Aseptic Technique

Tomasz Bykowski1 and Brian Stevenson2,3

1
Center for Medical Education, Warsaw, Poland
2
Department of Microbiology, Immunology, and Molecular Genetics, University of
Kentucky College of Medicine, Lexington, Kentucky
3
Corresponding author: [email protected]

This article describes common laboratory procedures that can reduce the risk
of culture contamination (sepsis), collectively referred as “aseptic technique.”
Two major strategies for aseptic work are described: using a Bunsen burner and
using 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 microorganisms on petri dishes. © 2020 by John Wiley & Sons, Inc.

Keywords: aseptic technique r bacteriology r Bunsen burner r cell culture

r laminar flow r laminar hood r sterilization r virology

How to cite this article:

Bykowski, T., & Stevenson, B. (2020). Aseptic technique. Current
Protocols in Microbiology, 56, e98. doi: 10.1002/cpmc.98

INTRODUCTION come second nature after practical laboratory

Aseptic technique is a set of routine experience.
measures that are taken to prevent cultures, CAUTION: The information in this article
sterile media stocks, and other solutions from is meant as an introduction to basic aseptic
being contaminated by unwanted microor- technique and does not cover safety aspects
ganisms (i.e., sepsis). While such actions are necessary for working with specific organ-
sometimes called “sterile technique,” that ter- isms. It is important that the reader understand
minology is appropriate only in reference to the appropriate methods of disposal and de-
preventing introduction of any organisms to contamination for the specific microorganism
laboratory or medical equipment and reagents with which he or she is working. Refer to
(e.g., during surgery). Since the goal of a biol- Current Protocols articles Coico & Lunn
ogist is to grow microorganisms or eukaryotic (2005) and Current Protocols (2005) for more
cells without introduction of extraneous or- information.
ganisms, aseptic techniques are crucial for
accurate and meaningful experimentation. STRATEGIC PLANNING
One should always remember that a com-
pletely sterile working environment does not Contamination Risk Assessment and
exist. However, there are a number of simple, Space Organization
common-sense procedures that will reduce Localizing potential sources of biological
the risk of culture contamination. Examples contamination is an important part of arrang-
of aseptic technique include cleaning and ing space when a room is used as a laboratory.
disinfecting lab surfaces prior to use, limiting Benches for aseptic work should be organized
the duration that cultures or media are un- away from air conditioning vents, cooling
capped and exposed to the air, keeping petri fans, open windows, or blowers from heating
dishes closed whenever possible, effectively or refrigerating systems. Containers used for
sterilizing inoculating loops and other equip- the disposal of biological material may con-
ment that comes into contact with cultures or tain large numbers of viable microorganisms,
media, and avoiding breathing on cultures or even when emptied. Some contaminants, e.g.,
sterile instruments. These precautions will be- sporulating bacteria, yeasts, and fungi, are
Bykowski and
Stevenson

