Aseptic Technique APPENDIX 4D

Tomasz Bykowski 1 and Brian Stevenson2 1Center for Medical Education, Warsaw, Poland 2Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, Kentucky

ABSTRACT This chapter describes common laboratory procedures that can reduce the risk of culture con- taminations (sepsis), collectively referred as “aseptic technique.” Two major strategies of aseptic work are described: using a Bunsen burner and a laminar flow hood. Both methods are presented in the form of general protocols applicable to a variety of laboratory tasks such as pipetting and dispensing aliquots, preparing growth media, and inoculating, passaging, and spreading microor- ganisms on petri dishes. Curr. Protoc. Microbiol. 11:A.4D.1-A.4D.11. C 2008 by John Wiley & Sons, Inc. Keywords: aseptic technique r sterilization r bacteriology r Bunsen burner r laminar flow r laminar hood r virology r cell culture

INTRODUCTION understand the appropriate methods of dis- Aseptic technique is a set of routine mea- posal and decontamination for the specific mi- sures that are taken to prevent cultures, sterile croorganism with which he or she is working. media stocks, and other solutions from being Refer to UNIT 1A.1 and APPENDIX 1B for more contaminated by unwanted microorganisms information. (i.e., sepsis). While such actions are some- times called “sterile technique,” that terminol- The Bunsen Burner ogy is appropriate only in reference to prevent- Probably the easiest way to create a ing introduction of any organisms to labora- relatively sterile environment on the lab- tory or medical equipment and reagents (e.g., oratory bench is by using a simple gas- during surgery). Since the goal of a biologist powered burner (see Fig. A.4D.1). This com- is to grow microorganisms or eukaryotic cells mon piece of equipment burns a continuous without introduction of extraneous organisms, stream of a flammable gas—usually natural aseptic techniques are crucial for accurate and gas (methane)—based upon a design made al- meaningful experimentation. most 150 years ago by the German chemist One should always remember that a com- Robert Wilhelm Bunsen (1811-1899). A ma- pletely sterile working environment does not jor purpose of the open flame in aseptic tech- exist. However, there are a number of sim- nique is to create a cone of hot air above and ple, common sense procedures that will reduce around the laboratory bench to reduce the vi- the risk of culture contaminations. Examples ability of organisms on suspended dust parti- of aseptic technique are cleaning and disin- cles. The ability of the Bunsen burner flame to fecting lab surfaces prior to use, limiting the heat things very quickly also makes it an ideal duration that cultures or media are uncapped choice for sterilizing inoculating loops, warm- and exposed to the air, keeping petri dishes ing glass bottle necks, or igniting alcohol on closed whenever possible, effectively steriliz- culture spreaders. ing inoculating loops and other equipment that A Bunsen burner is not practical in some sit- comes into contact with cultures or media, and uations, e.g., within a laminar flow unit where avoiding breathing on cultures or sterile instru- the heat will disrupt airflow. A microincinera- ments. These precautions will become second tor may be used as an alternative. This con- nature after practical laboratory experience. sists of a circular heating element. Placing CAUTION: The information in this an inoculating loop or needle within the ring APPENDIX is meant as an introduction to basic will quickly heat and sterilize the loop/needle. aseptic technique and does not cover safety Note that a microincinerator does not provide aspects necessary for working with specific other aseptic technique benefits of a lit Bunsen Commonly Used Methods for Cell organisms. It is important that the reader burner. Culture

Current Protocols in Microbiology A.4D.1-A.4D.11, November 2008 A.4D.1 Published online November 2008 in Wiley Interscience (www.interscience.wiley.com). DOI: 10.1002/9780471729259.mca04ds11 Supplement 11 Copyright C 2008 John Wiley & Sons, Inc. chimney

air regulating collar

gas

rubber tubing

Figure A.4D.1 Bunsen burner. The gas burner consists of a vertical metal tube through which a narrow jet of natural gas is directed. Air is drawn in via air holes located near the stand. The gas/air mixture burns above the upper opening. The regulating collar can be turned to cover or partly cover the air holes, which allows regulation of the amount of air sucked in and hence the temperature and shape of the flame.

