of lactose – acid production – absorped by Crystal violet 0.001 Lactose monohydrate 10.000 neutral red – when pH falls below 6.8 colour change to red

Neutral red 0.030 http://2.bp.blogspot.com/- XaGKyJzCBPk/UgS8FU96L3I/ AAAAAAAAAt8/7B6bSjxLjjI/s Agar 13.500 1600/lol.PNG pH after sterilization( at 25°C) 7.1±0.2 Chromogenic culture medium Coliform bacteria (MPN method) • One or more chromogenic substrates • For detection of specific enzyme, which are able to cleave out the chromogenic substrate, that a designed chromophore (responsible for

the colour change) is released https://encrypted- tbn3.gstatic.com/images?q=tbn:ANd9GcQp0jRYnm kNvmAadTkSzSsNYUpHuBzWmdbKoWQ79qbh3nLI hZ18ww

CHROMOGENIC MEDIUM (Colilert): Coliforms use β-galactosidase to metabolize ONPG and change it from colorless to yellow. Fluorogenic culture medium • One or more fluorogenic substrates • For detection of specific enzyme, which are able to cleave out the fluorogenic substrate, that a designed fluorophore (responsible for the UV fluorescence) is released

FLUOROGENIC MEDIUM (Colilert): E. coli use β-glucuronidase to metabolize MUG and create fluorescence (UV 360 nm). Since most non-coliforms do not have these enzymes, they are unable to E. coli (MPN grow and interfere method)

http://ga.water.usgs.gov/projects/bacteria/pictures/summaryanalysisecoli.jpg Preparation of culture media (1/4)

• water quality • distilled water •conductivity max. 25 µS cm-1 (25 °C), but preferred < 5 µS cm-1 • microbial contamination max. 103 cfu/ml, but max. 102 cfu/ml preferred (regularly verification, ISO 6222, incubation at 22 °C for 68 ± 4h)

Microbiological and chemical requirements of laboratory grade water

Benson, T. S., Dutko, T., Ilnicki, L., Lankford, M., Pentz, C., Salinsky, J., Teirab, B. and Ziemer, W., 1998. Equipment calibration, maintenance, and performance verification. Revision 1, 7/11/00. In: USDA/FSIS Microbiology Laboratory, Guidebook 3rd Edition/1998.chapter 36 revison 1, 6/8/00 pp 18 Preparation of culture media (2/4)

• weighing • balance with max. error 1 % • adding the designed volume of water (not to add up to the final volume) •mixing or solubilization • agar media to leave few minutes to soak • dispersing by repeating or continous stirring (e.g. (sterile) magnetic stirrer) • if neccessary, followed by heating to dissolving – to avoid overheating •bottle for preparation • for preparation use bottle with volume 20% upper than volume of prepared culture medium to avoid running over Preparation of culture media (3/4)

•pH • pH is designed for the final prepared media and reagents at 25 °C •pH adjustment to do before sterilisation in order to obtain the desired pH (±0.2 usually) in the final prepared medium • In the case of agar media, take a small volume and allow it to cool down to 25°C and carefully introduce a pH electrode into cooled agar and measure. •Adjustement of pH • c(NaOH) = 1 mol/l (~40 g/l) • c(HCl) = 1 mol/l (~36.5 g/l) • When adjusting of pH to be done after sterilisation, to use sterilised solutions Preparation of culture media (3/4)

http://1.bp.blogspot.com/- •pH meter 4AtMuBrl0e8/VVcaZBdAxII/AAAAAAAAAc4/gNmaSs6I0wY/s640/phmetre -Hanna-HI-99161.jpg

•stick pH meter Preparation of culture media (3/4)

• sterilization • autoclave: moist heat (steam) • used for the sterilization of heat-stabile culture media and aqueous solutions and the destruction of discarded cultures • air must first be removed in order to achieve the 121 °C ± 3 °C for 15 mins (for max. 1 l) necessary for successful sterilization. • the temperature 121 °C to destroy bacillus spores. •boiling in water bath • some media with heat-labile compounds • e.g. selective media for Enterobacteriaceae, the present inhibitors inhibits the growth of G+ microflora) •membrane filters (0.2 µm pores) • usually employed for heat-sensitive substances, e.g. vitamin solutions; • the filters are heat-sterilised before use • to check the correct kind of membrane filter (e.g. not binding proteins or antibitiocs) and moist before using

• the medium to be sterilised on the same day when prepared

Training of Shigella spp. and Yersinia enterocolitica detection, Prague, 19-23 October 2015 Preparation of culture media (3/4)

• supplements or enrichments • safety rules to work with and discard – it can be toxic substances (e.g. antibiotics, dyes) •when powdered, to be dissolved according to the manufacturer´s recommendation • e.g. antibiotics in sterile water or mixture of EtOH and sterile water •to be careful about storage •antibiotics solution looses its activity by a longer storage even at 5-8 °C • if not allowed, it should be consumed on the day of preparation • or if possible, to freeze (but not to refreeze after defreezing) • but the change in activity due to freezing must be determined by the producer or the consumer •adding into medium • for termolabile supplement into medium cooled at 50 °C or below • the supplement must be minimally at the room temperature (to avoid gelidifying of agar etc.) • to carefully and precisely mix Preparation of culture media (3/4)

• Preparation of Petri agar plates • after sterilisation/boiling to cool down to 47-50 °C in a regulated water bath (the time depends on the volume, kind of medium, number of bottles) •to add correctly supplements and enrichments •to pour out medium as soon as possible, maximally within 4 hours •not to let solidify and remelt again ! •to pour out in sterile Petri dishes • the height of agar minimally 3 mm • for Petri dishes with average 90 mm ~18-20 ml of medium • the higher volume (and the height) of agar is possible for plates • to be stored • to be cultivated more than 72 h • to be cultivated at the cultivation temperature higher than 40 °C Selectivity Culture media - selectivity • what is the ability of the selective medium to Target microorganism = what we want to grow on the agar medium/in the broth supress the growth of the non-target and what we want to detect – distiguish from other microorganisms ? microorganisms • what is the ability of target microorganism to grow Non-target microorganism = what we want to be inhibited and not grow or grow on the preferably in the presence of non-target agar medium/in the broth or what we want not to give the microorganism in the tested selective medium ? same reaction as the target Non-selective medium Different species or their strains have different ability to grow - some species or strains have the higher 0.2 ml growth rate than the others - the growth rate depends on the temperature, pH, present substances for nutrition, present inhibitors... - some species can inhibit the others – by True amount of production of antimicrobial compounds, by cells in a changing the physical properties of the sample medium (0.2 ml) Selective medium

Selectivity (SF) for demonstration that a medium suppresses the growth of a non-target microorganism Culture media - productivity selective media = selective pressure = tested media • chemicals – antibiotics, dyes etc. to selectively inhibit some groups of microorganisms • by cultivation conditions (temperature etc.) non-selective media = without selective pressure = reference media Non-selective medium (reference)

Productivity • what is the efficiency of the target microorganism to grow in the tested 0.2 ml medium • how many % of inoculated target microorganisms is able to grow (in comparison of reference media) • selective pressure inhibits partly also True amount of the target microorganisms (damaged cells in a cells mainly), also for them it is usually sample not very easy to accept the conditions (0.2 ml) Selective medium Culture media - specifity

Specifity • does the target microorganism grow specifically ? – to give the expected appearance of colonies (colour, surface....) ? - The specifity is used on some biochemical property = presence of enzyme - But different strains of the same species can behave differently !!! - No presence of tested enzyme... - The enzyme has the lower activity – it works slowly...