Current Protocols in Microbiology e98, Volume 56 1 of 11

Published in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpmc.98
© 2020 John Wiley & Sons, Inc.
 very difficult to eradicate from the working ology or molecular biology laboratories, they
environment. Waste should be kept very well should be carried out very carefully, because
covered or, if feasible, in a different room, there are no degrees of sterilization: an object
especially if using particularly harmful or is either sterile or not.
sturdy microorganisms.
Filter sterilization
General Cleanliness Reagents such as antibiotics, drugs, sugars,
As a matter of principle, keeping high stan- amino acids, vitamins, and complex media
dards of cleanliness has to be a universally that are either flammable or that would be
accepted norm in the biological laboratories. denatured by heat are usually filter sterilized.
People should wear lab coats that are worn A range of syringe-based or bottle-top filters
only in the laboratory. Researchers should of different pore sizes physically remove
wash their hands often during the course of (exclude) living organisms and can be used
the day, especially prior to any handling of according to the suppliers’ instructions. Filtra-
biological cultures, media, or sterile supplies. tion devices may use either negative (vacuum)
Dust and stains must be removed regularly, or positive pressure. An advantage of positive
and spills cleaned and decontaminated imme- pressure in filtering is that it reduces clogging
diately. from precipitated salts.
Bench tops and shelves should be always Most living organisms are retained by a
be washed immediately before any use with 0.45-μm filter. That size filter is often used as
10% (v/v) household bleach (containing a pre-filter because it also clears the liquid of
sodium hypochlorite). This solution will in- larger particulates. However, because many
activate all viruses, bacteria, fungi, and other bacteria can pass through 0.45-μm pores, a
potential contaminants. Alternatively, a solu- 0.22-μm filter should be used to ensure steril-
tion of antiseptic cleanser (e.g., Lysol), diluted ization of the fluids. Viruses will pass through
according to the manufacturer’s directions, 0.22-μm filters, although they generally stick
is adequate. Organic disinfectants such as to the membrane. Some bacteria, such as
70% (v/v) ethanol are generally less effective saprophytic Leptospira spp. commonly found
than bleach solutions because ethanol may in domestic water supplies, may also pass
evaporate too quickly to effectively sterilize through 0.22-μm filters. If such potential
surfaces and may not completely inactivate contaminants may cause difficulties, consider
all potential contaminants. autoclaving water before using it to prepare
Outerwear may be covered with dirt or solutions that finally need to be filter sterilized.
dust, and so should be kept in a cloakroom Filtering procedures are usually carried out
away from the work space. These simple in a laminar flow unit or on a clean bench that
rules of cleanliness will eliminate the bulk of has been wiped with decontaminant, 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 any
reagents are supplied by the manufacturer radioactive isotopes, cannot be autoclaved
as ready to use for cultures, while many under any circumstances.
others do not have to be sterilized at all (e.g., Autoclaving (using steam under pres-
ethanol, phenol, or concentrated detergents). sure) is a rapid method for sterilizing almost
However, some require sterilization (i.e., the anything except heat-labile substances. A
complete destruction or elimination of all temperature higher than the boiling point
viable organisms) before use. of water (superheating of liquids) inside the
A wide variety of sterilization methods autoclave is achieved because the system is
that are used in the process of food storage, under pressure. Typical autoclaving condi-
treatment of medical and surgical equipment, tions of 121°C (250°F) for 15 min at 103 kPa
and drug production, based on heat and high (15 psi) are sufficient to kill virtually all forms
pressure, liquid and gaseous chemicals, radia- of life, including bacterial endospores, and
tion, or physical removal of microorganisms, will inactivate viruses.
have been developed. Although only a few, However, the time of the sterilization cycle
Bykowski and quite straightforward protocols are actually should be modified according to the amount
Stevenson
applied to the handling of items in microbi- and type of loaded items. Examples of objects
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that have slower surface heating or steam pen- ature conditions are maintained for the
etration and that should be autoclaved longer desired period of time. At the end of the
are an unusually large bottle of medium, a cycle, a valve opens, and the chamber
bulky bag of biohazard waste, or a beaker full rapidly returns to atmospheric pressure.
of microcentrifuge tubes with aluminum foil Drying time may also be added to the cy-
covering. cle.
General principles for loading an autoclave 2. The “liquid” or “slow exhaust” cycle is
can be summarized as follows: used for liquids to prevent them from
1. Polypropylene and polycarbonate can be explosive boil-overs. Pressure is slowly
autoclaved, but polyethylene and high- released, which allows the super-heated
density polyethylene cannot. Initials im- fluids to cool gradually.
printed on bottom of containers can help CAUTION: It is acceptable to autoclave dry
in identification of the material type items using the liquid cycle, but liquids should
(PP, polypropylene; PC, polycarbonate; never be autoclaved using a fast exhaust cycle.
PE, polyethylene; HDPE, high density Sterilized goods can be denoted using a
polyethylene). special indicator tape which contains a chem-
2. The autoclave must not be overcrowded; ical that changes color when the appropriate
the steam needs to be able to penetrate autoclaving conditions have been met. Some
everywhere. All materials must be placed types of packaging have built-in indicators on
in an autoclavable tray (polypropylene them.
plastic or stainless steel tubs) with sides Autoclaves should be tested periodically,
at least 10 cm high to catch spills and using biological tests such as bacterial spore
avoid possible damage to the machine. test kits. Users should consult with their insti-
Dry items must be wrapped in aluminum tutions for guidelines on frequency of testing
foil to prevent them from being re- and record keeping.
contaminated after the procedure.
3. Liquids should be sterilized in a vessel Oven sterilization
of at least twice the volume of the liquid Selected goods such as glassware, metal,
to be autoclaved, to avoid boiling over and other objects that will not melt at tem-
and spilling. Containers must be loosely peratures between 121°C and 170°C can be
capped or stoppered to avoid shattering sterilized with dry heat (in a hot-air oven). As