The Laminar Flow Unit laboratories, where they help prevent spread A laminar flow unit (or hood) is a sophis- of viruses and some bacteria. Consult UNIT 1A.1 ticated appliance that can further help pre- as well as the resources in APPENDIX 1B vent contamination of reagents and biological for additional information on biosafety and cultures. Used correctly, it provides the work biocontainment. space with clean, ultrafiltered air. It also keeps room air from entering the work area and both suspends and removes airborne contaminants STRATEGIC PLANNING introduced into the work area by personnel. Contamination Risk Assessment and The most important part of a laminar flow Space Organization hood is a high-efficiency bacteria-retentive fil- Localizing potential sources of biological ter, i.e., the HEPA (high-efficiency particulate contamination is an important part of arrang- air) filter. A certified HEPA filter must cap- ing space when a room is used as a laboratory. ture a minimum of 99.97% of dust, pollen, Benches for aseptic work should be organized mold, bacteria, and any airborne particles with away from air conditioning vents, cooling asizeof>0.3 μm at 85 liters/min. The first fans, open windows, or blowers from heating HEPA filters were developed in the 1940s by or refrigerating systems. Containers used for the U.S.A. Atomic Energy Commission as part the disposal of biological material may con- of the Manhattan Project (the development of tain large numbers of viable microorganisms, the atomic bomb) to provide an efficient, effec- even when emptied. Some contaminants, e.g., tive way to filter radioactive particulate con- sporulating bacteria, yeasts, or fungi, are very taminants. HEPA filter technology was declas- difficult to eradicate from the working envi- sified after World War II, allowing extensive ronment. Waste should be kept very well cov- research and commercial use. ered or, if feasible, in a different room, espe- Laminar flow hoods are essential com- cially if using particularly harmful or sturdy ponents of many biosafety level (BSL)-2 microorganisms. Aseptic Technique A.4D.2

Supplement 11 Current Protocols in Microbiology General Cleanliness that are either flammable or would be de- In principle, keeping high standards of natured by heat are usually filter sterilized. cleanliness has to be a universally accepted A range of syringe-based or bottle-top fil- norm in the biological laboratories. People ters of different pore sizes physically remove should wear lab coats that are worn only in (exclude) living organisms and can be used the laboratory. Researchers should wash their according to the suppliers’ instructions. Filtra- hands often during the course of the day, es- tion devices may use either negative (vacuum) pecially prior to any and all handling of bi- or positive pressure. An advantage of positive ological cultures, media, or sterile supplies. pressure in filtering is that it reduces clogging Dust and stains must be removed regularly, from precipitated salts. and spills cleaned and decontaminated im- Most living organisms are retained by a mediately. Bench tops and shelves should be 0.45-μm filter. That size filter is often used washed immediately before all uses with 10% as a prefilter because it also clears the liquid (v/v) household bleach (containing sodium of larger particulates. However, because many hypochlorite). This solution will inactivate all bacteria can pass through 0.45-μm pores, a viruses, bacteria, fungi, and other potential 0.22-μm filter should be used to ensure steril- contaminants. Alternatively, a solution of an- ization of the fluids. Viruses will pass through tiseptic cleanser (e.g., Lysol), diluted accord- 0.22-μm filters, although they generally stick ing to the manufacturer’s directions, is ade- to the membrane. Some bacteria, such as quate. Organic disinfectants such as 70% (v/v) saprophytic Leptospira spp. commonly found ethanol are generally less effective than bleach in domestic water supplies, may also pass solutions because ethanol may evaporate too through 0.22 μm filters. If such potential quickly to effectively sterilize surfaces and contaminants may cause difficulties, consider may not completely inactivate all potential autoclaving water before using for solutions contaminants. Outerwear may be covered with that need to be filter sterilized. dirt or dust, and so should be kept in a cloak- Filtering procedures are usually carried out room away from the work space. These simple in a laminar flow unit or on a bench equipped rules of cleanliness will eliminate the bulk of with Bunsen burner (see below) to avoid potential contaminations from the work area. recontamination of just-sterilized material.