The target microorganism gives the expected reaction Horizontal method for the enumeration of microorganisms Horizontal method = by using Petri dishes SAMPLE PROCESSING • resuspension in diluent - decimal dilution - spreading, pourring/inoculation (MPN) • filtration ISOLATION ON SELECTIVE MEDIA

SUB-ISOLATION OF PRESUMPTIVE COLONIES (non-selective agar)

BACTERIA GROWING ON SELECTIVE MEDIA ARE NOT APPROPRIATE FOR BIOCHEMICAL TESTING – SELECTIVE MEDIA DECREASED „FITNESS“ OF BACTERIA AND INFLUENCES THEIR PRESENT BIOCHEMICAL BEHAVIOR (NOT PROPERTIES), WHICH CAN INTERFER WITH THE EXACTNESS AND RELIABILITY OF TESTING

CONFIRMATION/IDENTIFICATION Horizontal method for the enumeration of microorganisms Horizontal method = by using Petri dishes SAMPLE Sample: usually 10 g of solid or 10 ml of liquid food/feed Diluent: e.g. saline solution with peptone, peptone water etc. • solid food/feed: 10 g + 90 ml of appropriate diluent DECIMAL • liquid food/feed: 10 ml + 90 ml of appropriate diluent DILUTIONS = 1. dilution, to be done in a sterile special bag, sample to be resuspended or mixed well = further dilutions: in tubes (1 ml of suspension + 9 ISOLATION ml of diluent)

CULTIVATION

COLONY COUNTING 1:100 1:1000 1:10000 1:100000 1:1000000

1:10 SUB-ISOLATION OF CULTIVATION + PRESUMPTIVE CONFIRMATORY RESULT COLONIES TESTING Horizontal method for the enumeration of microorganisms Horizontal method = by using Petri dishes

Inoculation of some dilutions (chosen to cover the given limit) SAMPLE • by spreading suspension (usually 0.1 or 0.2 ml) onto medium in Petri dishes • by pourring over suspension (usually 1 ml) in an empty DECIMAL Petri dish by melted medium DILUTIONS

ISOLATION

CULTIVATION 1:100 1:1000 1:10000 1:100000 1:1000000

COLONY COUNTING

http://www.studyblue.com/notes/note/n/lab-practical-exam-review-1/deck/6074578 SUB-ISOLATION OF (upraveno) CULTIVATION + PRESUMPTIVE CONFIRMATORY COLONIES RESULT TESTS Plating method pourring over gives better results for facultative anaerobes than spreading

spreading used for aerobic microorganisms

http://department.monm.edu/chemistry/chem Streaking istry330/fall2004/abickert/images/streak.gif http://classes.midlandstech.com/carterp/Courses/bio225/chap06/Microbi al%20Growth%20ss5.htm Horizontal method for the enumeration of microorganisms Horizontal method = by using Petri dishes

SAMPLE Cultivation: time and temperature depends on the microorganisms Colony counting: limit of colony forming unit on a plate: DECIMAL 10-300 for counting all colonies, 10-150 for counting DILUTIONS presumptive colonies Confirmation: choice of up 5 presumptive/typical/atypical colonies per a plate to be confirmed by biochemical testing (see ISOLATION specifity/selectivity) Result: A.B x 10c CFU/g, ml (e.g. 5.2 x 104 CFU/g, ml)

CULTIVATION

COLONY COUNTING

http://www.studyblue.com/notes/note/n/lab-practical-exam-review-1/deck/6074578 (ADAPTED S.P.)

SUB-ISOLATION OF CULTIVATION + PRESUMPTIVE CONFIRMATORY RESULT COLONIES TESTS Colony counting C N (colonies/g sample) = If n1 = n2=2 then  V *2,2*d C  C If n1 = n2=1 then  V *1,1*d V (n1 0,1n2 ).d

• C… the number of all counted colonies on plates of two dilutions, following each other, when at least one plate is more colonies then 10 (the highest amount is 300 for total colonies/150 for typical and/or atypical colonies or according to used ISO method for analysis) • V…. the volume of the sample spread on the plate (ml) (if 1 ml is spread over 3 plates, it is taken as 1 plate with V=1 ml) • d…..the coeficient of the first dilution taken for calculation (10-x)

• n1 …the number of counted plates of the first dilution taken for calculation • n2….the number of counted plates of the second dilution taken for calculation Colony counting ISO 7218:2007 Microbiology of food and animal feeding stuffs – General requirements and guidance for microbiological examinations (if not determined by the used method differently) 1. Typical cases - Each dilution plated on one Petri dish (if not asked different, but plating two plates is widely asked and highly recommended) - Maximally 300 colonies per one P. dish (but typical and/or presumptive colonies maximally 150 per one P. dish) a) Counting (all colonies) - max. 300 colonies per one P.dish - at least one dish with minimally 10 colonies

C… all counted colonies on plates of two dilutions (one C per each) N  V *1,1*d V…. the volume of the inoculum d…..the coeficient relative to the first dilution taken for calculation= 10-x Colony counting b) Counting for confirmation- max. 150 (typical and/or atypical colonies per one P.dish) b C… presumptive colonies a  A *C A…..number of presumptive colonies taken for confirmation (typically A=5) a b….number of typical colonies positively confirmed a….the number of confirmed colonies at the plate – then V (n1 0,1n2 ).d used for the calculation according to a) Example of application: coagulase-positive staphylococci- typical and atypical colonies can be present bc bnc a  cc  cnc Ac Anc

Ac is the number of typical colonies submitted to the coagulase test

Anc is the number of atypical colonies submitted to the coagulase test

bc is the number of typical colonies which have been shown to be coagulase-positive

bnc is the number of atypical colonies which have been shown to be coagulase-positive

cc is total number of typical colonies seen on plate

cnc is total number of atypical colonies seen on plate Colony counting c) Low numbers for the first used dilution !!!!! (d1) • (4-10 colonies) On the plate with the first used dilution is less than 10 colonies, but minimally 4 colonies: According to the Poisson distribution for the 50 % acceptable of relative trueness - the lower limit is 4 colonies at one plate and it is calculated by using the formulae before (ISO/TR 13843:2000) • (1-3 colonies) On the plate with the first used dilution is 1-3 colonies: „Microorganisms are present, but in the number lower than 4/(V*d) per g or per ml.“ • 0 colonies „less than 1/(V*d) microorganisms per ml or per g” (note: if two parallel plates are used (each per 0.1 or 1 ml according to the method, V should be considered in the formula „less than….“ twice times (as 0.2 or 2 ml) – because the examined volume with no colonies is the volume spread over these two parallel plates)

d) More than upper limit for the last used dilution (d2) “more than 300/(V*d2) or more than 150*(b/A)/(V*d2) microorganisms per ml or per g (if confirmation is done)” Colony counting Counting all colonies 10 g + 90 ml PW , V= 1 ml N  C Dilution, V=1 ml 10-1 10-2 V *(n1 0,1n2 )*d

Colonies <300 >10 Maximally 300 colonies per one P. dish, at least one dish with minimally 10 colonies Colonies <300 <10

Colonies 255 38 Colonies 255 5

255  38 3 255  5 N   2.7*10 CFU / g N   2.4*103 CFU / g 1*(11*0.1)*0.1 1*(11*0.1)*0.1

Colonies >300 10-300 Colonies & 55

55 55 Only this used for N    5.5*103 CFU / g calculation 1*(1 0*0.1)*0.01 1*1*0.01 If using one general formula is C 55 confusing, the simpler formula is   5.5*103 CFU / g N  V *n*d  1*1*0.01 Enterobacteriaceae and coliform bacteria Family Enterobacteriaceae found in soil, water, plants and animals and their gastrointestinal tracts Gram-negative, straight rods, some of which motile Most grow well at 37 °C, some better at 25 - 30°C, facultatively anaerobic, fermenting glucose, oxidase-negative and catalase-positive (except Shigella dysenteriae type 1), resistant to bile salts Proteus sp., Hafnia sp., Salmonella sp., Shigella sp., Pantoea sp., Yersinia sp., etc. coliform bacteria = subgroup of Enterobacteriaceae similar to E. coli = coliform may be found also as normal intestinal flora of humans and/or animals,considered opportunistic pathogens - indicator for faecal contamination in water or bad hygiene/insufiecient storage conditions in food Escherichia sp., esp. Escherichia coli; Enterobacter sp., Klebsiella sp., Citrobacter sp. fermenting lactose (enzyme β-D-galactosidase) Escherichia coli β-D-glucuronidase (app. 97 % of strains), indole positive Enterobacteriaceae, coliform bacteria and E. coli Biochemical properties used for isolation on selective media and confirmation

Family Enterobacteriaceae Gram-negative, resistant to bile salts VRBD Agar fermenting glucose, oxidase-negative, cultivation temperature: 37 °C

coliform bacteria VRBL agar all features of Enterobacteriaceae MacConkey agar, ENDO agar, + fermenting lactose (enzyme β-D-galactosidase) Chromocult Coliform agar

Escherichia coli all features of Enterobacteriaceae and coliforms + enzyme β-D-glucuronidase (app. 97 % of strains, not present e.g. in E. coli O157) TBX indole positive VRBD (Violet Red Bile Dextrose) Agar Selective agar for the isolation and enumeration of all Enterobacteriaceae species in foodstufs Typical Composition (g/litre) Peptone from gelatine 7.0; yeast extract 3.0; sodium chloride 5.0; D(+)glucose 10.0; bile salt mixture 1.5; neutral red 0.03; crystal violet 0.002; agar-agar 13.0. Selective activity Crystal violet and bile salts inhibit the accompanying bacterial flora. Differential activity Degradation of glucose is accompanied by production of acid, which is indicated by a colour change to red and Escherichia coli ATCC 8739 by zones of precipitated bile acids surrounding the colonies. All Enterobacteriaceae are detected as they all degrade glucose to acid. The culture medium is not,however, absolutely specific for these organisms as some other accompanying bacteria (e.g. Aeromonas) also show these reactions.

