Chapter 5

Sterilization and
Sterile Technique
Masanobu Kawachi
National Institute for Environmental Studies

Mary-Hélène Noël
National Institute for Environmental Studies

Filtration, Microwave Sterilization, Pasteurization,

Contents Sterilization, Tyndallization, Ultraviolet Radiation

1.0. Introduction
2.0. Presterilization Cleaning Procedures
2.1. New Culture Vessels
2.2. Dirty Culture Vessels
1.0. Introduction
2.3. Reusable Glass Pipettes
3.0. Sterilization Methods Sterilization is a process for establishing an aseptic con-
3.1. Autoclaving dition, that is, the removal or killing of all microorgan-
3.2. Dry-Heat Sterilization isms. Sterilization is very important in phycological
3.3. Pasteurization and Tyndallization research, especially when maintaining living organisms
3.4. Sterilization by Filtration as isolated strains in culture. The use of sterile tech-
3.5. Microwave Oven Sterilization nique, in combination with sterile equipment and
3.6. Ultraviolet Radiation supplies, minimizes contamination, resulting in more
3.7. Bleach precise experiments free of potential variables caused
3.8. Ethylene Oxide Sterilization by unwanted organisms. Sterilization is not a difficult
4.0. Storage of Sterilized Materials procedure, but precautions must be taken when
5.0. Sterile Techniques working with sterilized material to avoid contamina-
5.1. Sterile Rooms and General Techniques tion. Indeed, once sterilized materials are exposed, they
5.2. Transferring Liquid Cell Cultures soon become contaminated from the ambient air, which
5.3. Transfer of Agar Culture contains dust, spores, and microorganisms.
6.0. Sterilization of Culture Media The term disinfect is sometimes confused with steril-
6.1. Stock Solutions ization. Disinfection is usually defined as an operation
6.2. Sterilization of Liquid Culture Media that kills or reduces the number of pathogenic microor-
6.3. Sterilization of Agar Culture Media ganisms in an environment or on a surface. For
7.0. Evaluating Sterile Conditions example, cleaning hands and surfaces such as tables and
8.0. References benches with detergents or 70% ethanol before han-
dling cultures or sterilized equipment is a disinfecting
Key Index Words: Autoclaving, Bleaching, Cell Culture procedure that is usually conducted as part of the sterile
Transfer, Culture Medium, Dry-Heat Sterilization, technique. The aim of this chapter is to describe dif-

Copyright © 2005 by Academic Press

Algal Culturing Techniques 65 All rights of reproduction in any form reserved.
66 Sterilization and Sterile Technique
ferent sterilization methods and basic sterile techniques
(including disinfecting) that are commonly used in
phycological studies.
There are various sterilization methods, which can be
roughly classified into four categories: heat sterilization,
electromagnetic wave sterilization, sterilization using
filtration, and chemical sterilization (Table 5.1). Heat
sterilization is the most common of the general cate-
gories and usually requires high temperatures (≥100°C),
implying that the materials to be sterilized can resist
high temperatures (e.g., glassware, metallic instru-
ments, and aluminum foil). Liquids are filter-sterilized
when the liquid contains fragile components that are
destroyed by high temperature. Electromagnetic waves
(e.g., ultraviolet [UV] rays, gamma rays, x-rays, and
microwaves) are used as an alternative for materials that
cannot be exposed to high temperature (e.g., many
plastic products or liquids with a labile component).
Today, disposable plastic supplies sterilized with gamma
rays are readily available. Finally, many different types
of chemicals have been used for the purpose of sterili-
zation (Table 5.1); however, chemical traces may remain
after the sterilization treatment, and those chemicals
may be detrimental to living algae and to the investiga-
tor. Therefore, chemical sterilization procedures have
fallen into disuse in the laboratory.
2.0. Presterilization
Cleaning Procedures
2.1. New Culture Vessels
New glass and plastic vessels, except ready-to-use steril-
ized products, should be cleaned before the first use,
because chemicals or other traces of the product manu-
facturing can remain in the vessels and may be harmful
to living cells. The procedure consists of immersing the
containers in a dilute hydrochloric acid bath (generally 1
M HCl) for 1 week, rinsing them several times with
running tap water, and rinsing with distilled or deionized
water before drying. For a less rigorous method, new
glass and plastic vessels can be washed with neutral deter-
gent commercialized for laboratory use (e.g., M-251L
without Phosphorus, Shapu Manufacturing System;
Neodisher FT with neutralizer, Dr. Weigert GmbH &
Co.), followed by thorough rinsing and drying.
Several types of caps for vessels are available on the
market: heat-resistant plastic caps, metal caps, and
expansive silicon rubber plugs. It is recommended to
check the safety of the material before use, because some
products can release toxic substances during the auto-
claving process. For example, black Bakelite caps should
be autoclaved in water several times before using with
culture medium. New black caps color the water with
phenolic compounds that are released when the caps are
first autoclaved. New polyethylene caps may also release
harmful substances when they are first autoclaved.
2.2. Dirty Culture Vessels
Culture vessels that were used to grow cells should be
autoclaved to kill all cells. Living cells, especially as
cysts, may cause contamination of local waters if dis-
carded improperly. After autoclaving, the liquid is
cooled and discarded; when agar is used, it should be
cooled to near the gelling point and then discarded. The
vessels should be briefly rinsed with running water and
then cleaned.
The standard cleaning method consists of immersing
the vessels overnight in a neutral detergent bath (com-
mercial detergent for laboratory use), followed by scrub-
bing with a brush and sponge. Vessels are then rinsed
several times with running water, ensuring that all deter-
gent is removed (i.e., no suds should be present [rinsing
>10 times ensures good removal]). The final rinse is with
distilled or deionized water, and the cleaned vessels are
dried in an area protected from dust (Fig. 5.1).
Other glassware such as petri dishes, watch glasses, and
microscope slides are also cleaned with this procedure. In
cases where the algal cells are strongly attached to the
inner surface of the glassware, it is often necessary to soak
the glassware in hot water several hours before proceed-
ing with the subsequent cleaning steps. For cleaning caps
on screw-type culture tubes, as well as the expansive type
of silicon rubber plugs for flasks (Fig. 5.2), a mild deter-
gent is used (with scrubbing when necessary), followed by
rinsing and drying as described previously.
2.3. Reusable Glass Pipettes
Reusable glass pipettes, used to transfer cells, should be
immediately rinsed with tap water after use so that cell
material does not dry onto the glass surfaces. The
cotton plug should be removed before the pipettes are
rinsed. When immediate rinsing is not possible, the
pipettes should be placed tip down into a plastic beaker
containing water and then rinsed later. For washing, the
pipettes can be immersed in a detergent bath (i.e.,
commercial detergent for laboratory use) (Fig. 5.3) for
 Sterilization and Sterile Technique 67