Sterilizing Equipment and Reagents as Autoclaving a Prelude to Effective Aseptic Work CAUTION: Items containing volatile sol- Starting aseptic work with material that vents or corrosive chemicals (e.g., phenol, is contaminated is a path to nowhere. Many trichloroacetic acid, ether, chloroform), or reagents are supplied by their manufacturer as any radioactive isotopes cannot be autoclaved ready to use for cultures, while many others under any circumstances. do not have to be sterilized at all (e.g., ethanol, Autoclaving (using steam under pressure) phenol, or concentrated detergents). How- is a rapid method for sterilizing almost any- ever, some require sterilization (i.e., the com- thing except heat-labile substances. A temper- plete destruction or elimination of all viable ature higher than the boiling point of water organisms) before use. inside the autoclave (super-heating of liquids) People have developed a wide variety of is achieved because the system is under pres- sterilization methods based on heat and high sure. Typical autoclaving conditions of 121◦C pressure, liquid and gas chemicals, radiation, (250◦F) for 15 minutes at 103 kPa (15 psi) or physical removal of microorganisms that are are sufficient to kill virtually all forms of used in the process of food storage, treatment life, including bacterial endospores, and will of medical surgery equipment, or drug pro- inactivate viruses. duction. Although only a few, quite straight- However, the time of the sterilization cycle forward protocols are actually applied to the should be modified according to the amount handling of goods in microbiology or molecu- and type of loaded items. Examples of ob- lar biology laboratories, they should be carried jects that have a slower surface heating or out very carefully, because there are no degrees steam penetration and should be autoclaved of sterilization: an object is either sterile or longer are an unusually large bottle of media, not. a bulky bag of biohazard waste, or a beaker full of microcentrifuge tubes with aluminum foil Filter sterilization covering. Commonly Used Reagents such as antibiotics, drugs, sug- General principles of loading an autoclave Methods for Cell ars, amino acids, vitamins, and complex media can be summarized as follows: Culture A.4D.3

Current Protocols in Microbiology Supplement 11 1. Polypropylene and polycarbonate can types of packaging have built-in indicators be autoclaved, but polyethylene and high- on them. density polyethylene cannot. Initials im- Autoclaves should be tested periodically, printed on bottom of containers can help in using biological tests such as bacterial spore identification of the material type (PP, poly- test kits. Users should consult with their insti- propylene; PC, polycarbonate; PE, polyethly- tutions for guidelines on frequency of testing lene; HDPE, high density polyethylene). and record keeping. 2. The autoclave must not be overcrowded; the steam needs to be able to penetrate every- Oven sterilization where. All materials must be placed in an au- Selected goods such as glassware, metal, and other objects that will not melt at temper- toclavable tray (polypropylene plastic or stain- ◦ ◦ less steel tubs) with sides at least 10 cm high atures between 121 C and 170 C can be ster- to catch spills and eliminate possible damage ilized with dry heat (in a hot-air oven). As the to the machine. Dry items must be wrapped heat takes much longer to be transferred to the organism, a temperature of 160◦Cfor≥2hr in aluminum foil to prevent them from being ◦ recontaminated after the procedure. or 170 C for 1 hr is routinely used. The pro- 3. Liquids should be sterilized in a vessel cedure certainly cannot be applied for liquids, of at least twice the volume of the liquid to be rubber, or any plastic objects. However, the autoclaved, to avoid boiling over and spilling. method has advantages; for instance, it can be To prevent bottle bottoms from breaking, the used for powders and other heat-stable items tub or tray holding them should be filled with that are adversely affected by steam, and it 3 to 5 cm of water. Heat resistant borosilicate does not cause rusting of steel objects. It is glass (Pyrex) vessels are preferred. also a method of choice for the treatment of 4. Containers must be loosely capped or glassware used for work with ribonucleic acid stoppered to avoid shattering during pressur- (RNA), since the process of baking not only ization and depressurization. For screw-cap kills organisms, but also inactivates any resid- containers, the lid should be hand tightened, ual RNA-degrading enzymes (RNases). then loosened one-half turn. Stoppers occa- General considerations sionally pop off during the autoclaving pro- Note that a standard microwave oven (i.e., cess, so loosely covering the stopper or cap the household appliance), although capable of with a piece of aluminum foil can help hold bringing water to boiling, does not provide a the cap in place. useful method for sterilizing laboratory goods CAUTION: Be aware that large bottles with and should only be used for warming up the narrow necks can simulate sealed containers if liquids. filled with too much liquid. Sterilized solutions and other reagents will To optimize autoclaving of common lab- need to cool down before use, which takes oratory goods, two general types of exhaust cycles has been designed: time. Thus sterilization should be performed 1. The “gravity” or “fast exhaust” cycle is well before the actual experimental work for all dry items, e.g., glassware, tips, flasks, begins. tubes. The chamber is charged with steam, and It is best for each researcher in the labo- a set pressure and temperature conditions are ratory to keep his/her own stocks of reagents maintained for the desired period of time. At (e.g., media, buffers, salts, glycerol) to pre- the end of the cycle, a valve opens, and the vent accidental contamination by a co-worker. chamber rapidly returns to atmospheric pres- In this manner, each worker is solely respon- sure. Drying time may also be added to the sible for the condition of his/her reagents and cycle. for any impact on experimental results. This 2. The “liquid” or “slow exhaust” cycle is strategy can be facilitated by preparing media used for liquids to prevent them from explo- or solutions in several small bottles (aliquots), sive boil-overs. Pressure is slowly released, one for each researcher, with only one con- which allows the super-heated fluids to cool tainer per worker opened at a time. gradually. Although growth media are more likely to CAUTION: It is acceptable to autoclave dry produce noticeable biological contamination items using the liquid cycle, but liquids should (by turning color, developing clumps, or be- never be autoclaved using a fast exhaust cycle. coming turbid), most other liquids used in the Sterilized goods can be denoted using a laboratories can also support the growth of or- special indicator tape which contains a chem- ganisms. Solutions should be checked before Aseptic Technique ical that changes color when the appropriate using by gently swirling and looking for the autoclaving conditions have been met. Some presence of cloudy material. A.4D.4