Shigella flexneri ATCC 29903 Enterobacteriaceae ISO 21528-2:2004 Microbiology of food and animal feeding stuffs -- Horizontal methods for the detection and enumeration of Enterobacteriaceae -- Part 2: Colony-count method

Sample 10 g sample 1 ml (-1. dilution) 1 ml (-(X+1). dilution Sample (if liquid) + 90 ml BPW + 9 ml BPW …. + 9 ml BPW preparation and 1. DAY 0. dilution - 1. dilution - 2. dilution - X. dilution decimal dilution

Pourring over of 1 ml by VRBD

Isolation on selective agar VRBD VRBD VRBD VRBD VRBD VRBD VRBD VRBD

Cultivation: 37 °C, 24 h±2 h Confirmation of 2. DAY typical/atypical Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if colonies present from each plate used for calculation • Inoculation on Inoculation on nutrient agar, 37˚C, 24±3 h non-selective agar

3. DAY Biochemical test: test for oxidase, glucose fermentation under anaerobic condition – • Confirmatory tests glucose agar (37 °C, 24 h)

Evaluation of biochemical tests Result 4. DAY Calculation of CFU/g, ml

***1 ml of tested suspension pourred over (up to 15 minutes after suspension preparation) by VRBD (at 44 °C – 47 °C) twice – semi-anaerobic conditions – at first by 10 ml, after solidification by other 15 ml Oxidase test Oxidase •abbreviated general name for some kind of cytochrome c oxidases (cytochrome oxidase or indophenol oxidase), enzymes involved in aerobic respiratory chain, when oxygen is used as the final receptor for hydrogen. •Oxidases are usually present only in aerobic organisms. •Presence or absence of oxidase(s) is an important feature of the bacterial metabolism, used mainly in the identification process of gram-negative bacteria.

Oxidase positive (OXI +) Aeromonas sp. Pseudomonas sp. Dark-blue to violet Oxidase negative (OXI -) Enterobacteriaceae Bacillus sp. Colourless

Detection: Reaction of the culture with redox indicator as e.g N,N-dimethyl-p-phenylenediamine (DMPD): dark-blue when oxidized and colorless when reduced. Different test performances are possible. VIOLET RED BILE AGAR WITH LACTOSE (VRBL) Selective medium for the detection and enumeration of coliforms in dairy products, water and food FORMULA IN g/l Lactose 10.00 Bile Salts 1.50 Gelatin Peptone 7.00 Neutral Red 0.03 Sodium Chloride 5.00 Crystal Violet 0.002 Yeast Extract 3.00 Bacteriological Agar 15.00 Final pH 7.4 ± 0.2 at 25ºC

Escherichia coli ATCC 11775 Selective action Bile salts and crystal violet inhibit Gram-positive bacteria, bile salts partially inhibits non-intestinal Gram-negative bacteria

Differential action Lactose fermentation detected by neutral red as a pH indicator. Lactose fermenters form red colonies with red-purple halos. Occasionally the cocci of the intestinal tract can develop as small, punctiform red colonies. Appearance of colonies Microorganisms red colonies with or without red-purple Coliform bacteria halos of precipiation small, punctiform red colonies Occasionally the cocci of the intestinal tract Escherichia coli Salmonella gallinarum colourless Lactose-negative Enterobacteriaceae ATCC 25922 NCTC 9240 and others Endo Agar used to confirm the detection and enumeration of coliform bacteria following presumptive test of drinking water, from milk, dairy products and food. Composition Selective activity: Ingredients Gms / Litre sodium sulphite and basic fuchsin inhibits partially Gram positive Peptic digest of animal tissue 10.000 bacteria Lactose 10.000 Dipotassium phosphate 3.500 Sodium sulphite 2.500 Basic fuchsin 0.500 Agar 15.000 Final pH ( at 25°C) 7.5±0.2

Shigella flexneri ATCC 12022

Differential activity: Coliforms – lactose-fermenter - pink colonies lactose non-fermenters - colourless colonies on the medium. E. coli – this reaction is very pronounced as the fuchsin crystallizes, exhibiting a permanent greenish metallic luster (fuchsin luster) to the colonies. 67 Endo Agar ChromoCult®Coliform Agar Selective agar for the simultaneous detection of total coliforms and E. coli in drinking water and processed food samples Typical Composition (g/litre) Peptone 3.0; sodium chloride 5.0; sodium dihydrogen phosphate 2.2; di-sodium hydrogen phosphate 2.7; sodium pyruvate 1.0; tryptophan 1.0; agar-agar 10.0; Sorbitol 1.0; Tergitol® 7 0.15; 6-chloro-3-indoxyl-beta-Dgalactopyranoside 0.2; 5- bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid 0.1; isopropyl-beta- Dthiogalactopyranoside 0.1. pH: 6.8 ± 0.2 at 25°C. Coliform identification Salmon-GAL – cleaved by β-D-galactosidase - characteristic for coliforms - a salmon to red color of the colonies. E. coli identification Escherichia coli ATCC 11775 X-glucuronide – cleaved by β-D-glucuronidase - characteristic for E. coli. E. coli cleaves also Salmon-GAL so the colonies take on a dark-blue to violet color.

Citrobacter freundii ATCC 8090 Escherichia coli ISO 16649-2:2001 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli -- Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide

Pourring over of 1 ml of TBX agar*

Isolation on selective agar TBX TBX TBX TBX TBX TBX TBX TBX

Cultivation: 44 °C, 18-24 h Confirmation of typical/atypical colonies ChromoCult ® TBX ( • Inoculation on Tryptone Bile X- non-selective agar glucuronide ) Agar • Confirmatory tests

Evaluation of typical colonies – the used biochemical reaction as taken to be Result 2. DAY so specific, that only colonies of E. coli are blue-green – no confirmatory step evaluation Calculation of CFU/g, ml

*about 15 ml temperated to 44-47 °C ChromoCult ® TBX ( Tryptone Bile X-glucuronide ) Agar Selective-differential chromogenic agar for the detection and enumeration of Escherichia coli in foodstuffs, animal feed and water. Typical composition ( g / Litre ) Peptone 20.0; bile salts No. 3 1.5; X- β - D - glucuronide 0.075; agar-agar 10.0. Selective activity: Growth of accompanying Gram-positive flora is largely inhibited by the use of bile salts and the high incubation temperature of 44 °C (also Gram-negative). Differential activity X-β-D-glucuronide (5-bromo-4-chloro-3- indolyl-β-D -Glucuronide) is cleaved by the β-D-glucuronidase (this enzyme differentiates most E.coli ssp. from other coliforms) to split the bond between the chromophore 5-bromo-4 - Escherichia coli ATCC 25922 chloro -3-indolyle and the β-D-glucuronide. E.coli colonies -blue-green.

Appearance Microorganisms blue-green E. coli β-D-glucuronidase positive (96 – 97 % of strains) colourless β-D-glucuronidase-negative E.coli strains (3 - 4 %) form colourless colonies, e. g. E.coli 0157, or they cannot grow at elevated temperature of 44 °C, e.g. E. coli 0157: H7.