during pressurization and depressuriza- the heat takes much longer to be transferred
tion. To prevent bottoms of bottles from to the organism, a temperature of 160°C for
breaking, the tub or tray holding them ≥2 hr or 170°C for 1 hr is routinely used. The
should be filled with 3 to 5 cm of water. procedure certainly cannot be applied for liq-
Heat-resistant borosilicate glass (Pyrex) uids, rubber, or any plastic objects. However,
vessels are preferred. the method has advantages; for instance, it
4. All containers (empty or containing liq- can be used for powders and other heat-stable
uids or solid apparatus) must be auto- items that are adversely affected by steam,
claved with loose lids, which will per- and it does not cause rusting of steel objects.
mit the super-heated steam to get into the It is also a method of choice for the treatment
container. For screw-cap containers, the of glassware used for work with ribonucleic
lid should be hand tightened, then loos- acid (RNA), since the process of baking not
ened one-half turn. Stoppers occasion- only kills organisms, but also inactivates any
ally pop off during the autoclaving pro- residual RNA-degrading enzymes (RNases).
cess, so loosely covering the stopper or
cap with a piece of aluminum foil can General considerations
help hold the cap in place. Note that a standard microwave oven (i.e.,
CAUTION: Be aware that large bottles with the household appliance), although capable
narrow necks can simulate sealed containers of bringing water to boiling, does not provide
if filled with too much liquid. a useful method for sterilizing laboratory
To optimize autoclaving of common lab- goods and should only be used for warming
oratory goods, two general types of exhaust up liquids.
cycles have been designed: Sterilized solutions and other reagents will
1. The “gravity” or “fast exhaust” cycle is need to cool down before use, which takes
for all dry items, e.g., glassware, tips, time. Thus, sterilization should be performed
flasks, tubes. The chamber is charged well before the actual experimental work Bykowski and
Stevenson
with steam, and set pressure and temper- begins.
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 It is best for each researcher in the labo- It is also impractical to use a Bunsen
ratory to keep his/her own stocks of reagents burner (or other flame source) when working
(e.g., media, buffers, salts, glycerol) to prevent in the field, e.g., when collecting specimens.
accidental contamination by a co-worker. In In such cases, applying general aspects of
this manner, each worker is solely respon- aseptic technique, such as minimizing time
sible for the condition of his/her reagents that culture tubes are open, will probably
and for any impact on experimental results. be adequate to prevent contamination. Note
This strategy can be facilitated by preparing also that collection/transfer devices and other
media or solutions in several small bottles apparatus need to be pre-sterilized, and care
(aliquots), one for each researcher, with must be taken to prevent introduction of
only one container per worker opened at a contaminants during use (as described further
time. below).
Although growth media are more likely to
produce noticeable biological contamination The Laminar Flow Unit
(by turning color, developing clumps, or A laminar flow unit (or hood) is a so-
becoming turbid), most other liquids used in phisticated appliance that can further help
the laboratories can also support the growth prevent contamination of reagents and bio-
of organisms. Solutions should be checked logical cultures. Used correctly, it provides
before use by gently swirling and looking for the work space with clean, ultra-filtered air.
the presence of cloudy material. It also keeps room air from entering the work
area and both suspends and removes airborne
The Bunsen Burner contaminants introduced into the work area by
A Bunsen burner (see Fig. 1) is a useful personnel.
tool for maintaining aseptic technique. This The most important part of a laminar flow
common piece of equipment burns a con- hood is a high-efficiency bacteria-retentive fil-
tinuous stream of a flammable gas—usually ter, i.e., the HEPA (high-efficiency particulate
natural gas (methane)—based upon a design air) filter. A certified HEPA filter must capture
made almost 150 years ago by the German a minimum of 99.97% of dust, pollen, mold,
chemist Robert Wilhelm Bunsen (1811-1899). bacteria, and any airborne particles with a size
It is useful for many purposes, including ster- of >0.3 μm at 85 L/min. The first HEPA filters
ilizing inoculating loops and needles, heating were developed in the 1940s by the United
lips of flasks and bottles to ensure sterility, States Atomic Energy Commission as part
burning off alcohol from culture spreaders, of the Manhattan Project (the development
and removing air bubbles from the surfaces of of the atomic bomb) to provide an efficient,
freshly poured Petri dishes of melted media. effective way to filter radioactive particulate
Traditionally, microbiologists have been contaminants. HEPA filter technology was
encouraged to have a lit Bunsen burner on the declassified after World War II, allowing
bench during all aspects of aseptic technique. extensive research and commercial use.
It is hypothesized that this creates a cone Laminar flow hoods are essential com-
of hot air above and around the laboratory ponents of many Biosafety Level (BSL)-2
bench to reduce the viability of organisms on laboratories, where they help prevent spread
suspended dust particles. However, there is of viruses and some bacteria. Consult Coico
evidence that air currents created by the burner & Lunn (2005) as well as the resources
may draw air and suspended contaminants in Current Protocols (2005) for additional
into the work area, but not impact viability information on biosafety and biocontainment.
of those contaminants. In our experience,
many aseptic techniques can be adequately Useful Materials to Have on Hand
performed without a lit burner on the bench. A list of simple materials that might be
A Bunsen burner is not practical in some useful in aseptic work and that should be kept
situations, e.g., within a laminar flow unit on hand is presented in Table 1.
where the heat will disrupt airflow. A mi-
croincinerator may be used as an alternative. SAFETY CONSIDERATIONS
This consists of a circular heating element.
Placing an inoculating loop or needle within Autoclaving
the ring will quickly heat and sterilize the Autoclaving produces steam, which can
loop/needle. Note that a microincinerator cause burns and requires a lot of caution in
Bykowski and does not provide other aseptic technique handling. The most important moment is
Stevenson
benefits offered by a lit Bunsen burner. opening the machine after completion of the
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Figure 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.