Supplement 11 Current Protocols in Microbiology Table A.4D.1 Common Laboratory Items Useful for Aseptic Work

Material Common use

Aluminum foil Widely used in laboratories for packing goods for autoclaving. It protects them from recontamination and facilitates storage. Simple lids can be made of it for beakers, flasks or bottles. Wrapping boxes of disposable tips, centrifuge tubes, and bundles of glass pipets or spatulas with aluminum foil not only protects, but also helps to keep them organized by size or type. Disposable rubber gloves Should be worn at all times during aseptic work; usually made of latex rubber, PVC (polyvinyl chloride), or nitrile. Recently a range of mild to severe allergic reactions from exposure to natural rubber latex has been widely recognized. Providing the laboratory with both powder-free low-protein latex disposable gloves and nonlatex gloves is recommended. After removing latex gloves, hands should be washed with a mild soap and dried thoroughly. Disposable wipes (lint-free) Since they do not leave unwanted particles behind, they are considered very useful for wiping bench tops, interior of the laminar flow hood, or any other object during cleaning and disinfection. 70% (v/v) ethanol or other Simple, flammable disinfectants used for cleaning items industrial methylated spirits and surfaces from microorganisms and stains. They are (IMS; denatured alcohols) also used for dipping culture spreaders. See Safety Considerations for usage instructions. Eye protection and thermal gloves Equipment for extra protection should be kept within reach and used during procedures such as opening an autoclave. Flint-on-steel striker Used to quickly ignite a Bunsen burner when needed. 10% (v/v) household bleach Chemical more effective for disinfecting surfaces than (containing sodium hypochlorite) quickly evaporating ethanol. It should not be stored for longer than 1 month at room temperature, since sodium hypochlorite will degrade. Laboratory coat Clean, long coat of the right size used to protect the researcher against toxic substances and biohazard material. It also protects samples from contamination from human skin and apparel. Spray or squirt plastic bottles Very useful for dispensing ethanol, water, and other liquids for disinfection and cleaning of large surfaces and places difficult to reach with a wipe.