Escherichia coli DSMZ 502 Coagulase-positive staphylococci ISO 6888-1:1999 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -- Part 1: Technique using Baird-Parker agar medium

Spreading of 0.1 ml onto Baird-Parker agar (BP)*

Isolation on selective agar BP BP BP BP BP BP BP BP

Cultivation: 37 °C, 24 h/48 h (colony counting after both time) Confirmation of 2./3.DAY typical/atypical Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if colonies present from each plate used for calculation • Inoculation on Inoculation in BHI and blood agar, 37˚C, 24±3 h non-selective agar 3./4. DAY Biochemical test: plasma-coagulase test (incubation of culture grown in BHI with rabbit plasma) – 37 °C , 24 h; detection of haemolysis • Confirmatory tests

Evaluation of biochemical tests Result 4./5. DAY Calculation of CFU/g, ml

***instead of 0.1 ml on one Petri dishes (average 90 mm) also 1 ml on three Petri dishes by 90 mm, when low count expected, Czech Republic ČR – ČSN EN ISO 6888-1:1999 – national version – spreading of 0.2 ml BAIRD-PARKER AGAR (BP)

Typical Composition (g/litre) Peptone from casein 10.0; meat extract 5.0; yeast extract 1.0; sodium pyruvate 10.0; glycine 12.0; lithium chloride 5.0; agar-agar 15.0. Also to be added (supplement): Egg-yolk tellurite emulsion 50 ml; if required, sulphamethazine 0.05 g/l. pH: 6.8 ± 0.2 at 25°C Nutrient: peptone, meat extract, yeast extract Selective stimulation of the growth of staphylococci (resuscitation partial): pyruvate and glycine Inhibition of accompanying microflora: lithium chloride and Staphylococcus aureus ATCC 25923 tellurite Differential mode for Staphylococcus colonies show two characteristic features when grown in this opaque medium (opaque, because of its egg-yolk content) a. characteristic zones and rings are formed as a result of lipolysis and proteolysis, b. reduction of tellurite to tellurium produces a black colouration. The egg-yolk reaction and tellurite reduction are usually found to occur together with a positive coagulase reaction and can thus serve as an index for the latter (coagulase positive staphylococci – S. aureus and other species)http://www.bacteriainphotos.com/photo%20gallery/staphylococcus%20aureus%20baird%20parker.jpg BAIRD-PARKER AGAR (BP)

Staphylococcus aureus ATCC 25923 Coagulase-positive staphylococci ISO 6888-1:1999 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) -- Part 1: Technique using Baird-Parker agar medium Confirmation of 2./3.DAY Choice of typical (or atypical) colonies to be confirmed – up to 5 colonies, if typical/atypical present from each plate used for calculation colonies Inoculation in BHI and blood agar, 37˚C, 24±3 h • Inoculation on non-selective agar Biochemical test: plasma-coagulase test (incubation of culture grown in BHI 3./4. DAY with rabbit plasma) – 37 °C , 24 h; detection of haemolysis • Confirmatory tests

Typical colonies: black or grey, convex, shiny, 1-5 mm in average, with the clearance zone wide by 2-5 mm, after at least 24 h incubation there can be an opaque ring Atypical colonies: grey, without the clearance zone, or black with narrow white edge or without, the clearance zone is missing or badly visible. The atypical colonies are mainly formed by coagulase-positive strains of staphylococci from dairy products, seafood or harslet

β - Plasma- haemolysis coagulase S. aureus + + S. epidermidis - - Coagulase-positive staphylococci Coagulase test : coagulase is an enzyme with the ability to coagulate plasma It differentiates S. aures from the other species, with less or without impact on human health and food safety (enterotoxins production) as (S. epidermidis, S. saprophyticus), other coagulase-positive staphylococci (S. schleiferi, S. intermedius) are not so common, coagulase production usually correlates with the pathogenity Staphylococcus aureus forms two types of coagulase the free coagulase is an extracellular enzyme the bound coagulase on the surface of the cell wall Slide test: detect only bound coagulase Dense culture mixed in saline solution mixed with EDTA-rabbit plasma = „clumping“, a screening test Tube test (false-positive reactions and http://www.mgm.ufl.edu/~gulig/mmid/mmid-lab/images/coag-ex.jpg autoagglutination can occur) • Grown liquid culture (grown in BHI) mixed with rehydrated EDTA-rabbit plasma (for 4, 18 and 24 h) • Detection of free and bound coagulase • Free coagulase reacts with plasmatic factor to produce staphylotrombin, which catalyzes changes of fibrinogen to fibrin (clumps, sluts or completely congelation in a tube – positive when ¾ http://faculty.mc3.edu/jearl/ML/coagulase.jpg congelates) Total count of microorganisms ISO 4833-1:2013 Microbiology of the food chain -- Horizontal method for the enumeration of microorganisms - Part 1: Colony count at 30 degrees C by the pour plate technique

Pourring over of 1 ml of PCA agar*

Isolation on non- selective agar PCA PCA PCA PCA PCA PCA PCA PCA

Cultivation: 30 °C, 72 h±3 h Confirmation of typical/atypical colonies • Inoculation on non-selective agar

• Confirmatory tests Colony counting of all grown colonies Result 4. DAY Calculation of CFU/g, ml evaluation

*1 ml of tested suspension to pour over with 12-15 ml of melted PCA (44-47 °C) Yeasts and moulds ISO 21527-1:2008 Microbiology of food and animal feeding stuffs -- Horizontal method for the enumeration of yeasts and moulds Part 1: Colony count technique in products with water activity greater than 0,95 (DRBC to be used) Part 2: Colony count technique in products with water activity less than or equal to 0,95 (DG18 to be used)

Sample 10 g sample 1 ml (-1. dilution) 1 ml (-(X+1). dilution Sample (if liquid) + 90 ml SP + 9 ml SP …. + 9 ml SP preparation and 1. DAY 0. dilution - 1. dilution - 2. dilution - X. dilution decimal dilution

Spreading of 0.1 ml onto DRBC/DG18 agar* DRBC/ DG18 Isolation on selective agar DRBC/ DRBC/ DRBC/ DRBC/ DRBC/ DRBC/ DRBC/ DG18 DG18 DG18 DG18 DG18 DG18 DG18

Confirmation of Cultivation: 25 °C, 3-5 days (by the bottom down – typical/atypical not in the usual reverse position) colonies • Inoculation on non-selective agar

• Confirmatory tests Colony counting of all grown colonies Result Calculation of CFU/g, ml evaluation 3.-6. DAY

* Diluent – peptone water with 0.1 % peptone, for low counts also 3 plates by 0.3 ml each Cultivation in an open plastic bag to avoid the dissemination of spores from plates . Dichloran Rose Bengal Selective agar for the enumeration of food spoiling Chloramphenicol (DRBC) Agar yeasts and moulds. Typical Composition (g/litre) Peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0; dichloran 0.002; magnesium sulfate 0.5; Rose Bengal 0.025; chloramphenicol 0.1; agar-agar 15.0. pH: 5.6 ± 0.2 at 25°C. Low pH- inhibition of most bacteria Chloramphenicol - broad-spectrum antibiotic inhibitory to a wide range of Gram-negative and Gram-positive bacteria. Rose Bengal • to inhibit bacterial growth and restricts the Saccharomyces cerevisiae ATCC 9763 growth of rapidly growing molds • incorporated in the cells of yeasts and molds, turning these colonies pink. Dichloran - to inhibit the rapid spreading of mucoraceous fungi and restricts colony sizes of other genera, easing the colony count. Magnesium Sulfate provides trace elements. Glucose, peptone - nutrition

Mucor racemosus ATCC 42647 Dichloran Rose Bengal Selective agar for the enumeration of food spoiling Chloramphenicol (DRBC) Agar yeasts and moulds.