TABLE 5.1. Summary of sterilization types, including applicability and limits

Category Sterilization Effective method Application Limitation

method

Heat Flame Direct heat with fire Surface sterilization Non–heat-resistant

(Bunsen burner) (test tube openings, materials (e.g., most
transfer loops, glass plastics)
pipettes)
Heat Autoclaving 2 atm (steam pressure), For general use: Non–heat-resistant
121°C; time varies (10, liquids and agar, glass materials; pH change;
20 min for small liquid and metal vessels, metal contamination
vol; 1 h for large vol) equipment
Heat Dry heat 250°C, 3 to 5 h; current Dry goods: glass and Non–heat-resistant
protocol at 150°C for 3 metal vessels and materials; liquids
to 4 h equipment
Heat Pasteurization 66–80°C for at least 30 min, Liquids with heat- Not complete sterilization
followed by quick cooling labile components (originally for killing food
(4–10°C) germs)
Heat Tyndallization 60–80°C, 30 min, followed Liquid with heat-labile Requires time
by quick cooling; cycle components
repeated 3 times in 3 d
Filtration Filtration £0.2 mm pore size filter Liquid with heat-labile Small volumes, high-
components viscosity liquids, viruses
not eliminated
Electromagnetic Microwave 10 min at 700 W; 5 min with Liquids: small volume Small liquid volumes; dry
waves intervals at 600 W. For dry of media; dry goods: goods with water require
goods: 20 min at 600 W glassware, vessels elimination of water
with water, 45 min without
water
Electromagnetic Ultraviolet 260 nm, 5–10 min Surface of materials, Ultraviolet-sensitive plastics
waves radiation working area
Chemical Bleach (sodium 1–5 mL for 1 L water, Large volume of water Cysts may survive;
hypochlorite) several hours for aquaculture neutralization required
(e.g., sodium thiosulfate,
250 g · L-1 stock solution;
1 mL for 4 mL of bleach)
Chemical Ethanol 50–70% solution Popular, general Some resistant
disinfection microorganisms
Chemical Ethylene oxide Airtight room or pressure Plastic and rubber Explosive; chemical residue
cabin products, non–heat- is problematic or toxic
resistant products
Chemical Corrosive 0.1%; add same amount of Antiseptic and Poison; not for materials
sublimate, NaCl and dissolve with disinfectant contacting live cells
HgCl2 distilled water
Chemical Phenol (carbolic 3% solution Antiseptic and Poison; not for materials
acid) disinfectant contacting live cells
Chemical Saponated 3–5% solution Antiseptic and Poison; not for materials
cresol solution disinfectant contacting live cells
Chemical Formaldehyde 2–5% solution Antiseptic and Poison; not for materials
(formalin) disinfectant contacting live cells
68 Sterilization and Sterile Technique
FIGURE 5.1. A dust-proof drying cabinet for storing clean
or sterile materials and supplies.
FIGURE 5.2. Erlenmeyer flasks with silicon rubber plugs.
Left, covered with aluminum foil; center, a standard silicon rubber
plug; right, a silicon rubber plug with an extended shield over the
lip of the flask.
1 or 2 days. An ultrasonic bath and siphon-type pipette
washer are used to completely remove the detergent
(Fig. 5.4). Pipettes are then rinsed with distilled water
and dried in an oven at 150°C. Cotton plugs are then
inserted into the top of the pipettes, the pipette end is
briefly flamed to remove loose cotton fibers, and then
FIGURE 5.3. Detergent baths for cleaning glass inoculation
pipettes (inset: top view of bath).
the pipettes are placed into their canister and autoclaved
or sterilized by dry-heat technique.
3.0. Sterilization Methods
3.1. Autoclaving
Autoclaving is the most effective and popular way to ster-
ilize heat-resistant materials and is generally used to ster-
ilize liquids. An autoclave is a specialized apparatus
consisting of a heavy-walled closed chamber (Fig. 5.5)
within which high steam pressure produces a high-
sterilizing temperature (ca. 121°C; Table 5.2) without
boiling liquids (under ideal conditions). Duration of the
autoclaving depends on the volume of liquid to be steril-
ized; for example, 10 minutes of autoclaving at 121°C is
sufficient to sterilize test tubes of 18 mm diameter,
whereas 1 hour is required to sterilize 10 liters of liquid.
When properly operated, an autoclave kills all microor-
ganisms, even the heat-resistant spores of bacteria and
fungi. After autoclaving, the surface and the inside of
sterilized materials are often wet, especially materials
containing paper or cotton, and in most cases a dry-heat
 Sterilization and Sterile Technique
FIGURE 5.4. An ultrasonic siphon-type pipette washer.
69
step in an oven at 150°C is required unless the autoclave
is capable of a dry cycle for vessels resistant to 150°C.
Some precautions must be taken for correct auto-
claving. For liquids, it is important to leave a free space
in the containers (e.g., flasks and test tubes) of at least
one quarter of the total volume to guarantee space for
steam and potential boiling. The caps of the containers,
especially screw caps of test tubes, should be loose to
prevent an excessive buildup of pressure.
Steam- and heat-resistant material is required for
labeling vessels to be autoclaved. Autoclave tape that is
commercially available is recommended, and the date of
sterilization should be added to the label. There should
be space between the individual items within the
autoclave; that is, do not overfill the autoclave. Before
starting the autoclave, make sure that the water level in
the autoclave is sufficient to ensure steam generation
throughout the autoclaving process, unless the
autoclave is connected to a utility steam supply. The
autoclave door should be closed without excessive tight-
ening, because the steam pressure pushes outward on
the door, forming a tight seal. If tightening is excessive,
then it may be difficult to open the door.
After completion of the autoclaving process, the
autoclave door should not be opened until the pressure
is completely reduced and the temperature is less than

Pasteurization
Heat 66 to 80°C
30 min

Room temperature
20°C
Quick cool down
< 10°C

Tyndallization
Heat 60 to 80°C
30 min

Day 1 Day 2 Day 3

Room temperature
20°C

Quick cool down Quick cool down Quick cool down

4 to 10°C 4 to 10°C 4 to 10°C

FIGURE 5.5. Schematic representation of the temperature cycles for pasteurization and tyndallization.
70 Sterilization and Sterile Technique