Useful Materials to Have on Hand ing the machine after completion of the cy- A list of simple materials that might be use- cle. Consider some basic rules that should be ful in aseptic work and should be kept on hand applied while opening autoclave: is presented in Table A.4D.1. Wear a lab coat, eye protection, loose fitting thermal (insulating) gloves, and closed- SAFETY CONSIDERATIONS toe shoes. Autoclaving Check that the chamber pressure is zero Autoclaving produces steam, which can before attempting to open the door. Stand behind the door when opening and Commonly Used cause burns and requires a lot of caution in Methods for Cell handling. The most important moment is open- use it as a shield. Slowly open a crack and Culture A.4D.5

Current Protocols in Microbiology Supplement 11 wait for steam to be released. Beware of a rush Ultraviolet Light of steam. Open the door fully to remove tray. Many laminar flow hoods are equipped with After the slow exhaust cycle, open the au- ultraviolet (UV) lights, which can help re- toclave door and allow liquids to cool 10 to duce contamination by inducing DNA damage 20 min before removing. Superheated liquids to potential contaminants. Such light is also may suddenly boil over if the container is harmful to laboratory workers. Never look di- moved. rectly at a UV light, as that can cause eye Do not move hot bottles if the liquid is bub- damage. The UV light can also cause skin bling or boiling. burns (sun burns) to anyone in the room. Con- When removing liquids from the autoclave, sequently, UV lights should always be turned point the opening of the container away from off whenever the room is occupied. yourself and other people because the liquid may suddenly boil and spray out the opening. TECHNIQUES FOR MAINTAINING ASEPTIC CONDITIONS Working with Open Flame Usually, there is no need to equip the lab- Burners should be on only for as long as oratory with expensive paraphernalia just for needed for a particular procedure. The blue culture of hardy bacteria such as Escherichia flame of a hot Bunsen burner can be difficult coli, since that bacteria’s rapid growth rate to see. Never leave an open flame unattended, generally allows it to out-compete any con- not even for a few seconds. taminants. A typical laboratory bench supplied Since burns are among the most common with a Bunsen burner is sufficient. Working laboratory accidents, making sure that face, aseptically while using a cone of heat pro- clothing, and hair are not above or near the duced by the burning gas has been the practice opening of the burner tube is a crucial safety in laboratories worldwide since the 19th cen- measure. Temperatures in the hottest region tury. It is straightforward and does not require of the burner flame approach 1500◦C, and ob- considerable financial outlays. jects will heat extremely quickly. Any heated However, a laminar flow hood is the method object, glass or metal, needs time to cool down of choice when a more rigid aseptic technique before it can be safely touched. is required. The rich media used for eukary- It is also absolutely necessary to remove otic cell cultures and many microorganisms, flammable and combustible materials from coupled with their slow growth rates, necessi- the vicinity of the flame. Nitrocellulose mem- tates high levels of security from contamina- branes, commonly used for blotting tech- tion. For some organisms, work must always niques, are extremely flammable. be performed inside a laminar flow unit to meet Ethanol used for dipping culture spread- biosafety standards and to protect personnel ers is an exception to the rule regarding against potentially harmful microorganisms or flammables, but only small volumes (≤20 ml) viruses. in glass beakers should be used at a time. Any aseptic work should be completed as An accidental alcohol fire in a beaker can quickly as is comfortable to minimize the risk be quickly extinguished simply by covering of contamination. Consider that working in an the top of the beaker with a piece of alu- aseptic manner may take longer than when be- minum foil. Keeping the alcohol beaker cov- ing less cautious. Reserve extra time to avoid ered with such a piece of foil when not in use being rushed, which may result in spilling will help reduce accidental fires and will slow or breaking important samples, dishes, or evaporation. solutions. A fire extinguisher should always be kept nearby. Bench-Top Aseptic Work Using a The tubing connecting the Bunsen burner to Bunsen Burner the laboratory gas supply should be checked Before starting work, locate the gas and air regularly. Latex rubber tubing tends to harden controls on the burner. They can be in different and crack after exposure to air, making it a poor places on different models. In some cases, it tubing choice. Silicone tubing (e.g., Tygon) is is necessary to use the laboratory gas valve generally more durable. to control the gas flow rate. Once a Bunsen Never use a Bunsen burner in a Class II burner has been adjusted for optimal air and laminar flow hood. The heated air may disrupt gas flow, those settings can be left in place, air flow and possibly damage the hood. making future usage more efficient. Aseptic Technique A.4D.6