cycle. Consider some basic rules that should and wait for steam to be released.
be applied while opening autoclave: Beware of a rush of steam. Open the
door fully to remove tray.
Wear a lab coat, eye protection, After the slow exhaust cycle, open
loose-fitting thermal (insulating) the autoclave door and allow liquids
gloves, and closed-toe shoes. to cool 10 to 20 min before remov-
Check and make sure that the chamber ing. Superheated liquids may sud-
pressure is zero before attempting to denly boil over if the container is
open the door. moved.
Stand behind the door when opening and Do not move hot bottles if the liquid is Bykowski and
use it as a shield. Slowly open a crack bubbling or boiling. Stevenson

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 Table 1 Common Laboratory Items Useful for Aseptic Work

Material Common use

Aluminum foil Widely used in laboratories to pack 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
them, but also helps to keep them organized by size or type.
Disposable rubber Should be worn at all times during aseptic work; usually made of latex rubber,
gloves 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 non-latex gloves is recommended. After removing latex gloves, hands
should be washed with a mild soap and dried thoroughly.
Disposable wipes Since they do not leave unwanted particles behind, these are considered very
(lint-free) useful for wiping bench tops, the interior of the laminar flow hood, or any other
object during cleaning and disinfection
70% (v/v) ethanol or Simple, flammable disinfectants used for cleaning items and surfaces from
other industrial microorganisms and stains. They are also used for dipping culture spreaders. See
methylated spirits Safety Considerations for usage instructions.
(IMS; denatured
alcohols)
Eye protection and Equipment for extra protection should be kept within reach and used during
thermal gloves procedures such as opening an autoclave
Flint-on-steel striker Used to quickly ignite a Bunsen burner when needed
10% (v/v) household This chemical is more effective for disinfecting surfaces than quickly
bleach (containing evaporating ethanol. It should not be stored for longer than 1 month at room
sodium hypochlorite) temperature, since sodium hypochlorite will degrade.
Laboratory coat A clean, long coat of the right size is used to protect the researcher against toxic
substances and biohazardous material. It also protects samples from
contamination from human skin and apparel.
Spray or squirt plastic Very useful for dispensing ethanol, water, and other liquids for disinfection and
bottles cleaning of large surfaces and places difficult to reach with a wipe