Mucor racemosus ATCC 42647 Saccharomyces cerevisiae ATCC 9763 Dichloran Glycerol (DG18) Agar

Selective agar with low water activity (aw) for the enumeration and isolation of xerophilic moulds in dried and semi-dried foods as well as a general purpose medium for counting yeast and moulds in foodstuffs. Typical Composition (g/litre) Peptone 5.0; glucose 10.0; potassium dihydrogen phosphate 1.0; dichloran 0.002; magnesium sulfate 0.5; chloramphenicol 0.1; agar-agar 15.0. pH:5.6 ± 0.2 at 25°C. Preparation Suspend 31.6 g in 1 litre of demin water and heat to boiling until completely dissolved. Add 175 ml of glycerol p.a. to the medium, mix Wallemia sebi and autoclave at 121°C for 15 min. Cool to approx. 50°C, mix well and pour plates. By reducing the water activity from approx. 0.99 to 0.95 with 18 % (w/w) glycerol and addition of chloramphenicol growth of bacteria is prevented. Low pH- inhibition of most bacteria Chloramphenicol - broad-spectrum antibiotic inhibitory to a wide range of Gram-negative and Gram-positive bacteria. Magnesium Sulfate provides trace elements. Glucose, peptone - nutrition

Eurotium rubrum Dichloran Glycerol (DG18) Agar YGC Agar (Yeast Extract Glucose Chloramphenicol Agar Selective agar for isolating and counting yeasts and moulds in milk and milk products.

Typical Composition (g/litre) Yeast extract 5.0; D(+)glucose 20.0; chloramphenicol 0.1; agar-agar 14.9. pH: 6.6 ± 0.2 at 25°C.

Saccharomyces cerevisiae ATCC 9080 Chloramphenicol to suppress accompanying bacterial flora (unlike other antibiotics e.g. oxytetracycline, it has the advantage of being fully autoclavable)

Yeast extract, glucose - nutrion

Aspergillus niger ATCC 16404 YGC Agar (Yeast Extract Glucose Chloramphenicol Agar

Saccharomyces cerevisiae ATCC 9080

Aspergillus niger ATCC 16404 Sabouraud Dextrose Agar Sabouraud Dextrose Agar is used for the cultivation of yeasts, moulds and aciduric bacteria. Composition Ingredients Gms / Litre Dextrose 40.000, Mycological, peptone 10.000, Agar 15.000 Final pH ( at 25°C) 5.6±0.2 Mycological peptone provides nitrogenous compounds. Dextrose provides an energy source. High dextrose concentration and low pH favours fungal growth and inhibits contaminating bacteria from test samples. Malt Extract Agar Base(w/ Mycological Peptone) Composition Ingredients Gms / Litre Malt extract 30.000, Mycological peptone 5.000, Agar 15.000 Final pH ( at 25°C) 5.4±0.2 Malt extract - an acidic environment and nutrients favourable for growth and metabolism of yeasts and moulds. Mycological peptone rapidly gives a luxuriant growth with typical morphology and pigmentation. For mycological count, it is advisable to adjust the reaction of medium more acidic with addition of 10% lactic acid. Antibiotics may be added as sterile solutions to the molten medium in order to suppress bacterial growth. 3M Petrifilm™ Count Plates - principle

an all-in-one plating system made by the Food Safety Division of the 3M Corporation

cost-effectiveness, simplicity, convenience, and ease of use 3M Petrifilm™ Count Plates - principle

3M Petrifilm™ Count Plates: Coating technology

1. Top Film Plastic Film coated with adhesive, indicator and cold water soluble gel.

2. Bottom Film Plastic coated paper printed with a grid, adhesive standard methods nutrients, cold water soluble gel. 3M Petrifilm™ Coliform Count Plates Using the same typical biochemical features of coliform bacteria as in ISO 4832:2006 =bacteria resistant to crystal violet and bile salts, fermenting lactose to acid and gas

The dehydrated medium is based on VRBL agar principles • crystal violet inhibits G+ bacteria • bile salts inhibit most G- bacteria • fermentation of lactose to acid – decreased pH is detected by acidobazic indicator neutral red, which changes in some range of pH its colour to red Enumeration of coliforms (total or •but fermentation of lactose to gas is thermotolerant). detected for all grown colonies Reading facilitated by an indicator, which immediately by trapping gas bubbles in colours colonies the gel red. Upper film traps the gas produced by lactose fermentation. MEMBRANE FILTRATION

MEMBRANE FILTRATION • used for capturing bacteria from larger volume of sample • filter is then tranferred onto a plate to let them grow • It also can be used for sterilisation of liquids

Pore size Particles that pass through them in (µm) Erythrocytes, yeast cells, bacteria, viruses, 10 http://microbiologyon- molecules line.blogspot.cz/2009/08/filtration.html 5 Yeast cells, bacteria, viruses, molecules 3 Some yeast cells, bacteria, viruses, molecules 1.2 Most bacteria, viruses, molecules 0.45 A few bacteria, viruses, molecules 0.22 Viruses, molecules 0.10 Medium-sized to Small Viruses, molecules 0.05 Small viruses, molecules http://www.flickriver.com/photos/artsyscience/2345558461/ 0.025 Only the very smallest viruses, molecules Ultra-filter Small molecules MEMBRANE FILTRATION

MEMBRANE FILTRATION • used for capturing bacteria from larger volume of sample • filter is then tranferred onto a plate to let them grow • It also can be used for sterilisation of liquids

http://microbiologyon- line.blogspot.cz/2009/08/filtration.html • filtrable samples - (dilution in order to obtain countable amount of colonies) – filtration=capturing bacteria on a membrane filter • nonfiltrable samples – diluting – pourring over or spreading on according to the tested microorganisms properties Enumeration of Escherichia coli and coliform bacteria in water

ISO 9308-1:2014 Water quality -- Enumeration of Escherichia coli sample and coliform bacteria -- Part 1: Membrane filtration method for day waters with low bacterial background flora Membrane filtration (filter 0,45μm) → 100 ml

1. Tranfer of filter onto TTC agar

Cultivation 36±2 °C, 24 - 48 h

2. Lactose + colonies Lactose - colonies

Isolation on TSA → 36±2 °C, 21±3 h

° 3. OXI test IND → 44±0,5 C, 21±3 hod - OXI- 4. IND+ Coliform bacteria Escherichia coli

Pitná voda - normy - Vyhl. č.252/2004 91 Lactose TTC Agar with Tergitol® 7 Selective differential medium for the detection and enumeration of E. coli und coliform bacteria in water using the membrane filtration method. Typical Composition (g/litre) Lactose 20.0; peptone 10.0; yeast extract 6.0; meat extract 5.0; bromothymol blue 0.05; Tergitol®7 0.1; agar-agar 12.7. Additive: TTC 0.025. pH: 7.2 ± 0.2 at 25°C.

Selectivity action : sodium heptadecylsulfate (Tergitol®7) and 2,3,5-Triphenyltetrazoliumchloride (TTC) to inhibit most Gram- Citrobacter freundii positive bacteria. ATCC 8090 from above Differential action Degradation of lactose to acid is indicated by the pH indicator bromothymol blue, which changes the colour of the medium under the membrane to yellow. The reduction of TTC by lactose-negative bacteria - dark red colonies. Lactosepositive E. coli and coliform bacteria reduce TTC weakly - yellow-orange colonies.

Escherichia coli ATCC 25922 from below Lactose TTC Agar with Tergitol® 7 Real samples – well water

• TTC (accompanying microflora– brown-violet CFU)

93 Lactose TTC Agar with Tergitol® 7

TTC – from From below above

Real sample

94 MPN MPN - Most Probable Number - The end point dilution method preparation a series of dilutions from a sample Presumption: in the liquid medium the distribution of cells is uniform, cells do not cluster together and that there is detectable growth even if only one viable microbial cell is present in the tube/well.

estimation of bacterial count is based by using statistical tables

the number of positive tubes/wells (showing characteristic microbial growth: change in optical density, shifting of pH, inoculation of each incubation dilution into liquid media changing of redox values and subsequently using 3-5 parallels colour, etc.) MPN – coliform bacteria/ E. coli MPN - Most Probable Number - The end point dilution method Lauryl Sulfate Broth Nutrients: tryptose, lactose, L-tryptophan + phosphate bufferring system Inhibition: lauryl sulfate (G+, non-coliform G-)

positive tubes for coliform bacteria = gas formation due to lactose fermentation to gas in Durham tubes positive tubes for E. coli = gas formation in Durham tubes and cleaving of fluorogenic substrate (MUG) - fluorescence (and positive indol reaction – confimatory test) ISO 7251:2005-MPN principle Estimation of MPN values 1) Calculation by formulae – see IS0 7218:2007 10.5.6.1 2) Software programs 3) Using MPN tables Table B.5 ISO 7218:2007 MPN index for 3 following dilutions with 3 tubes starting from 1 g in a tube – (43 possibilities) - if x g in the first tube used for calculation – to multiply the result by 1/x (for 10 g = to multiply by 0.1, for 0.1 g = to multiply by 10)-Tables are for 1 g in the first tube Table B.7 ISO 7218:2007/ Table A.1 ISO 7251:2005(E) (more detailed) MPN index for 3 following dilutions with 5 tubes starting from 1 g in a tube (63 possibilities)