TABLE 5.2. Sterilization perature than the preset temperature, possibly leading
temperature and steam pressure to damage of some materials.
for autoclaving. The materials to be sterilized should be dry and
covered (e.g., with aluminum foil or an autoclave bag)
or placed inside a container (e.g., a stainless steel box or
Temperature Steam pressure glass dish) to avoid contamination once the sterilization
process is finished. The door of the oven should not be
°C °F atm psi opened before the oven temperature has decreased less
then 60°C, because quick cooling of the sterilized mate-
100 212 1.0 14.7 rial enhances contamination by convection currents.
110 230 1.4 20.6
115 239 1.7 25.0
121 250 2.0 29.4
134 273 3.0 44.1 3.3. Pasteurization and Tyndallization

During the nineteenth century, Tyndall and Pasteur

100°C. Opening the autoclave prematurely may cause
boiling of superheated liquids and may result in con-
tamination from spores in the ambient room air that cir-
culate by convection currents and enter the autoclave;
however, a liquid culture medium should not be left to
cool to room temperature in the autoclave, because this
often results in the formation of precipitants. This is
particularly important when autoclaving seawater or a
culture medium containing silicate. Rapid cooling
outside the autoclave minimizes the formation of pre-
cipitants. For screw-top culture tubes, the loose caps
should be tightened after the liquid is cool. Hot glass-
ware should not be placed directly into cold storage,
because this may cause fine cracks to form in the glass.
When trace metal contamination must be avoided,
other methods of sterilization should be used (Price et
al. 1989).
3.2. Dry-Heat Sterilization
A hot air oven is generally used for dry-heat steriliza-
tion of dry goods (i.e., not liquids). This mode of ster-
ilization is advantageous to eliminate unacceptable
autoclave residues (e.g., for certain isolation techniques;
see Chapter 6) and to dry the cotton in cotton-plugged
pipettes. Dry-heat sterilization requires higher temper-
atures and longer heating than sterilization by auto-
claving. The most rigorous method involves heating to
250°C for 3 to 5 hours; however, in many instances,
heating to 150°C for 3 or 4 hours is sufficient. If the
oven is not equipped with a fan, then the temperature
in the oven may not be uniform; caution is required,
because some areas of the oven may reach a higher tem-
developed, for other purposes, procedures for steaming
solutions, such as food items; hence, the procedure is
called pasteurization and tyndallization. Phycologists
have adopted and modified the techniques for liquids
that should not be exposed to temperatures more than
100°C or that cannot be autoclaved. Heat is usually pro-
vided by unpressurized steam; thus the term steaming is
often applied in phycology (Starr and Zeikus 1993; see
also Chapter 11). Pringsheim (1946) recommended
steaming for the preparation of biphasic soil-water
culture medium, and steaming is often used to prepare
enriched seawater culture media. Various methods are
practiced, although no clear definitions are assigned to
these, but they can be summarized as methods for
heating a liquid solution to a high temperature and
holding it at this temperature for a period, followed by
a quick cooling.
For pasteurization, the liquid is traditionally raised to
a temperature between 66°C and 80°C, held at this tem-
perature for at least 30 minutes, and then rapidly cooled
to a temperature less than 10°C (Fig. 5.6). However,
pasteurization of seawater is usually carried out at 95°C
for 1 hour. For tyndallization, a process similar to
pasteurization is applied on the first day (heating
to 60°C–80°C for 30 minutes, quickly cooling to
~4°C–10°C); the material is kept cool until the follow-
ing day, when it is heated and cooled as before; on the
third day, the cycle is again repeated (Fig. 5.6). The
repeated process is designed to kill cysts that germinate
after the first or second heating.
In aquaculture, where large containers of seawater or
freshwater are used, flash sterilization is used. Steam is
produced, often by a home furnace, and passed between
the titanium plates of a heat exchanger. The seawater
(or freshwater) is pumped through the titanium plates,
and the water is almost instantly heated to ~70°C. This
 Sterilization and Sterile Technique
a
b in an oven at a temperature of up to 120°C. After
FIGURE 5.6. Autoclave for laboratory use. (a) Front view.
(b) Top view.
procedure does not kill the most resistant spores, but it
does kill most life in the water.
3.4. Sterilization by Filtration
Sterilization by filtration is required for heat-labile
components, such as vitamins, or volatile components
of liquids, such as organic solvents. This method is also
used for convenience and rapidity when only a small
volume of liquid is to be sterilized. A variety of filters
are available (i.e., different pore sizes, composition,
color, and size). Membrane filters that can be autoclaved
are generally used for sterilizing culture medium. The
filters should have a pore size less than 0.2 mm; however,
it is important to note that viruses can pass through such
filters. When the solution has a high viscosity or con-
tains suspended particles, prefiltration with a 1-mm pore
71
FIGURE 5.7. A reusable filtration apparatus inside an
autoclavable bag.
size filter is required. Both disposable and reusable types
of filtration equipment are commercially available.
Sterile, disposable filtration sets are convenient and
ready to use, but they are relatively expensive. Reusable
filtration equipment (e.g., glass or polycarbonate mate-
rials) can usually be autoclaved; the membrane filter and
its assembly unit are either placed in an autoclave bag
(Fig. 5.7) or wrapped in aluminum foil before autoclav-
ing. After autoclaving, the filtration set should be dried
cooling, the filtration apparatus can be used on a bench