When removing liquids from the heated object, glass or metal, needs time to
autoclave, point the opening of the cool down before it can be safely touched.
container away from yourself and It is also absolutely necessary to remove
other people because the liquid may flammable and combustible materials from
suddenly boil and spray out the the vicinity of the flame. Nitrocellulose
opening. membranes, commonly used for blotting
techniques, are extremely flammable.
Working with Open Flame Ethanol used for dipping culture spread-
Burners should be on only for as long as ers is an exception to the rule regarding
needed for a particular procedure. The blue flammables, but only small volumes (≤20
flame of a hot Bunsen burner can be difficult ml) in glass beakers should be used at a time.
to see. Never leave an open flame unattended, An accidental alcohol fire in a beaker can be
not even for a few seconds. quickly extinguished simply by covering the
Since burns are among the most common top of the beaker with a piece of aluminum
laboratory accidents, making sure that face, foil. Keeping the alcohol beaker covered with
clothing, and hair are not above or near the such a piece of foil when not in use will
opening of the burner tube is a crucial safety help reduce accidental fires and will slow
measure. Temperatures in the hottest region evaporation.
of the burner flame approach 1500°C, and A fire extinguisher should always be kept
Bykowski and objects will heat extremely quickly. Any nearby.
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 The tubing connecting the Bunsen burner risk of contamination. Consider that working
to the laboratory gas supply should be checked in an aseptic manner may take longer than
regularly. Latex rubber tubing tends to harden when being less cautious. Reserve extra time
and crack after exposure to air, making it to avoid being rushed, which may result
a poor tubing choice. Silicone tubing (e.g., in spilling or breaking important samples,
Tygon) is generally more durable. dishes, or solutions.
Never use a Bunsen burner in a Class II
laminar flow hood. The heated air may disrupt Using a Bunsen Burner
air flow and possibly damage the hood. Before starting work, locate the gas and
air controls on the burner. They can be in
Ultraviolet Light different places on different models. In some
Many laminar flow hoods are equipped cases, it is necessary to use the laboratory
with ultraviolet (UV) lights, which can help gas valve to control the gas flow rate. Once a
reduce contamination by inducing DNA dam- Bunsen burner has been adjusted for optimal
age in potential contaminants. Such light is air and gas flow, those settings can be left in
also harmful to laboratory workers. Never place, making future use more efficient.
look directly at a UV light, as that can cause 1. Close the burner’s air control. If the
eye damage. UV light can also cause skin burner has its own gas control valve, shut
burns (sunburn) to anyone in the room. Con- it and then open it, allowing only small
sequently, UV lights should always be turned amount of gas to get into the burner tube.
off whenever the room is occupied. 2. To ignite the gas using a flint-on-steel
NOTE: Ultraviolet light in a laminar flow striker, hold it 2 to 3 cm above and
hood is not likely to create a sterile environ- slightly to the side of the burner tube
ment. At best, the light will reduce numbers top. Squeeze and release striker repeat-
of extraneous organisms, but not eliminate all. edly until a spark ignites the flame. If us-
NOTE: Ultraviolet light cannot pass ing a match, light it and slowly bring it
through solid objects, so bottoms of con- up from the bottom along the side of the
tainers and other items in shade will not burner tube until the flame ignites. If a
be affected by the ultraviolet light. This is match is struck in the middle of the gas
another reason to limit the amount of items stream, the high-pressure gas stream of-
kept in a laminar flow hood. ten blows out the flame before the burner
can light.
TECHNIQUES FOR MAINTAINING 3. If the initial flame is low on oxygen, it is
ASEPTIC CONDITIONS bushy orange in appearance and not very
Usually, there is no need to equip the lab- hot (Fig. 2A,B). Open the air-control
oratory with expensive paraphernalia just for vent until the central blue cone of the
culture of hardy bacteria such as Escherichia flame forms and a slight buzzing sound
coli, since that orgainism’s rapid growth is audible (Fig. 2C). If the flame is too
rate generally allows it to out-compete any small, gradually increase both the sup-
contaminants. A typical laboratory bench sup- ply of gas and air flow to the burner. The
plied with a Bunsen burner is sufficient. Work- hottest part of the flame is just above the
ing aseptically on an open bench has been the central blue cone.
practice in laboratories worldwide since the 4. When finished using the burner, turn it
19th century. It is straightforward and does off at the laboratory gas valve. Do not
not require considerable financial outlays. leave a lit burner unattended.
However, a laminar flow hood is the CAUTION: Never leave a lit burner unat-
method of choice when a more rigid aseptic tended.
technique is required. The rich media used
for eukaryotic cell cultures and many mi- Handling and Pipetting Liquids
croorganisms, coupled with their slow growth All growth media, cultures, and sterile
rates, necessitate high levels of security from reagents should be manipulated on a clean,
contamination. For some organisms, work decontaminated bench or within a decontami-
must always be performed inside a laminar nated laminar flow hood. Arrange bottles, test
flow unit to meet biosafety standards and to tubes, plates, etc., in convenient locations.
protect personnel against potentially harmful Historically, a lit Bunsen burner has also
microorganisms or viruses. been placed on the work bench. Mouths of
Any aseptic work should be completed opened tubes and bottles are passed briefly Bykowski and
Stevenson
as quickly as is comfortable to minimize the through the flame immediately upon opening
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 Figure 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). This flame 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 inner, 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.

