Understanding and Managing Cell Culture Contamination


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Understanding and Managing Cell Culture Contamination
Technical Bulletin

Life Sciences

John Ryan, Ph.D. Corning Incorporated Life Sciences Acton, MA 01720
Table of Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
What Are the Major Cell Culture Contaminants? . . . . . . . . . . . . . . . . . . . . . . . . . . 2
What Are the Sources of Biological Contaminants? . . . . . . . . . . . . . . . . . . . . . . . . . 8
How Can Cell Culture Contamination Be Controlled? . . . . . . . . . . . . . . . . . . . . . . . . 11
A Final Warning . . . . . . . . . . . . . . . . . . . . . . . 20
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Cell Culture Protocols and Technical Articles . . . . . . . . . . . . . . . . . . . . . . .22

Introduction
No cell culture problem is as universal as that of culture loss due to contamination. All cell culture laboratories and cell culture workers have experienced it. Culture contaminants may be biological or chemical, seen or unseen, destructive or seemingly benign, but in all cases they adversely affect both the use of your cell cultures and the quality of your research. Contamination problems can be divided into three classes:
◗ Minor annoyances — when up to several plates or flasks are occasionally lost to contamination;
◗ Serious problems — when contamination frequency increases or entire experiments or cell cultures are lost;
◗ Major catastrophes — contaminants are discovered that call into doubt the validity of your past or current work.

Table 1. Some Consequences of Contamination
◗ Loss of time, money, and effort
◗ Adverse effects on the cultures
◗ Inaccurate or erroneous experimental results
◗ Loss of valuable products
◗ Personal embarrassment
The most obvious consequence of cell culture contamination is the loss of your time, money (for cells, culture vessels, media and sera) and effort spent developing cultures and setting up experiments. However, the less obvious consequences are often more serious (Table 1). First there are the adverse effects on cultures suffering from undetected chemical or biological contaminants. These hidden (cryptic) contaminants can achieve high densities altering the growth and characteristics of the cultures. Worse yet are the potentially inaccurate or erroneous results obtained by unknowingly working with these cryptically contaminated cultures. Products, such as vaccines, drugs or monoclonal antibodies, manufactured by these cultures will probably be useless. For some researchers the most serious consequence of contamination is suffering the embarrassment and damage to their reputation that results when they notify collaborators or journals that their experimental results are faulty and must be retracted due to contaminants in their cultures.
Preventing all cell culture contamination has long been the dream of many researchers, but it is an impractical, if not impossible, dream. Contamination cannot be totally eliminated, but it can be managed to reduce both its frequency of occurrence and the seriousness of its consequences. The goal of this bulletin is to review the nature of cell culture contamination and the problems it causes, and then to explore some of the key concepts and practical strategies for managing contamination to prevent the loss of valuable cultures and experiments.

What Are the Major Cell Culture Contaminants?
A cell culture contaminant can be defined as some element in the culture system that is undesirable because of its possible adverse effects on either the system or its use. These elements can be divided into two main categories: chemical contaminants and biological contaminants.
Chemical Contamination
Chemical contamination is best described as the presence of any nonliving substance that results in undesirable effects on the culture system. To define further is difficult; even essential nutrients become toxic at high enough concentrations. Nor is toxicity the only concern since hormones and other growth factors found in serum can cause changes that, while not necessarily harmful to cultures, may be unwanted by researchers using the system. (Reviewed in references 1-3.)
Media The majority of chemical contaminants are found in cell culture media and come either from the reagents and water used to make them, or the additives, such as sera, used to supplement them. Reagents should always be of the highest quality and purity and must be properly stored to prevent deterioration. Ideally, they should be either certified for cell culture use by their manufacturer or evaluated by the researcher before use. Mistakes in media preparation protocols, reading reagent bottle labels, or weighing reagents are other common sources of chemical contamination.
Sera Sera used in media have long been a source of both biological and chemical contaminants. Due to cell culture-based screening programs currently used by good sera manufacturers, it is unusual to find a lot of fetal bovine sera that is toxic to a majority of cell cultures. However, it is common to find substantial variations in the growth promoting abilities of different lots of sera for particular cell

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culture systems, especially for cultures that have specialized or differentiated characteristics. Uncontrollable lot-to-lot variation in hormone and growth factor concentrations makes this problem inevitable; careful testing of sera before purchase, or switching to serum-free media can avoid these problems.
Table 2. Types and Sources of Potential Chemical Contaminants
◗ Metal ions, endotoxins, and other impurities in media, sera, and water
◗ Plasticizers in plastic tubing and storage bottles
◗ Free radicals generated in media by the photoactivation of tryptophan, riboflavin or HEPES exposed to fluorescent light
◗ Deposits on glassware, pipettes, instruments etc., left by disinfectants or detergents, antiscaling compounds in autoclave water, residues from aluminum foil or paper
◗ Residues from germicides or pesticides used to disinfect incubators, equipment, and labs
◗ Impurities in gases used in CO2 incubators
Remember also that serum proteins have the ability to bind substantial quantities of chemical contaminants, especially heavy metals, that may have entered the culture system from other sources, rendering them less toxic. As a result, switching from serum-containing medium to a serum-free system can unmask these toxic chemical contaminants, exposing the cells to their adverse effects.
Water The water used for making media and washing glassware is a frequent source of chemical contamination and requires special care to ensure its quality. Traditionally, double or triple glass distillation was considered to be the best source of high quality water for cell culture media and solutions. Newer purification systems combining reverse osmosis, ion exchange and ultrafiltration are capable of removing trace metals, dissolved organic compounds and endotoxins and are increasingly popular. However, these systems must be properly maintained and serviced to ensure continued water quality. Because of its