top (i.e., without requiring a laminar flow hood), as long
as the filtration reservoir is not exposed to contamina-
tion. After the liquid is filtered, the sterile solution in
the reservoir bottle can be dispensed (using sterile tech-
nique) into other appropriate sterilized vessels.
For very small volumes, sterile syringes with dispos-
able filter units (Fig. 5.8) are commonly used, although
reusable filter units are also available. Disposable units
are manufactured for immediate use, whereas reusable
filter units must be autoclaved and dried before use as
described previously.
Filter sterilization should be used with caution,
however. Membrane filters, such as intertwined organic
fibers or perforated polycarbonate sheets, sometimes
have openings larger than the nominal size. For
example, Stokner et al. (1990) found that 0.2-mm filters
had numerous surface defects visible by electron
microscopy and that both particles and algae that were
several micrometers were identified in the filtrates.
Small heterotrophic flagellates are able to squeeze
through pore openings that are much smaller than their
normal cell diameter.
72 Sterilization and Sterile Technique
FIGURE 5.8. Disposable syringe filtration unit.
3.5. Microwave Oven Sterilization
Sterilization with a microwave oven is quicker than
steam or dry sterilization. The heat produced by
microwaves is of two types: ionic polarization and
dipole rotation. In practice, cells may be killed more by
the heat from boiling liquids than by microwaves,
and indeed, the killing mechanism is not known
(Keller et al. 1988). But nevertheless, the microwave
sterilization technique is effective and nontoxic. It is
recommended to use a microwave oven that is equipped
with a rotating table and has up to 700 watts (W) of
power.
For the sterilization of liquids in previously cleaned
culture vessels, there are different protocols. Keller et
al. (1988) showed that, for 1 to 1.5 liters of seawater,
microalgae were killed in 5 minutes, bacteria in 8
minutes, and fungi in 10 minutes. Microwave ovens
manufactured today are more powerful than those pro-
duced in 1988, so the times may vary depending on the
power of the microwave oven. Furthermore, the effec-
tiveness of a microwave oven often diminishes over
time, and therefore an older oven requires more time.
Leal et al. (1999) describe a quick intermittent proto-
col, such as a 5-minute total microwave time (2, 1, and
2 minutes of treatment, separated by intervals of 30
seconds) at 600 W.
For sterilizing glassware, Boye and van den Berg
(2000) described a protocol in which a small volume of
distilled or deionized water was added and then
microwaved for 20 minutes at 600 W. After steriliza-
tion, the water was discarded in a laminar flow hood
with use of sterile technique. When water was not
added to the glassware, sterilization by microwave
required 45 minutes or more to destroy bacterial spores
(Jeng et al. 1987), analogous to time differences
between autoclave and dry-heat oven sterilization
methods (see previous text).
3.6. Ultraviolet Radiation
Although x-rays and gamma rays (ionizing radiation)
are widely used for commercial manufacturing (e.g.,
sterile plasticware), they are not suitable for use in lab-
oratories for safety reasons. UV radiation is suitable for
laboratory use, including sterilization of culture facility
hoods and benches (see later text). UV radiation is
damaging to humans (especially eyes), and care must
be taken to avoid exposure. In addition, UV radiation
produces ozone, and some people find this offensive
(Hamilton 1973). UV radiation has its primary lethal
effect at 260 nm and forms covalent bonds between
adjacent thymines in DNA. These thymine-thymine
dimers, in turn, cause DNA replication errors, which
cause potentially lethal mutations.
UV lamps typically produce radiation with wave-
lengths between 240 and 280 nm. The energy varies
with the bulb, ranging from 40 to 40,000 microwatt-
seconds per cm2, and the choice of bulb depends on the
application. Low-energy bulbs are used for sterilizing
hoods and benches, whereas high-energy bulbs are used
for sterilizing water. UV radiation does not penetrate
ordinary glass, so for sterilizing liquids, especially large
volumes (e.g., carboys), a waterproof submersible bulb
is usually required. Quartz test tubes allow UV radia-
tion to penetrate, but these test tubes are too expensive
for routine culture work. Furthermore, quartz glass
easily scratches, which greatly reduces its efficiency
(Hamilton 1973; for additional details, see Hamilton
and Carlucci 1966). With regard to sterilizing large
volumes of water, it must be remembered that UV light
does not penetrate water uniformly—it is absorbed, and
the effectiveness diminishes with distance from the
bulb. Continual mixing of the water and sufficient expo-
sure time usually circumvents this problem for the
water, but if organisms are attached to the inner surface
of the container, then sterilization may be ineffective
unless more powerful lamps are used with longer
exposure times.
3.7. Bleach
Bleach (sodium hypochlorite) is widely used in aqua-
culture hatcheries where very large volumes of water
require sterilization. Although the process may not kill
all cysts when smaller amounts of bleach are added, it
is very effective in killing most organisms. The amount
of bleach added depends on the organic matter in the
water. Typically, 1 to 5 mL of commercial bleach is
 Sterilization and Sterile Technique 73
added per liter of water, and after gentle mixing, the
water is left to stand (no mixing and no aeration) for
several hours. For shorter times, add more bleach. The
solution should not be exposed to direct sunlight during
treatment. After the bleach treatment is finished,
the solution is neutralized with sodium thiosulfate
(Na2S2O3 · 5H2O). If 250 g of sodium thiosulfate are dis-
solved in 1 liter of water, then 1 mL of the sodium thio-
sulfate solution is added for each 4 mL of bleach used.