and just before closing the containers. More 1. Loosen closures (i.e., lids, caps, etc.) of
recently, the value of “flaming” has been all containers prior to any manipulations.
questioned. If one is prompt with transferring This will ensure that a procedure will not
liquids, and minimizes the amount of time have to be stopped midway.
that containers are open, the practice of flam- 2. Hold the container in your left hand at a
ing is not necessary. Moreover, studies are 45° angle, so that dust cannot fall in when
indicating that flaming the mouth of a culture it is open.
tube introduces carbon dioxide, which can 3. Hold the instrument to be used for ma-
significantly change the pH. For those rea- nipulation (inoculating loop, pipet, nee-
sons, we have omitted flaming steps in these dle, toothpick, etc.) in the right hand.
protocols. 4. Grasp the container closure using the lit-
If users choose to include flaming, then tle finger of your right hand, and lift from
a brief (1- to 2-s) pass of opened tubes, etc. container. Do not set the container closure
through the burner flame should be added down. Doing so increases the risk of it be-
immediately after opening and just before coming contaminated from the bench top.
closing containers. Remember that even a cleaned bench top
Lips of plastic containers and containers of is not sterile.
flammable solutions should never be flamed. 5. Quickly manipulate the instrument into
Autoclaved beakers containing sterile wooden the container, and then withdraw it.
toothpicks do not have to be flamed. 6. Replace the container closure immedi-
ately.
Manipulating vessels containing liquids
NOTE: The following instructions assume Transferring liquids from one test tube
that the experimenter is right-handed. In the to another
case of a left-handed individual, switch the 1. Hold both tubes in the left hand.
hands in the instructions from right to left, 2. Remove the closures from both tubes
and vice versa. with little finger of right hand.
Most manipulations of cultures or sterile 3. Take culture, reagent, etc., from first tube.
Bykowski and reagents in tubes, bottles, or flasks should be 4. Add culture, reagent, etc., to second tube.
Stevenson
performed as follows: 5. Replace both closures.
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 NOTE: If using an inoculating loop, close where a Bunsen burner should never be used.
all containers before re-flaming the loop. Like- Plastic, disposable loops are available either
wise, if using a pipet to transfer liquids, close individually wrapped or packaged in bulk.
all containers before disposing of the pipet.
Using Petri Dishes (Plates)
Storing and Using Pipets Before pouring medium into sterile petri
Cans of sterile glass pipets should be kept dishes, first remove the plates from their
in the horizontal position to reduce introduc- container (e.g., plastic sleeve) and arrange
tion of dust and other airborne contaminants. them on the clean lab bench. A lit burner will
Pipets should be removed in a manner that be useful during plate-pouring processes.
prevents the tips from contacting any poten- Remove petri dish lids only when needed
tially contaminated surfaces. Remove pipets for pouring the medium, and close immedi-
by inserting as little of the fingers as possible, ately after. When the lid is removed, it should
and as the pipet is withdrawn, hold it away be held over the plate as a shield and never
from any surface to prevent its contamination placed on the bench top. Periodically heating
(including the ends of other pipets, since they the lip of the container of liquefied medium
have come into contact with your hands, and with the burner flame can help ensure sterility.
are therefore considered nonsterile). It is best A lit burner is also useful for removing air
to fill pipet cans only 2/3 full, to permit the bubbles that form on the surface of liquefied
above-described withdrawal of the first pipets. medium. If bubbles form, immediately pass
Individually wrapped, presterilized plastic the burner flame over the opened dish (burner
pipets are an alternative to glass pipets, which pointed at a 45° angle downward). The com-
are generally inexpensive when purchased bination of heat and the stream of burning gas
in small quantities but expensive over the will pop bubbles, leaving a smooth surface.
long term. Remember that the outsides of This process must be as brief as possible, to
the wrappings are considered contaminated. avoid damaging the medium with excess heat
Open from one end, and then peel the wrap- and to avoid melting of plastic dishes.
ping backward, allowing removal of the pipet For all manipulations of cultures in petri
without contact with the outer surface of the dishes, lids should be lifted for as short a time
wrapper. Bulk-wrapped, presterilized pipets as possible. Lids are never to be placed on the
are somewhat less expensive, but it is often bench top. Do not walk around the room with
difficult to maintain sterility of the large an open plate. As with all other media, do not
quantities of pipets in each package. breathe on open plates.