Number of Index Category Limits of confidence Category: positive results MPN (95%) If the analysis results are Lower Upper exact, then 95 % limit limit combinations should be 2 0 0 0.92 1 0.15 3.5 the category 1; 1.4% the category 2; 0.9 % the 2 0 1 1.4 2 0.4 3.5 category 3 and 0.1 % the 2 0 2 2.0 0 0.5 3.8 category 0. 2 2 1 2.8 3 0.9 9.4 ISO 7251:2005-MPN principle Using MPN tables – more than three dilutions for 3 tubes Sa Liquid sample 1 ml 10-1 ml 10-2 ml 10-3 ml MPN mp 10 ml le Other 10-1 g 10-2 g 10-3 g 10-4 g L. sample O.sampl. samples 1 g (ml-1 ) (g -1 ) 1 3 3 2 1 0 1.1x101 1.1x102 Lower limit 0.2x101 0.2x102 Upper limit 4.0x101 4.0x102 Number of positive Index Category Limits of 1 – better category results MPN confidence (95%) 3-3-2: Lower Upper higher total Table B.5 ISO 7218:2007 limit limit number 3 3 2 110 1 20 400 of the positive tubes 3 2 1 1.4 2 0.4 3.5 2 1 0 1.5 1 0.4 3.8 Horizontal method for the detection of microorganisms Horizontal method = by using Petri dishes SAMPLE PROCESSING + PRE-ENRICHMENT + ENRICHMENT • Sample amount according to EU legislative usually 25 g (or 10 g) • resuspension in pre-enrichment non-selective or selective medium (X g of sample + 9X ml of enrichment medium) – cultivation – in order to allow stressed cells to start to grow • Transfer the enriched suspension into selective enrichment medium (usually 0.1-1 ml into 10 ml volume) – in order to perform selective enrichment of target microorganism ISOLATION ON SELECTIVE MEDIA (usually 2 kinds of agar by different mode of action)

SUB-ISOLATION OF PRESUMPTIVE COLONIES (non-selective agar) BACTERIA GROWING ON SELECTIVE MEDIA ARE NOT APPROPRIATE FOR BIOCHEMICAL TESTING – SELECTIVE MEDIA DECREASED „FITNESS“ OF BACTERIA AND INFLUENCES THEIR PRESENT BIOCHEMICAL BEHAVIOR (NOT PROPERTIES), WHICH CAN INTERFER WITH THE EXACTNESS AND RELIABILITY OF TESTING CONFIRMATION/IDENTIFICATION Detection of Salmonella spp. according to ISO 6579:2002 25 g of sample + 225 ml of buffered peptone water; homogenization ISO 6579:2002 Microbiology of Pre-enrichment food and animal feeding stuffs Incubation at 37±1°C for 18±2 h -- Horizontal method for the 1. day detection of Salmonella spp.

1 ml of homogenate to 10 ml MKTTn 0.1 ml of homogenate to 10 ml RVS Selective incubation at 37±1°C for 24±3 h incubation at 41.5°C for 24±3 h enrichment 2. day

Plating out by 10 µ on two selective media and incubation at 37±°C for 24± 3h : Isolation on •Xylose Lysine – Desoxycholate Agar (XLD) selective agar plates •Any other selective medium (BGA,HE, BS, SS, DC, chromogenic media…) 3. day

Isolation of a charasteristic colony on Nutrient Agar and incubation at 37±°C for 24± 3h Confirmation

Confirmation 4.-6. day Detection of Salmonella spp. according to ISO 6579:2002: Pre-enrichment Pre-enrichment • Why? • in dried, processed foods are often present in low numbers and in debilitated condition • for the repair of cell damage, dilute toxic or inhibitory substances

NON-SELECTIVE PREENRICHMENT= RESUSCITATION BEFORE TRANSFER INTO SELECTIVE MEDIA

• What is important? • sampling • higher amount of sample – BWP 37 ± 1°C • pH • sour food – minimal pH 4.5 Buffered Peptone Water (BPW) For the preliminary, non-selective enrichment of bacteria, particularly pathogenic Enterobacteriaceae, from foodstuffs and other materials. Typical Composition (g/litre) Peptone from casein 10.0; sodium chloride 5.0; disodium hydrogen phosphate dodecahydrate 9.0; potassium dihydrogen phosphate 1.5.

Mode of Action The broth is rich in nutrients and produces high resuscitation rates for subletally injured bacteria and intense growth. The phosphate buffer system prevents bacterial damage due to changes in the pH of the medium. pH: 7.0 ± 0.2 at 25 °C. Detection of Salmonella spp. according to ISO 6579:2002: Selective enrichment Selective enrichment • Why? • to favor a higher ratio of Salmonella to non- Salmonella • allow Salmonella to multiplicate to level detectable after plating: need 103 - 104 CFU Salmonella /ml • MKTTn (37±1°C for 24±3 h): Muller-Kauffmann Tetrathionate- Novobiocin Broth • growth of coliform and other enteric bacteria (tetrathionate) • primarily Gram-positive bacteria (brillant green, novobiocin) • the accompanying bacteria (bile salts) • RVS (41.5°C for 24±3 h): Rappaport-VASSILIADIS Salmonella Enrichment Broth • high osmotic pressure (magnesium chloride concentration) • low pH (pH = 5.2) • malachite green (106 mg/L) • minimal nutritional requirements (peptone: 5 g/L) • selenite Rappaport-Vassiliadis Salmonella Enrichment Broth

Typical Composition (g/litre) Peptone from soymeal 4.5; magnesium chloride hexahydrate 29.0; sodium chloride 8.0; dipotassium hydrogen phosphate 0.4; potassium dihydrogen phosphate 0.6; malachite-green 0.036. pH: 5.2 ± 0.2 at 25°C cultivation: 41˚C, 24±3 h dipotassium hydrogen phosphate + potassium dihydrogen phosphate = phosphate buffering system http://www.kisanbio.com/shopimages/kisanbiotech/0390100000492.jpg Selectivity action: Selective conditions for preferably growth of Salmonella spp. favor especially other Enterobacteriaceae – Salmonella spp. are more resistant • malachite green • higher osmotic pressure by adding magnesium chloride • low pH 5.2 ± 0.2 • lower nutrition (peptone from soymeal)

Rappaport-VASSILIADIS Salmonella Enrichment Broth is not suitable for selective enrichment of S. typhi and S. paratyphi A. Detection of Salmonella spp. according to ISO 6579:2002: Isolation on selective agar plates Isolation on selective agar plates • non-lactose fermenting and produce hydrogen sulfide • XLD • second selective medium • Hektoen Enteric Agar • Deoxycholate citrate agar • Salmonella-Shigella agar • Brillant Green Agar • Bismuth Sulfite Agar • Chromogenic Agar: Rambach agar (Chromagar Co.); SMID agar (bioMérieux); CSE Agar (PPR Diagnostics); Compass Salmonella agar (Biokar Diagnostics).; Salmonella Chromogenic agar (Oxoid); Chromagar Salmonella (Chromagar Co.); ABC medium (Lab M) Detection of Salmonella spp. according to ISO 6579:2002: Isolation on selective agar plates • XLD (37±°C for 24± 3h)

Xylose-lysine-deoxycholate citrate; lactose + sucrose: phenol red, sodium thiosulfate and ferric ammonium citrate

• little or no production of hydrogen sulphide: S. Typhi, S. Senftenberg, S. Pullorum

• slowly produce of lactose: S. Choleraesuis, S. Arizona

• poor growth of S. Typhimurium

• Citrobacter, Proteus, Pseudomonas XLD (Xylose LysineDeoxycholate) Agar Typical Composition (g/litre) Yeast extract 3.0; sodium chloride 5.0; D(+)xylose 3.75; lactose 7.5; sucrose 7.5; L(+)lysine 5.0; sodium deoxycholate 1.0; sodium thiosulfate 6.8; ammonium iron(III) citrate 0.8; phenol red 0.08; agar-agar 14.5. pH: 7.4 ± 0.2 at 25 °C Differential activity: Degradation of xylose, lactose and sucrose to acid = phenol red to change its colour to yellow. Production of hydrogen sulfide is indicated by thiosulfate and iron(III) salt - to form a precipitate of black iron sulfide in the colonies. Decarboxylation of lysine to cadaverine - a purple colouration around the Salmonella enteritidis NCTC 5188 colonies due to an increase in pH.