Supplement 11 Current Protocols in Microbiology 1. Close the burner’s air control. If the 4. When finished using the burner, turn it burner has its own gas control valve, shut it off at the laboratory gas valve. Do not leave a and then open it allowing only small amount lit burner unattended. of gas to get into the burner tube. CAUTION: Never leave a lit burner unat- 2. To ignite the gas using a flint-on-steel tended. striker, hold it 2 to 3 cm above and slightly to the side of the burner tube top. Squeeze and release striker repeatedly until a spark ignites Handling and Pipetting Liquids the flame. If using a match, light it and slowly All growth media, cultures, and sterile bring it up from the bottom along the side of reagents should be manipulated inside the cone the burner tube until the flame ignites. If a of heat that is created above and around a lit match is stuck in the middle of the gas stream, Bunsen burner or within a laminar flow hood. the high-pressure gas stream often blows out Bottles and test tubes should be arranged near the flame before the burner can light. the burner but not directly within the flame. 3. If the initial flame is low on oxygen, it Brief (1 to 2 sec) flaming of the lips of tubes is bushy orange in appearance and is not very and flasks should only be done when caps hot (Fig. A.4D.2A,B). Open the air control are removed during transfer of liquids and vent until the central blue cone of the flame cultures. This is especially important when forms and a slight buzzing sound is audible the content is to be poured from a container (Fig. A.4D.2C). If the flame is too small, grad- (e.g., pouring petri dishes or aliquots of large ually increase both the supply of gas and air volumes of liquid). flow to the burner. The hottest part of the flame The purpose of flaming is not to actually is just above the central blue cone. sterilize, but to warm the opening and create

yellow / orange flame

outer nonluminous flame

inoculating loop hottest region (up to 1500 C))

outer yellow cone

inner blue cone inner blue cone

A B C D

Figure A.4D.2 Adjusting the Bunsen burner and heating an inoculation loop. A lighted Bunsen burner with its air holes closed produces a cool, yellow-orange flame (A). It is not particularly useful for heating things. The more the air holes are open, the more blue and fierce the flame becomes (B,C). A properly adjusted Bunsen burner gives a blue flame containing two cones: an outer, nonluminous pale blue cone and an inner, deeper blue cone (D). There is no combustion inside the central cone. Temperatures in the part of the flame just above the central inner cone (where the inoculating loop is heated) approach 1500◦C. See text for more details. Commonly Used Methods for Cell Culture A.4D.7

Current Protocols in Microbiology Supplement 11 air convection currents up and away from the dust and other airborne contaminants. Pipets opening. This canopy of warm, rising air helps should be removed in a manner that prevents prevent the entrance of dust particles. Flaming the tips from contacting any potentially con- should be performed immediately upon open- taminated surfaces. Remove pipets by insert- ing and just before closing tubes and bottles. ing as little of the fingers as possible, and as Lips of disposable plastic containers and con- the pipet is withdrawn, hold it away from any tainers of flammable solutions should never be surface to prevent its contamination (including flamed. Autoclaved beakers containing sterile the ends of other pipets, since they have come wooden toothpicks do not have to be flamed. into contact with your hands, and are therefore NOTE: The following instructions assume considered nonsterile). the experimenter is right-handed. In the case Individually wrapped, presterilized plastic of a left-handed individual, switch the hands pipets are an alternative to glass pipets, gen- in the instructions from right to left, and vice erally inexpensive when purchased in small versa. quantities, but expensive over long terms. Re- member that the outsides of the wrappings are Manipulating vessels containing liquids considered contaminated. Open from one end, Most manipulations of cultures or sterile and then peel the wrapping backward, allow- reagents in tubes, bottles, or flasks should be ing removal of the pipet without contact with performed as follows: the outer surface. Bulk-wrapped, presterilized 1. Loosen closures (i.e., lids, caps, etc.) of pipets are somewhat less expensive, but it is all containers prior to any manipulations. This often difficult to maintain sterility of the large will ensure that a procedure will not have to quantities of pipets in each package. be stopped midway. 2. Hold the container in your left hand at a 45 degree angle, so that dust cannot fall in Using an Inoculating Loop Before each use, an inoculating loop must when it is open. be sterilized using a burner, as follows: 3. Hold the instrument to be used for manip- 1. Place the junction between the loop wire ulation (inoculating loop, pipet, needle, tooth- and the handle just above the inner blue cone pick, etc.) in the right hand. (hottest point) until the wire turns red. 4. Grasp the container closure using the lit- 2. Slowly draw the wire through the blue tle finger of your right hand, and lift from con- flame, making sure that every part of the wire tainer. Do not set the container closure down. is heated to glowing red. The loop tip is heated Doing so increases the risk of it becoming con- last. taminated from the bench top. Remember that 3. Cool the loop by making contact with even a cleaned bench top is not sterile. another sterile surface, e.g., an unused section 5. While continuing to hold the closure with of an agar plate. Do not blow on the loop or the little finger, lightly flame the container wave it in the air to cool it. Such actions will opening. contaminate the loop. 6. Quickly manipulate the instrument into 4. The loop is now ready for immediate the container, and then withdraw it. use. Do not put the loop down or touch it to a 7. Lightly flame the container opening nonsterile surface before using. again. 5. Flame the loop again immediately after 8. Replace the container closure immedi- use before setting it down. ately. As an alternative, presterilized inoculating Transferring liquids from one test tube loops may be purchased. These are particu- to another larly handy for use within a laminar flow hood, 1. Hold both tubes in the left hand. where a Bunsen burner should never be used. 2. Remove the closures from both tubes Plastic, disposable loops are available either with little finger of right hand. individually wrapped or packaged in bulk. 3. Flame the lips of both tubes. 4. Take culture, reagent, etc., from first tube. Using Petri Dishes (Plates) 5. Add culture, reagent, etc., to second tube. Before pouring medium into sterile petri 6. Flame the lips of both tubes again. dishes, first remove the plates from their 7. Replace both closures. container (e.g., plastic sleeve), and arrange them on the lab bench convenient to the Storing and Using Pipets burner. Flame the lip of the container of lique- Aseptic Technique Cans of sterile glass pipets should be kept in fied medium immediately before pouring the horizontal positions, to reduce introduction of medium into the plates, and again after each A.4D.8