Using an Inoculating Loop Working in the Laminar Flow Unit

Before each use, an inoculating loop must An additional layer of security against
be sterilized using a burner, as follows: contamination is the use of a laminar flow
1. Place the junction between the loop wire hood. Although several modifications of the
and the handle just above the inner blue two major flow hood design types (horizontal
cone (hottest point) until the wire turns and vertical) are commercially available (see
red. Fig. 3), the vertical hood design is probably the
2. Slowly draw the wire through the blue most commonly encountered nowadays. The
flame, making sure that every part of the major design concepts of both types of hoods
wire is heated to glowing red. The loop are similar. Room air is taken into the unit
tip is heated last. and passed through a prefilter to remove gross
3. Cool the loop by making contact with an- contaminants (lint, dust, etc.). The air is sub-
other sterile surface, e.g., an unused sec- sequently compressed and channeled through
tion of an agar plate. Do not blow on the the HEPA filter, which removes nearly all of
loop or wave it in the air to cool it. Such the bacteria from the air. Purified air flows
actions will contaminate the loop. out over the entire work surface in parallel
4. The loop is now ready for immediate use. layers at a uniform velocity with no disrup-
Do not put the loop down or touch it to a tion between the layers. A cluttered hood or
nonsterile surface before using. poor technique can easily overcome the de-
5. Flame the loop again immediately after sired airflow and reverse currents, potentially
use before setting it down. introducing contaminants into the work area.
As an alternative, presterilized inoculating Thoroughly read through the laminar flow
loops may be purchased. These are particu- unit supplier’s manual for detailed information Bykowski and
Stevenson
larly handy for use within a laminar flow hood, on the operation of your particular appliance,
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 Figure 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.