Klebsiella pneumoniae ATCC 13883 Salmonella – BG agar Typical Composition (g/litre) Peptone from meat 10.0; meat extract 5.0; yeast extract 3.0; disodium hydrogen phosphate 1.0; sodium dihydrogen phosphate 0.6; lactose 10.0; sucrose 10.0; phenol red 0.09; brilliant green 0.0047; agar-agar 12.0.pH: 6.9 ± 0.2 at 25 °C. Selectivity action: Brilliant green – inhibition of G+ bacteria, Salmonella Typhi and Shigella By addition of 0.2 % deoxycholate sodium – inhibition of Proteus colonies to swarm Differential activity: Salmonella enteritidis NCTC 5188 Fermentation of lactose – phenol red changes colour to yellow from red (in alkaline)

Appearance of Colonies Microorganisms Lactose- and sucrose-negative: Red, surrounded by a Salmonella, Proteus (no bright red swarming), Pseudomonas zone (small, crenate colonies) and others Lactose- or sucrose-positive: Yellow, surrounded by a E. coli, Enterobacter, possibly yellow zone Citrobacter, Klebsiella and others. Salmonella typhimurium ATCC 14028 Detection of Salmonella spp. according to ISO 6579:2002: Confirmation • Isolation of charasteristic colony onto non-selective media – Nutrient agar (37±°C for 24± 3h) • Confirmation: • Biochemical (37±°C for 24± 3h) • TSI • Urea agar (Christensen) • L-lysine decarboxylase https://classconnection.s3.amazonaws.com/74/flashcards/1275074/jpg/biochemical_test_re • β-galactosidase (ONPG) sults-_shigella1332821047536.jpg • VP test • Indole test • Motile (semi-solid nutrient agar) • Serological (37±°C for 24± 3h) • O antigens (somatic or cell wall) • H antigens (flagellar) • Vi antigens (polysaccharide virulence) • Phase I http://www.keydiagnostics.com.au/images/stories/virtuemart • Phase II /product/microgen-salmonella-latex-kit.jpg Confirmation of genus Salmonella

Strains Salmonella spp. S. Typhi S. Parathypi A S. Parathypi B S. Parathypi C Ostatní kmeny reaction % reaction % reaction % reaction % reaction %

TSI: glucose (acid + 100 + 100 + + + 100 formation) TSI: glucose (gas - 0 + 100 + + + 92 formation) TSI: lactose (acid - 2 - 100 - - - 1 formation) TSI: succrose (acid - 0 - 0 - - - 1 formation) TSI: hydrogen + 97 - 10 + + + + 92 sulfide Urea splitting - - 0 - - - - 1 Lysine + - 0 + + + + 95 decarboxylation β-galactosidace - - 0 - - - - 2 formation Voges-Proskauer - - 0 - - - - 0 reaction Indole production - - 0 - - - - 1 Biochemical confirmation of Salmonella spp. • TSI Triple Sugar Iron Agar (use a straight wire to stab the butt and streak the agar surface) - to determine whether organisms can ferment glucose, sucrose and/or lactose with or without production of gas and the ability of the organism to produce hydrogen sulphide from thiosulphate in an acid environment is also tested. inoculation in the butt by stab inoculation on the slant on the Formula / Liter surface Enzymatic Digest of Casein .. 5 g Enzymatic Digest of Animal • Fermentation of dextrose Tissue.... 5 g (glucose) to acid Yeast Enriched Peptone ..... 10 g • anaerobically - yellow butt Dextrose ...... 1 g • aerobically leads to products, Lactose ..... 10 g which are later oxidised Sucrose ..... 10 g by air and pH remains alkaline (red) Ferric Ammonium Citrate ... 0.2 g Sodium Chloride ...... 5 g • Fermentation of glucose to gas Sodium Thiosulfate ...... 0.3 g (anaerobically - in the stab) Phenol Red ...... 0.025 g Cracks, splits, or bubbles in medium Agar ...... 13.5 g indicate gas production Results Final pH: 7.3 ± 0.2 at 25 °C • Production of hydrogen sulfide An alkaline slant-acid butt (H2S) – reaction with ferrous (red/yellow) - fermentation of sulphate and sodium thiosulphate - dextrose only. a black precipitate in butt An acid slant-acid butt • Lactose and/or succrose (yellow/yellow) - fermentation of utilisation to acid dextrose, lactose and/or sucrose. Acid production – yellow butt and An alkaline slant-alkaline butt slant (red/red) - dextrose or lactose not fermented (non-fermenter). Bacterium fermenting lactose Phenol red – alkaline = red, and/or succrose are able to acid = yellow ferment glucose Biochemical confirmation of Salmonella spp. • TSI Triple Sugar Iron Agar (use a straight wire to stab the butt and streak the agar surface) - to determine whether organisms can ferment glucose, sucrose and/or lactose with or without production of gas and the ability of the organism to produce hydrogen sulphide from thiosulphate in an acid environment is also tested. Biochemical confirmation of Salmonella spp.

Urea agar (Christensen) (use a 1 μl loop full) - to determine whether an organism can split urea into ammonia and carbon dioxide by the action of the enzyme urease.

Phenol red: Composition Gms / Litre Medium is acidic - yellow Peptic digest of animal tissue 1.000 (pH 6.8±0.2) Dextrose 1.000 Alkaline reaction by urea Sodium chloride 5.000 hydrolysis - pink colour Disodium phosphate 1.200 Monopotassium phosphate 0.800 Nutrition: Phenol red 0.012 Peptic digest of animal tissues, dextrose Agar 15.000 Sodium chloride - osmotic equilibrium Final pH ( at 25°C) 6.8±0.2 http://image.slidesharecdn.com/lab17-141201085153-conversion- gate01/95/-10-638.jpg?cb=1417424219 Phosphates - buffering of the medium.

35-37 °C

http://image.slidesharecdn.com/helicobacterpylori-copy-110516201901- phpapp01/95/campylobacter-jejunihelicobacter-pylori-39-728.jpg?cb=1305577391 Biochemical confirmation of Salmonella spp.

• Indole test (use a 1 μl loop full) - to determine whether an organism can split indole from tryptophan. FORMULA IN g/l Indole Kovacs Reagent: Tryptone 10.00 L-Tryptophan 1.00 p-Dimethylaminobenzaldehyde 50.0 gm Sodium Chloride 5.00 Hydrochloric Acid, 37% 250.0 ml Final pH 7.5 ± 0.1 at 25ºC Amyl Alcohol 750.0 ml

Inoculation below the surface

indole production from tryptophan Production of indole is detected by Kovac´s reagent

http://www.microbiologyinfo.com/indole-test-principle-reagents-procedure-result- interpretation-and-limitations/ Biochemical confirmation of Salmonella spp. • L-lysine decarboxylase (use a 1 μl loop full) - to determine whether an organism can decarboxylate an amino acid (lysine) leading to formation of an amine. After inoculation both tubes are overlayed with sterile paraffin oil. Two steps detection: 1. step Dextrose is fermented – H+ is released Acidic conditions • bromocresol purple changes colour to yellow • in acidic conditions lysine decarboxylase is activated 2. step L-lysine is decarboxylated and OH- released Alkaline conditions http://fce-study.netdna- ssl.com/images/upload- • bromocresol purple changes colour back to Lysine Decarboxylase Broth flashcards/1029530/23326 Typical Composition (g/litre) 21_m.jpg purple Peptic digest of animal tissue 5.000; Yeast extract 3.000;Dextrose 1.000 (glucose prepared from starch, chemically equivalent to glucose); L-Lysine hydrochloride 5.000; Bromocresol purple 0.020 Final pH ( at 25 °C) 6.8±0.2 Inoculate below the surface, cover by sterile paraffin oil