3.8. Ethylene Oxide Sterilization

Historically, non-heat-tolerant materials (plastic and

rubber) were sterilized with ethylene oxide (Hamilton
1973). However, problems arose after sterilization,
because chemical traces remained, often killing the
FIGURE 5.9. A stainless steel basket for holding items in
living cells used in the experiment. Consequently, eth-
an autoclave. The basket contains Pasteur pipette canisters
ylene oxide sterilization has gradually fallen into disuse, covered with autoclavable bags. Note that the cap junction of a
and modern heat-tolerant reusable materials or sterile pipette canister is covered by aluminum foil.
disposable materials have become popular.

small, closed room, isolated from the normal laboratory

4.0. Storage of Sterilized Materials
Once it is sterilized, regardless of method, the main dif-
ficulty is keeping the material sterile. After cooling,
sterilized materials can be stored in clean, dust-free con-
tainers, cabinets, or shelves (Fig. 5.1), and refrigerated
storage is often used for culture media. It should
be remembered that at least the outer surface of the
container (e.g., metal box, canister, or aluminum
package) is contaminated. To prevent contamination
of the sterile contents, the junction of the container
and its cover should be wrapped with aluminum foil
before sterilization (Fig. 5.9). The body of the container
should be cleaned externally with ethanol or passed
through a flame if the container is small and made of
glass or metal before opening to remove the sterilized
contents.
5.0. Sterile Techniques
5.1. Sterile Rooms and General Techniques
A laminar flow hood, placed in a small, closed room, is
the best choice for manipulating cultures and sterile
materials. If a laminar flow hood is unavailable, then a
room, is the next best choice. These small rooms are
usually equipped with UV lamps, which can sterilize the
room when not in use, and a Bunsen burner (or alcohol
lamp). When small rooms are not available, sterile tech-
nique can still be performed in a normal laboratory
room if precautions are taken. Regardless of the setting,
the primary source of contamination will be from the
air, the working surface, and the external surfaces of
containers, vessels, and so on. Therefore, all manipula-
tions should be conducted in a way to avoid these risks.
In the United States, Lysol is sprayed in the air 10
minutes before sterile work (R. A. Andersen, personal
communication). The spray neutralizes charges on col-
loidal particles in the air, such as dust and spores, allow-
ing them to fall onto surfaces, such as tables and floors.
Before beginning the sterile work, the surfaces are
wiped with 70% ethanol to remove the dust and spores
and to ensure a good state of the environment for
conducting sterile work.
The working area and materials should be arranged
so that air circulation is reduced as much as possible
(Fig. 5.10; order of placement of the materials is from
a to f ), that is, the operator should make minimum
movements and any motion should be slow. The oper-
ator should avoid crossing hands or waving pipettes
through the air, and pipetting action should be con-
ducted slowly. A laminar flow hood ensures that air-
borne dust does not enter the working area, but
74 Sterilization and Sterile Technique
FIGURE 5.10. A laminar flow hood with Bunsen burner
and supplies organized for use by a right-handed person. Place-
ment and use order of the materials is indicated with the letters
(a) to (f). Note the stopper for the cylindrical canister. Test tube
arrangement is to facilitate handling and to avoid mistakes during
manipulations.
attention must be given to maintenance of the hood
filters. If a filter becomes contaminated, then the
laminar flow hood becomes a very effective contami-
nating machine. The condition of sterile materials
should be checked, and if any doubt exists, then the
material should not be used. The person may wear latex
gloves, which should be changed often, or alternatively
the person should wipe his or her hands with 70%
ethanol. Sleeves of clothing are a major source of con-
tamination; therefore, sleeves should be rolled up and
the person’s arms should be wiped with 70% ethanol.
Alternatively, the person may wear clean, UV
light–sterilized work clothes that are not worn outside
the transfer room. The work area should be wiped with
70% ethanol before the task is begun.
The Bunsen burner is usually placed in the center for
subculturing manipulations or inoculations, so that all
operations are conducted within a 40-cm circle around
the flame (Fig. 5.10). A receptacle for used pipettes, dis-
posable tips, and so on, should be placed on the right
side for a right-handed person (or on the left side for
a left-handed person) so that contamination risk is
limited. Once the work area is well organized, the
Bunsen burner can be ignited, and the person should
once again wipe his or her hands with 70% ethanol,
using care to avoid ignition of the alcohol. Thereafter,
the worker should not place his or her hands outside of
the hood (Fig. 5.10). If a sterile item (e.g., a pipette or
disposable tip) accidentally touches the outer surface of
a bottle or container, then it should be discarded and
replaced with a new sterile implement. The contents of
a glass vessel should not be poured directly into another
vessel unless the outer surface near the rim is passed
through a flame, because the outer surface is a major
source of contamination. Under no circumstances
should a pipette be reused. Similarly, if a portion of
sterile medium is poured into one cell culture, then the
remainder should be discarded; microscopic droplets
often splash from the cell culture back into the previ-
ously sterile medium.
5.2. Transferring Liquid Cell Cultures
The following example is a common procedure for
transferring established cultures into new culture
medium. Although many variations exist in different
laboratories, this provides a general procedure for
proper sterile technique. The example assumes that the
worker is using a laminar flow hood. If you do not
have a laminar flow hood, then certain steps are to be
deleted. The example also assumes the worker is using
reusable glass pipettes; if you use disposable plastic
pipettes or pipette tips, then some minor modifications
are necessary. Finally, the directions are for right-
handed people; left-handed people should reverse the
movements and the placement of items (see Fig. 5.10).
For additional details and other manipulations, see
Barker (1998).
1. Wear protective clothing that is sterilized by UV
light. If clothing is maintained inside the room,
then carefully do this after turning off the UV
lamp and entering the room.
2. Turn off the UV sterilizing light in the room,
open the door using a minimum opening, enter
the room with slow motion, and gently close the
door.
3. Turn on the laminar flow hood air circulator and
then turn off the UV sterilizing light in the
laminar flow hood. If there is an external gas
valve for the hood, then open it, but only if the
valve on the Bunsen burner is closed.
4. Wipe the working surface with 70% ethanol.
5. Place the Bunsen burner in the center.
6. Place the canister with sterile pipettes or
disposable pipette tips on the most left side (Fig.
5.10). Spray and wipe the surface of the container
with 70% ethanol.
7. Place the bottles, Erlenmeyer flasks, or test tubes
on the left side (i.e., second position for
 Sterilization and Sterile Technique
chronological order of use; see Fig. 5.10, with
order of materials from a to f ) at the periphery of
the 40-cm circle from the Bunsen burner.
8. Place the pipette bulb on the right side of the
Bunsen burner. Regularly clean the inner part of
the bulb with 70% ethanol and wipe carefully the
inner rim; loose cotton fibers in its opening
prevent a seal from forming, causing a pipette to
drip. Never use a bulb that has been used for
manipulation of chemicals (e.g., osmium,
glutaraldehyde).
9. Place the container for discarding used pipettes
outside the 40-cm circle, on the most right side of
the working area.
10. Make sure that all inoculation vessels are correctly
labeled.
11. After organizing all materials, turn on the
(secondary) gas valve and light the Bunsen burner.
Spray hands with 70% ethanol (or put on sterile
disposable gloves). Sterile manipulation can now
begin; make sure to stay within the 40-cm circle
around the Bunsen burner.
The following steps describe the procedure for trans-
ferring liquid cultures with a glass Pasteur pipette that
will be cleaned and reused.
12. With your left hand, pick up the pipette canister
and bring it close to the Bunsen burner.
Remaining in the 20-cm circle of the Bunsen
75
burner, remove the cap with your right hand by
putting pressure on the cap in between your palm
and two outer fingers (Fig. 5.11a). When the
canister cap is too large to be held as described,
place the cap at the base of the Bunsen burner
and flame it before replacing it onto the pipette
canister.
13. With your left hand, gently shake the canister so
that one pipette is smoothly extruded a few
centimeters from the canister opening (Fig.
5.11b).
14. With your right hand, catch the pipette with your
thumb and index and middle fingers, and then
remove it from the canister with a slow, uniform
motion (Fig. 5.11c). The pipette tip should never
contact the container opening.
15. Keep your right hand near the Bunsen burner; a
20-cm circle is the preferred distance to maintain
pipettes and to open vessels.
16. Replace the cap onto the canister, being very
careful that the pipette never touches the
container surface or anything else (Fig. 5.11d). To
avoid manipulations and risks during repeated
culture transfers, it is recommended to keep the
pipette canister open within the 40-cm circle and
to extract the pipettes with a stainless forceps
flamed before each extraction. In this case, the
canister content should be completely used or, if
not, should be resterilized before the next
use.