Supplement 11 Current Protocols in Microbiology 5 to 10 plates that are poured. Remove petri allel layers at a uniform velocity with no dis- dish lids only when needed for pouring the ruption between the layers. A cluttered hood medium, and close immediately after. When or poor technique can easily overcome the de- the lid is removed, it should be held over the sired airflow and reverse currents, potentially plate as a shield and never placed on the bench introducing contaminants into the work area. top Thoroughly read through the laminar flow For all manipulations of cultures in petri unit supplier’s manual for detailed information dishes, lids should be lifted for as short a time on the operation of a particular appliance, es- as possible. Lids are never to be placed on the pecially with regard to the position of air ducts bench top. Do not walk around the room with and the track of laminar flow. an open plate. As with all other media, do not breathe on open plates. Cleaning, disinfecting, and arranging the space inside hood (before use) Working in the Laminar Flow Unit Before starting work or re-entering the An additional layer of security against hood, remove jewelry from the hands and contamination is the use of a laminar flow wrists, tie long hair back, button up the labo- hood. Although several modifications of the ratory coat, and roll up the coat sleeves. Wash two major flow hood design types (horizontal your hands and forearms and don fresh dis- and vertical) are commercially available (see posable gloves. Spray the exterior of gloves Fig. A.4D.3), the vertical hood design is prob- with disinfectant and then rub the hands to- ably the most commonly encountered nowa- gether before putting them inside the hood. days. The major design concepts of both types Keep hands within the cleaned area of the hood of hoods are similar. Room air is taken into as much as possible and do not touch hair, the unit and passed through a prefilter to re- face, or clothing. Hand cleanliness is reduced move gross contaminants (lint, dust, etc.). The each time bottles and other nonsterile items are air is subsequently compressed and channeled touched, so the disinfecting procedure should through the HEPA filter, which removes nearly be repeated periodically. all of the bacteria from the air. Purified air To maintain efficient air flow, the Class II flows out over the entire work surface in par- laminar flow hood should remain turned on

A horizontal B vertical

HEPA filter (exhaust) HEPA filter

HEPA filter (air supply)

glass shield

work surface

intake

prefilter

work surface

Figure A.4D.3 Laminar flow hoods. Hoods with vertical (A) or horizontal (B) flow are the two major types of laminar flow units. See text for details. Commonly Used Methods for Cell Culture A.4D.9