- + CO2 + OH Biochemical confirmation of Salmonella spp. • β-galactosidase (ONPG) (use a 1 μl loop full) - to determine whether an organism has the enzyme ß-galactosidase by using the compound o- nitrophenyl-beta-D-galactopyranoside (ONPG). - this test is used to differentiate between lactose-negative and lactose-delayed organisms. - ONPG is hydrolysed by the enzyme to o-nitrophenol, which is yellow, and galactose. Lactose fermenting bacteria (ONPG Late lactose fermentering bacteria positive): Permease – not present two enzymes delayed in the production of acid from lactose Permease – transferring of the lactose because of sluggish permease activity molecule into the bacterial cell β-galactosidase – present β-galactosidase - cleaving the galactoside - give a positive ONPG test bond, producing glucose and galactose - Citrobacter spp., Arizona spp, inducible enzyme - made ONLY in the presence of the lactose substrate Non lactose fermenter (ONPG Coliform bacteria as E.coli, Negative): Klebsiella spp., Enterobacter spp. Permease – not present β-galactosidase – not present O-Nitrophenyl-β-D-galactopyranoside (ONPG) is Salmonella spp; Shigella spp; Proteus spp; Providencia spp. and Morganella spp. structurally similar to lactose (i.e. ONPG is an analog of lactose), except that orthonitrophenyl has been substituted for glucose. ONPG is colorless compound: O- nitrophenol is yellow, visual evidence of hydrolysis.

hydrolysis Biochemical confirmation of Salmonella spp. β-galactosidase (ONPG)

Procedure Results and Interpretations Culture The rate of hydrolysis of ONPG Bacteria grown in medium containing lactose (to induce the to o-nitrophenol may be rapid production of the galactosidase enzyme) , such as Kligler for some organisms; producing iron agar (KIA) or Triple sugar Iron (TSI) agar, (Note: β- a visible yellow color reaction galactosidase enzyme (inducible enzyme) is made ONLY within 5 to 10 minutes. in the presence of the lactose substrate). Most tests are positive within 1 Inoculation hour; however, reactions should 1.A loopful of bacterial growth is emulsified in 0.05mL of not be interpreted as negative physiologic saline to produce a heavy suspension before 24 hours of incubation. 2.One drop of toluene is added to the suspension and The yellow color is usually vigorously mixed for a few seconds to release the enzyme distinct and indicates that the for bacterial cells. organism has produced o- 3.An equal quantity of buffered ONPG solution is added to nitrophenol from the ONPG the suspension. substrate through the action of 4.The mixture is placed in a 37 oC water bath β-galactosidase 5.When Using ONPG Tablets 6.A loopful of bacterial suspension is added directly to the ONPG substrate resulting from adding 1mL of distilled water to a tablet in a test tube. 7.This suspension is also placed in a 37oC water bath Biochemical confirmation of Salmonella spp. • Voges–Proskauer (VP) test (use a 1 μl loop full) - to determine whether an organism can produce acetylmethylcarbinol (acetoin) from fermentation of glucose. If present, acetylmethyl carbinol is converted to diacetyl in the presence of ∝- naphthol (to be added first), strong alkali (40% KOH), and atmospheric oxygen. The diacetyl and quanidine- containing compounds found in the peptones of the broth then condense to form a pinkish red polymer. Positive Reaction: A pink-red color at the surface Examples: Viridans group streptococci (except Streptococcus vestibularis), Listeria, Procedure of Voges–Proskauer (VP) Test Enterobacter, Klebsiella, Serratia marcescens, 1.Prior to inoculation, allow medium to Hafnia alvei, Vibrio eltor, Vibrio alginolyticus, etc. equilibrate to room temperature. Negative Reaction: A lack of a pink-red color 2.Using organisms taken from an 18-24 Examples: Streptococcus mitis, Citrobacter sp., hour pure culture, lightly inoculate the Shigella, Yersinia, Edwardsiella, Salmonella, Vibrio furnissii, Vibrio fluvialis, Vibrio vulnificus, and Vibrio medium. parahaemolyticus etc. 3.Incubate aerobically at 37 °C, 24 hours. A copper color should be considered negative. A 4.Following 24 hours of incubation, aliquot rust color is a weak positive reaction. Quality Control of Voges–Proskauer (VP) Test MRVP broth (pH 6.9) 2 ml of the broth to a clean test tube. VP positive: Enterobacter aerogenes (ATCC13048) 5.Re-incubate the remaining broth for an VP negative: Escherichia coli (ATCC25922) Ingredients per liter of deionized water: additional 24 hours. buffered peptone= 7.0 gm 6.Add 6 drops of 5% alpha-naphthol, and glucose= 5.0 gm mix well to aerate. dipotassium phosphate= 5.0 gm 7.Add 2 drops of 40% potassium hydroxide, Voges-Proskauer Reagent A: Barritt’s reagent A and mix well to aerate. Voges-Proskauer Reagent B: Barritt’s reagent B 8.Observe for a pink-red color at the surface Alpha-Naphthol, 5% 50 gm within 30 min. Shake the tube vigorously Potassium Hydroxide 400 gm 1000 during the 30-min period. Absolute Ethanol ml Deionized Water 1000 ml Biochemical confirmation of Salmonella spp.

• Commercional delivered kits • API 20E - Identification system for Enterobacteriaceae and other non-fastidious Gram- negative rods (BioMerieux) - a plastic strip holding 20 mini-tests with dehydrates substrates

http://www.retroscope.eu/wordpress/wpcontent/uploads/2010/10/SalmonellaAPI20E.jpg

After incubation in a humidity chamber for 18-24 hours at 37°C, the color reactions are read (some with the aid of added reagents), and the reactions (plus the oxidase reaction done separately) are converted to a seven-digit code which is called the Analytical Profile Index, from which name the initials "API" are derived. The code can be fed into the manufacturer's database via touch-tone telephone, and the computerized voice gives back the identification, usually as genus and species. An on-line database can also be accessed for the identification. Serological confirmation of Salmonella spp.

• Serological confirmation: slide agglutination • O antigens • H antigens • Vi antigens • Phase I • Phase II https://classconnection.s3.amazonaws.com/ 664/flashcards/1813664/jpg/gram_neg_anti • Elimination of auto-agglutinable strains gens1358634331589.jpg • Place one drop of saline onto a clean glass slide. Disperse in this drop part of the colony to be tested or a colony from a pure culture, so as to obtain a homogenous and turbid suspension. • Rock the slide gently for 30 60 seconds. Observe the result against a dark background, preferably with the aid of a magnifying glass. • If the bacteria have clumped together into more or less distinct units, the strain is considered auto-agglutinable, and the detection of antigens will be impossible. • In practice, auto - agglutinating strains of Salmonella are rare; it is more economical to perform poly O, H and Vi serology first. Serological confirmation of Salmonella spp.

• Examination for O antigen • Using one pure colony, recognised as non-autoagglutinable, proceed as above, using one drop of the anti O serum instead of saline solution. • If agglutination occurs, the reaction is considered positive for the presence of that antigen.

•Examination for H antigens • Inoculate a Semi-Solid Nutrient Agar (SSNA) slope with a pure nonautoagglutinable colony from the XLDA or BGA plate. Incubate at 37± 1°C for 24± 3 hours. • Use this culture for examination for H antigens, proceeding as above, but using one drop of the anti H serum instead of saline solution. • If agglutination occurs, the reaction is considered positive for the presence of H antigen.

•Examination for Vi antigen • Use this culture for examination for Vi antigens, proceeding as above, but using one drop of the anti Vi serum instead of saline solution. Interpretation of serological confirmation

A B

Slide Agglutination

Sample A is a positive reaction and sample B is a negative reaction.

http://www.ssi.dk/~/media/Admin/Diagnostica%20 Downloads/Downloads%20UK/Brochures/BrochureS almonella%20antisera%20ver%202%20high1411201 Salmonella on swarm agar 1145552.ashx Thank you for your attention

[email protected]