a b

c d
FIGURE 5.11. Removal of a Pasteur pipette from a canister. Note that the canister cap, though large, is held in the right hand
between the palm and two outer fingers. All work is close to the Bunsen burner flame. (a), Opening the pipette canister; (b), Shaking
a pipette partially out of the canister; (c), Extraction of a pipette; (d), Replacing the cap on the canister.
76 Sterilization and Sterile Technique

17. With your left hand, place the canister
in its normal position on the working
surface.
18. With your left hand, pick up the pipette with
your thumb and index finger at the cotton-
plugged end of the pipette and remain close to
the flame (Fig. 5.12a).
19. With your right hand, pick up the bulb.
20. Insert the pipette bulb onto the pipette
(Fig. 5.12b).
21. It should not be necessary to flame the sterile
pipette; however, if you choose to flame the
pipette before use, then cool the pipette with a
small amount of sterile medium.
22. Handle the pipette in your right hand near the
bulb (Fig. 5.12c) and slowly pick up the cell
culture vessel with your left hand.
23. Bring the cell culture vessel to your right hand,
and using your palm and small finger of the right
hand while still holding the pipette, remove the
cap from the vessel (Fig. 5.13a). The pipette
should remain close to the Bunsen burner and
should not touch anything.
24. With the left hand, briefly flame the opening of
the cell culture vessel while slowly rotating the lip
of the vessel in the flame at an angle of at least 45
degrees; avoid breathing into the vessel if the
laminar flow hood window is not pulled down
(Fig. 5.13b; the hood window is up only for
photographic purposes). Note: do not flame
plasticware.
25. With the left hand, move the vessel slowly away
from the flame but keep it oriented at a 45 degree
angle to reduce the possibility of contamination.
26. Slowly insert the tip of the pipette into the liquid,
being careful not to touch the pipette against the
opening of the vessel.
27. Slowly draw a cell suspension into the pipette by
carefully controlling the pressure exerted on the
bulb, making certain that the liquid level does not
come near the cotton plug of the pipette (Fig.
5.13c). Collect only the necessary volume of cell
suspension. If extra material is removed, then it
must be discarded. To avoid any risk of spilling or
dripping, the bulb should be completely expanded
at the end of the collection.
28. After drawing up the appropriate amount of cell
suspension, the pipette is slowly removed while
avoiding contact with the vessel.
29. Orient the pipette to a nearly horizontal position
without exerting pressure on the bulb, and maintain
the pipette within 20 cm of the Bunsen burner.
30. Flame the mouth of the vessel again, using a
rotating motion (Fig. 5.13d). Do not touch the
pipette against anything.
31. Using your left hand, slowly bring the vessel to
the cap that has been maintained between the
small finger and palm of the right hand (Fig.
5.13e). Note: for larger caps (e.g., Erlenmeyer
large soft plugs, Fig. 5.2) that cannot be held
during manipulations, the cap is placed on the
working area and flamed before replacement.

a b

c
FIGURE 5.12. (a) Transferring the pipette from the right hand to the left hand. (b) Holding the pipette and attaching the bulb.
Note that the tip of the pipette remains near the Bunsen burner. Also note the cotton plug in the pipette that retains any contami-
nating material from the pipette bulb. (c) Holding the pipette in the right hand, slowly move the left hand (not visible) to pick up the
culture vessel containing cells.
 Sterilization and Sterile Technique 77

a b

c d

e
FIGURE 5.13. Demonstration with a test tube, in which the cap is held between the palm and small finger during aseptic work:
opening the test tube cap (a), flaming the test tube mouth (b), drawing up a cell suspension for transfer (c), the second flaming of
the test tube mouth (d), and replacing the test tube cap (e).