especially with regard to the position of air equipment (e.g., pipets, loops, spreaders, cell
ducts and the track of laminar flow. scrapers) at the edge of the work area just
before the sterile contents are pulled into the
Cleaning, disinfecting, and arranging hood. To help reduce introduction of contam-
the space inside hood (before use) inants from outside the hood, always keep a
Before starting work or re-entering the set of clean pipettors inside for exclusive use.
hood, remove jewelry from your hands and Leave a wide, clear space in the center of
wrists, tie long hair back, button up your the hood (not just the front edge) as a working
laboratory coat, and roll up the coat sleeves. surface. Arrange the area to have easy access
Wash your hands and forearms and don to all of it without having to reach over one
fresh disposable gloves. Spray the exterior item to get another (especially over an open
of gloves with disinfectant and then rub your bottle or flask). Reaching over items increases
hands together before putting them inside the chances of knocking things over, or transfer-
hood. Keep hands within the cleaned area ring contamination through the workplace
of the hood as much as possible and do not Never place large objects near the back
touch hair, face, or clothing. Hand cleanliness of the hood or clutter the area. Not only can
is reduced each time bottles and other non- these objects contaminate everything down-
sterile items are touched, so the disinfection stream, but they also disrupt the laminar flow
procedure should be repeated periodically. pattern of air, which normally suspends the
To maintain efficient air flow, the Class II contaminants and removes them from the area.
laminar flow hood should remain turned on A common mistake is to keep waste, old
24 hr a day. If turned off for any reason, it cultures and medium, empty boxes, note-
should be turned on at least 30 min before use. books, manuals, protocols, pencils, and other
The hood should be thoroughly cleaned before unnecessary items inside the hood. Such ob-
any use. Spray the working surface liberally jects should never be kept inside the laminar
with 70% (v/v) alcohol solution (denatured flow unit.
ethanol or other industrial methylated spirit)
and swab immediately with lint-free wipes. Working in a Class I or II laminar flow
Any object that is introduced into the hood
laminar hood environment, even glassware or Perform all work at a distance of no less
plastic containers marked as sterile, should than 6 in. from the front edge of the work
be disinfected by liberally spraying the surface. At a lesser distance, laminar-flow air
outside with 70% ethanol. Always remove begins to mix with the outside air, and risk of
Bykowski and outer pouches and wraps from disposable contamination is increased.
Stevenson

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 To ensure the proper air flow, maintain so they are not affected by the harmful
a direct path between the filter and the area conditions.
inside the hood where the manipulations are
being performed (nothing should touch the TROUBLESHOOTING
filter). Undesired turbulence may also be Most lapses in aseptic technique will be-
produced by coughing or sneezing into the come apparent by contamination of cultures
hood, quick movements, rotating, talking, etc. or supposedly sterile media and reagents.
To help keep air disturbances to a minimum, A simple method for checking the qual-
the hood should be located out of the stream ity of your aseptic technique is to perform
of traffic in the lab. manipulations of medium without intention-
Remember that a laminar flow hood is ally adding bacteria. Incubate the medium
not a completely sterile environment. All the overnight at 37°C, and then observe for signs
rules of aseptic work on the bench top apply of contaminant growth (e.g., turbidity).
to work in a hood. Minimize the time bottles A small amount of contamination is not
or plates are open, avoid touching extraneous always evident until the medium is incubated
surfaces with pipets, etc. at an appropriate temperature or atmosphere
In case of any spill, clean the work surface conditions.
immediately with sterile, distilled water and Some bacterial cultures may grow briefly,
follow by spraying liberally with 70% alcohol. then lyse. This may be due to bacteriophage
Use a side-to-side motion, starting at the back contamination resulting from poor aseptic
of the hood and working forward. technique. If this is suspected, thoroughly
If you fully comply with all of the above clean all laboratory benches and equipment
instructions, chances for the introduction of with disinfectants, and strengthen use of
sepsis into the cultures or reagents are low. aseptic techniques.
Never become so engrossed in your work that Many contaminations occur through the
you forget these basic rules. poorly designed or defective air-handling
systems, possibly transferring microorgan-
Cleaning and disinfecting the space isms from neighboring laboratories. Your
inside hood (after use) facility’s physical plant management should
When finished using the hood, remove all
be consulted if you suspect such possibilities,
unnecessary items, and then clean all surfaces as your laboratory’s air-handling system may
by liberally spraying with 70% alcohol. Most need to be re-engineered.
laminar flow hoods are equipped with a UV
lamp, which helps kill introduced microor- LITERATURE CITED
ganisms by irradiation. Since UV light is also Coico, R., & Lunn, G. (2005). Biosafety: Guide-
harmful to humans, do not switch on the light lines for working with pathogenic and infec-
when anyone is working in the laboratory. tious microorganisms. Current Protocols in Mi-
Turn on the UV light at the end of the work crobiology, 00, 1A.1.1–1A.1.8. doi: 10.1002/
9780471729259.mc01a01s00.
day to help keep down levels of potential
contaminants. Current Protocols. (2005). Resources for in-
ternational biosafety guidelines and regula-
IMPORTANT NOTE: Before switching on
tions. Current Protocols in Microbiology, 00,
the UV light, remove all important samples A.1B.1–A.1B.1. doi: 10.1002/9780471729259.
of living organisms to a sheltered location, mca01bs00.

Bykowski and
Stevenson

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