Current Protocols in Microbiology Supplement 11 24 hr a day. If turned off for any reason, it Remember that a laminar flow hood is not should be turned on at least 30 min before use. a completely sterile environment. All the rules The hood should be thoroughly cleaned before of aseptic work on the bench top apply to work any use. Spray the working surface liberally in a hood. Minimize the time bottles or plates with 70% (v/v) alcohol solution (denatured are open, avoid touching extraneous surfaces ethanol or other industrial methylated spirit) with pipets, etc. and swab immediately with lint-free wipes. In case of any spill, clean the work surface Any object that is introduced into the lami- immediately with sterile, distilled water and nar hood environment, even glassware or plas- follow by spraying liberally with alcohol. Use tic containers marked as sterile, should be dis- a side to side motion, starting at the back of infected by liberally spraying the outside with the hood and working forward. 70% ethanol. Always remove outer pouches If you fully comply with all the above in- and wraps from disposable equipment (e.g., structions, chances for the introduction of sep- pipets, loops, spreaders, cell scrapers) at the sis into the cultures or reagents are low. Never edge of the work area just before the sterile become so engrossed in your work that you contents are pulled into the hood. To help re- forget these basic rules. duce introduction of contaminants from out- side the hood, always keep a set of clean pipet- Cleaning and disinfecting the space tors inside for exclusive use. inside hood (after use) Leave a wide, clear space in the center When finished using the hood, remove all of the hood (not just the front edge) as a unnecessary items, and then clean all surfaces working surface. Arrange the area to have by liberally spraying with 70% alcohol. Most easy access to all of it without having to laminar flow hoods are equipped with a UV reach over one item to get another (especially lamp, which helps kill introduced microor- over an open bottle or flask). Reaching over ganisms by irradiation. Since UV light is also items increases chances of knocking things harmful to humans, do not switch on the light over, or transferring contamination through the when anyone is working in the laboratory. Turn workplace on the UV light at the end of the work day to Never place large objects near the back of help keep down levels of potential contami- the hood or clutter the area. Not only can these nants. objects contaminate everything downstream, IMPORTANT NOTE: Before switching on but they also disrupt the laminar flow pattern of the UV light, remove all important samples air, which normally suspends the contaminants of living organisms to a sheltered location, so and removes them from the area. they are not affected by the harmful conditions. A common mistake is to keep waste, old cultures and media, empty boxes, notebooks, manuals, protocols, pencils, and other unnec- essary items inside the hood. Such objects TROUBLESHOOTING should never be kept inside the laminar flow Most lapses in aseptic technique will be- unit. come apparent by contaminations of cultures or supposedly sterile media and reagents. WorkinginaClassIorIIlaminarflow A simple method for checking the quality of hood your aseptic technique is to perform manipula- Perform all work at a distance of no less tions of medium without intentionally adding than 6 in. from the front edge of the work bacteria. Incubate the medium overnight at surface. At a lesser distance, laminar flow air 37oC, and then observe for signs of contami- begins to mix with the outside air and contam- nant growth (e.g., turbidity). ination is possible. A small amount of contamination is not al- To ensure the proper air flow, maintain a ways evident until the medium is incubated direct path between the filter and the area in- at an appropriate temperature or atmosphere side the hood where the manipulations are be- conditions. ing performed (nothing should touch the fil- Some bacterial cultures may grow briefly, ter). Undesired turbulence may also be pro- then lyse. This may be due to bacteriophage duced by coughing or sneezing into the hood, contamination as a result of poor aseptic tech- quick movements, rotating, talking, etc. To nique. If this is suspected, thoroughly clean all help keep air disturbances to a minimum, the laboratory benches and equipment with disin- hood should be located out of the stream of fectants, and strengthen use of aseptic tech- Aseptic Technique traffic in the lab. niques. A.4D.10

Supplement 11 Current Protocols in Microbiology Many contaminations occur through the poorly designed or defective air handling sys- tems, possibly transferring microorganisms from neighboring laboratories. Your facility’s physical plant management should be con- sulted if you suspect such possibilities, as your laboratory’s air handling system may need to be re-engineered.

Commonly Used Methods for Cell Culture A.4D.11

Current Protocols in Microbiology Supplement 11