32. Replace the cap, always being careful that the
pipette does not touch anything. Be careful not to
bring the pipette too close to the flame, because
the heat may kill the cells.
33. Return the capped vessel to its previous position.
Your left hand is now free. Note: it is good
practice to place the drawn vessels (or,
alternatively, newly inoculated vessels) in a new
location so that accidental reinoculation does not
occur. In the case of test tubes held in a rack,
a row free of tubes can be maintained
between uninoculated and inoculated tubes
(See Fig. 5.10).
34. Slowly move your left hand to the vessel that will
be inoculated.
35. Using the same procedure described above, open
the new vessel, flame the opening, and insert the
pipette into the new vessel without touching the
mouth.
36. Slowly discharge the cell suspension into the
vessel and carefully remove the pipette. If the tip
of the pipette is placed below the surface, then
avoid bubbling into the liquid.
37. Flame the mouth of the vessel and replace the cap
as described above, using precautions as before.
38. The newly inoculated vessel, with cap attached,
should be placed onto the bench again.
39. Discard the used pipette in the storage container,
using caution to avoid any spillage of cell
suspension onto the bench. If any spill has
occurred, then wipe up the liquid with a Kimwipe
or towel, spray the area with 70% ethanol, and
wipe the surface again with a new tissue paper. If
wearing gloves, then change to a new pair of
sterile gloves; otherwise, spray hands with 70%
ethanol.
40. Bring the pipette to the discard container (Fig.
5.14a). With the free left hand, pick up the
pipette with your thumb and index finger at
the cotton-plugged end of the pipette, and
carefully remove the pipette bulb with your right
hand.
41. With your left hand, place the pipette in the
container, making sure that the tip is well
immersed in the water (Fig. 5.14b). Return the
bulb to its normal position. The working area is
78 Sterilization and Sterile Technique
a b
FIGURE 5.14. After transfer, the pipette is directed to the water-filled discard container (a). The bulb is removed once the
pipette is above the discard container to avoid spilling on the working area (not shown). (b) The discard container should be stable
enough to store the appropriate number of pipettes without risk of falling. A sufficient amount of water stabilizes the container and
ensures that the pipette tips remain wet.
back to its initial stage and ready for the next
transfer process.
42. Once all transfers are completed, turn off the Bunsen
burner, remove all the materials from the working
place, and wipe the surface with 70% ethanol.
43. Close the front window of the hood and close the
external gas valve, if present. Switch off the
ventilation system and turn on the UV lamp of
the hood. Exit the room by carefully opening and
closing the door. Turn off the room light and turn
on the UV lamp for the room.
44. Remove your protective clothing and place it in
the UV cabinet for sterilization. Exit the airlock
room carefully and switch on the UV sterilization
of this room.
45. In most cases, clean shoes or slippers are
recommended for the clean area, and these should
be changed when entering and exiting the clean
area. A sticky pad or carpet placed on the floor at
the door location is very helpful to keep dirt from
clean areas; dirt on shoes is perhaps the primary
source of contamination entering a room, where
later it may become airborne.
46. Appropriate signs should be in place to ensure a
safe work area. Custodians should be generally
prohibited from servicing sterile facilities unless
informed and instructed.
5.3. Transfer of Agar Culture
Refer to Section 5.2, with modifications as follows:
1–5. Follow steps 1 through 5, described previously.
6. Place the transfer loop at the base of the Bunsen
burner.
7. Place the test tubes or petri dishes on the left
side at the periphery of the 40-cm circle from
the Bunsen burner.
8. After organizing all materials, turn on the
(secondary) gas valve and light the Bunsen
burner. Spray hands with 70% ethanol, or put
on sterile disposable gloves. Sterile manipulation
can now begin, but make sure to stay within the
40-cm circle around the Bunsen burner.
9. Flame the transfer loop in the blue part of the
Bunsen burner flame until the metal is red (Fig.
5.15a).
10. Cool the loop while maintaining it within the
20-cm circle around the Bunsen burner (Fig.
5.15b).
11. Using the left hand, bring the cell culture vessel
toward the Bunsen burner within the 20-cm
circle.
12. When using a test tube, follow the instructions
given for transfer of liquids (steps 23 through 25
in the previous section). When using a petri
dish, open the cover with your left hand, to an
angle of 45 degrees; avoid touching the inner
part of the cover (Fig. 5.15c).
13. Slowly insert the loop and pick up the cells
without scratching the agar (Fig. 5.15d).
14. Replace the petri dish cover or the cap of the
test tube (see steps 30 to 32 in Section 5.2).
Keep the loop end within the 20-cm circle of the
Bunsen burner without touching any surfaces
and without getting too close to the flame.
15. Slowly move your left hand to the vessel that
will be inoculated.
16. Using the same procedure described previously,
open the new vessel and gently brush the surface
of the agar (without scratching the surface). Use
 Sterilization and Sterile Technique 79

a b

c d
FIGURE 5.15. Cell culture transfer onto agar medium: flaming a platinum loop in a Bunsen burner flame (a); cooling the loop
in the sterile zone near the Bunsen burner (b); opening a petri dish cover at a 45 degree angle (c); 45 degree angle opening for spread-
ing cells with a glass rod); and using a transfer loop to remove cells before transfer (d). Note that the transfer loop is centered to
avoid touching the test tube and that the test tube is oriented at a 45 degree angle.

the appropriate technique of transfer (i.e.,
horizontal lines in zigzag or quadrants system
for a petri dish). In any case, the loop end should
not touch the opening of the test tube or sides of
the petri dish.
17. After a test tube transfer, flame the mouth of the
test tube and replace the cap as described
previously, using precautions as before.
After a Petri dish transfer, replace the cover
gently.
18. Flame the end of the loop in the blue part of the
Bunsen burner until the loop is red. After
cooling the loop within 20 cm of the Bunsen
burner, the loop is ready for the next transfer.
19. Follow instructions given in Section 5.2., steps
42 to 46.
6.0. Sterilization of Culture Media
6.1. Stock Solutions
In most cases, stock solutions of each component of the
medium are prepared in autoclavable bottles. To
sterilize, each stock solution should be autoclaved or
sterile filtered and stored at 4°C. (Vitamins are
stored at -20°C.) To maintain sterility of stock
solutions, they should be handled aseptically. If
fungal or bacterial growth is discovered, then the stock
should be autoclaved and the contents discarded.
For details on preparing stock solutions, see Chapters 2
to 4.
6.2. Sterilization of Liquid Culture Media
Sterilization is typically carried out by autoclaving
(121°C for 15 minutes), followed by a cooling period.
The appearance of the culture medium should be
checked; if the color has changed or if precipitates have
formed, then the medium may not be suitable for
normal use. Reasons for such changes are multiple, so
the preparation procedure should be checked step by
step. Heat-labile components, like vitamins, may be
added after autoclaving by using a filter sterilization
method. Tyndallization is used in place of autoclaving
when destruction of fragile components by high tem-
perature must be avoided (see previous section on
pasteurization and tyndallization).
80 Sterilization and Sterile Technique
Using a microwave oven can also perform the steril-
ization of media. The temperature reached during this
type of sterilization is less than 84°C (Keller et al. 1988,
Hoff and Snell 2001). This allows the direct addition of
some labile components before microwave treatment.
Nevertheless, sterile addition of vitamins is still recom-
mended after microwave treatment. Microwave oven
sterilization of media is quick (only 10 minutes or less),
and it avoids both metal contamination, which occurs
during autoclaving, and carbonate precipitation (Price
et al. 1989). However, microwave sterilization can be
used only for small volumes of media (£1–1.5 liters),
whereas autoclave sterilization can be used for large
volumes of media (e.g., 20 liters).
6.3. Sterilization of Agar Culture Media
(See Also Chapter 2)
As for liquid culture media, sterilization is usually
obtained by autoclaving (121°C for 15 minutes). For
agar slants in test tubes, the tubes should be cooled at
an angle so that the agar solidifies with a smooth slope
that is not too close to the opening of the tube. When
petri dishes are used, the agar is dispensed into sterile
petri dishes under sterile conditions and then cooled. To
reduce contamination, the agar should not exceed half
the height of the dish.
For agar culture media containing heat-labile com-
ponents, the fragile components are added aseptically
after autoclaving but before the agar begins to gel
(approximately 50°C–60°C for standard-melting-point
agars).
7.0. Evaluating Sterile Conditions
Despite careful sterile technique, contamination may
sometimes occur. It may not be obvious how or why the
contamination occurs, or a less rigorous protocol may be
necessary when suboptimal conditions exist (e.g., at a
field station). Therefore, it is often helpful to employ
sterility tests to evaluate the conditions. Bacteria and
fungi grow in various nutrient agars and broths, although
it is important to note that not all bacteria or fungi grow
in any specific organic medium. For general testing pur-
poses, a 0.1% peptone agar or broth is usually satisfac-
tory. For best results, the peptone should be dissolved in
culture medium rather than distilled water. When fungal
contamination is a specific concern, malt extract is pre-
ferred, because it grows fungi better than peptone. In
specific cases, for example, testing for methyl-
aminotrophic bacteria, methylamine is the preferred
substrate; a peptone medium tests negative, because
there is insufficient substrate to detect bacterial growth.
To test for air-borne contamination over a working
surface, a series of the test medium is exposed over
specific periods. For example, petri dishes with sterile
peptone agar can be exposed at intervals of 1 minute, 5
minutes, 15 minutes, 30 minutes, and 1 hour. To achieve
this, the petri dish cover is removed for the period (e.g.,
1 minute) and then replaced. The petri dish is then
incubated for 1–3 days, and the number of bacterial or
fungal colonies is counted. For a laminar flow hood,
exposure for 1 hour should not result in any contami-
nating growth. For a still hood in a small room, often
no contamination occurs for short periods, and only one
or two colonies appear after 15–30 minutes. If numer-
ous colonies appear after incubation on the nutrient
agar plate that was exposed for a short period, then the
environment is heavily contaminated, and sterile proce-
dures are likely to fail. Similarly, a test tube of broth can
be uncapped for the period, recapped, and incubated.
Colonies cannot be counted when a broth test medium
is used, and the only results that can be measured are
positive or negative contamination. However, if one
exposes 10 test tubes at intervals similar to those used
for transferring a culture, if no positive growth occurs
in the test medium, and if environmental conditions
remain unchanged, then the worker can have confi-
dence that at least most of the transfers will be con-
taminant-free. If environmental conditions change
between the test and the actual sterile work, then the
test results no longer predict the sterile conditions. Two
examples of changing conditions are given. If the
tests are conducted at a field station when there are no
other people present, but the transfers are carried out
when several people are active in the room, then the
additional activity increases contamination above that of
the tests. If the tests are conducted in a small laboratory
room without activity, and then a custodian operates a
vacuum cleaner a few hours before cultures are trans-
ferred, then the room contains more air-borne spores,
and contamination is much higher than that of the tests.
Variations of these simple tests can be used to test
culture medium, vitamins, stock solutions, pipettes, and
so on. For example, if a stock solution used to prepare
a culture medium has been opened several times, then
the stock solution may be unsterile. By taking the stock
solution and test medium into a laminar flow hood
(see earlier procedures), a small amount of the stock
solution can be added to the test medium. If the
 Sterilization and Sterile Technique
test medium produces bacterial growth during incuba-
tion, then the stock solution is contaminated and
should be discarded. Similarly, if a culture medium is
prepared with sterile filtration and there is concern
about the effectiveness of the filtration (Stokner et al.
1990), then a small amount of the sterile medium can
be added to a test medium. A positive bacterial growth
demonstrates that bacteria have passed through the
filter. If the concern is about contamination by a het-
erotrophic flagellate that feeds phagotrophically on bac-
teria, then the test medium should contain bacteria.
These simple tests, with nearly endless modifications,
can be performed to help evaluate sterile conditions. The
tests must be carried out under conditions that show pos-
itive results when contamination exists. Also, if the real
work is to be conducted following the testing period,
then the test conditions must be representative of the
working conditions. The tests should not be considered
as an absolute measure of sterility, but they provide an
evaluation of sterility when no other means is available.
In conclusion, all sterilization protocols vary for each
person and laboratory practice. Whatever protocol is
used, good sterile technique is required for culturing
algae. Different protocols and modifications of available
materials are often required, and the goals of each indi-
vidual determine the exact protocols. Control methods
should be employed to verify sterile techniques, and a
careful, routine work protocol should be established to
minimize contamination.
8.0. References
Barker, K. 1998. At the Bench: A Laboratory Navigator. Cold
Spring Harbor Laboratory Press, New York, 460 pp.
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Boye, M., and van den Berg, C. M. G. 2000. Iron availability
and the release of iron-complexing ligands by Emiliania
huxleyi. Mar. Chem. 70:277–87.
Hamilton, R. D. 1973. Sterilization. In: Stein, J. R., ed. Hand-
book of Phycological Methods. Culture Methods and Growth
Measurements. Cambridge University Press, Cambridge,
181–93.
Hamilton, R. D., and Carlucci, A. F. 1966. Use of the ultra-
violet irradiated seawater in the preparation of culture
media. Nature. 211:483–4.
Hoff, F. H., and Snell, T. W. 2001. Plankton Culture Manual.
5th ed. Florida Aqua Farms, Inc., Dade City, Florida, USA,
162 pp.
Jeng, D. K. H., Kaczmarek, K. A., Woodworth, A. G., and
Balasky, G. 1987. Mechanism of microwave sterilization in
the dry state. Appl. Environ. Microbiol. 53:2133–7.
Keller, M. D., Bellows, W. K., and Guillard, R. R. L. 1988.
Microwave treatment for sterilization of phytoplankton
culture media. J. Exp. Mar. Biol. Ecol. 117:279–83.
Leal, M. F. C., Vasconcelos, M. T. S. D., and van den Berg,
C. M. G. 1999. Copper-induced release of complexing
ligands similar to thiols by Emiliania huxleyi in seawater
cultures. Limnol. Oceanogr. 44–7:1750–62.
Price, N. M., Harrison, G. I., Hering, J. G., Hudson, R. J.,
Nirel, P. M., Palenik, B., and Morel, F. M. M. 1989. Prepa-
ration and chemistry of the artificial algal culture medium
Aquil. Biol. Oceanogr. 6:443–61.
Pringsheim, E. G. 1946. Pure Cultures of Algae. Their
Preparation and Maintenance. Cambridge University Press,
Cambridge. 119 pp.
Starr, R. C., and Zeikus, J. A. 1993. UTEX—The culture
collection of algae at the University of Texas at Austin. J.
Phycol. 29(2, Suppl):1–106.
Stokner, J. G., Klut, M. E., and Cochan, W. P. 1990. Leaky
filters: a warning to aquatic ecologists. Can. J. Fish. Aquat.
Sci. 47:16–23.