aggressive solvent characteristics, highly purified water can leach potentially toxic metal ions from glassware or metal pipes, and plasticizers from plastic storage vessels or tubing. These contaminants can then end up in media or deposited on storage vessels and pipettes during washing and rinsing. Water used to generate steam in autoclaves may contain additives to reduce scale buildup in pipes; these potentially toxic additives can also end up on glassware.
Endotoxins Endotoxins, the lipopolysaccaridecontaining by-products of gram negative bacteria, are another source of chemical contaminants in cell culture systems. Endotoxins are commonly found in water, sera and some culture additives (especially those manufactured using microbial fermentation) and can be readily quantified using the Limulus Amebocyte Lysate assay (LAL).
These highly biologically reactive molecules have major influences in vivo on humoral and cellular systems. Studies of endotoxins using in vitro systems have shown that they may affect the growth or performance of cultures and are a significant source of experimental variability (Reviewed in references 6 and 39). Furthermore, since the use of cell culture produced therapeutics, such as hybridomas and vaccines, are compromised by high endotoxin levels, efforts must be made to keep endotoxin levels in culture systems as low as possible.
In the past, sera have been a major source of endotoxins in cell cultures. As improved endotoxin assays (LAL) led to an increased awareness of the potential cell culture problems associated with endotoxins, most manufacturers have significantly reduced levels in sera by handling the raw products under aseptic conditions. Poorly maintained water systems, especially systems using ion exchange resins, can harbor significant levels of endotoxin-producing bacteria and may need to be tested if endotoxin problems are suspected or discovered in the cultures.

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Figure 1. Photomicrograph of a low level yeast infection in a liver cell line (PLHC-1, ATCC # CRL-2406). Budding yeast cells can been seen in several areas (arrows). At this low level of contamination, no medium turbidity would be seen; however, in the absence of antibiotics, the culture medium will probably become turbid within a day.
Figure 2. Photomicrograph of a small fungal colony growing in a cell culture. At this point, this colony would still be invisible to direct visual observation. If this culture was subcultured at this point, all of the cultures or experiments set up from it would soon be lost to fungal contamination.
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Storage Vessels
Media stored in glass or plastic bottles that have previously contained solutions of heavy metals or organic compounds, such as electron microscopy stains, solvents and pesticides, can be another source of contamination. The contaminants can be adsorbed onto the surface of the bottle or its cap (or absorbed into the bottle if plastic) during storage of the original solution. If during the washing process they are only partially removed, then once in contact with culture media they may slowly leach back into solution. Residues from chemicals used to disinfect glassware, detergents used in washing, or some aluminum foils and wrapping papers for autoclaving or dry heat sterilization can also leave potentially toxic deposits on pipettes, storage bottles and instruments.
Fluorescent Lights
An important but often overlooked source of chemical contamination results from the exposure of media containing HEPES (N-[2-hydroxylethyl] piperazineN'-[2-ethanesulfonic acid]) — an organic buffer commonly used to supplement bicarbonate-based buffers), riboflavin or tryptophan to normal fluorescent lighting. These media components can be photoactivated producing hydrogen peroxide and free radicals that are toxic to cells; the longer the exposure the greater the toxicity (4,5). Short term exposure of media to room or hood lighting when feeding cultures is usually not a significant problem; but leaving media on lab benches for extended periods, storing media in walk-in cold rooms with the lights on, or using refrigerators with glass doors where fluorescent light exposure is more extensive, will lead to a gradual deterioration in the quality of the media.
Incubators
The incubator, often considered a major source of biological contamination, can also be a source of chemical contamination. The gas mixtures (usually containing carbon dioxide to help regulate media pH) perfused through some incubators may contain toxic impurities, especially oils or other gases such as carbon monoxide, that may have been previously used in the

same storage cylinder or tank. This problem is very rare in medical grade gases, but more common in the less expensive industrial grade gas mixtures. Care must also be taken when installing new cylinders to make sure the correct gas cylinder is used. Other potential chemical contaminants are the toxic, volatile residues left behind after cleaning and disinfecting incubators. Disinfectant odors should not be detectable in a freshly cleaned incubator when it is placed back into use.
Keep in mind that chemical contaminants tend to be additive in cell culture; small amounts contributed from several different sources that are individually nontoxic, when combined together in medium, may end up overloading the detoxification capabilities of the cell culture resulting in toxicity-induced stress effects or even culture loss.
Biological Contamination
Biological contaminants can be subdivided into two groups based on the difficulty of detecting them in cultures:
◗ those that are usually easy to detect — bacteria, molds and yeast;
◗ those that are more difficult to detect, and as a result potentially more serious culture problems, — viruses, protozoa, insects, mycoplasmas and other cell lines.
For a comprehensive review, see references 7 and 8.
Ultimately, it is the length of time that a culture contaminant escapes detection that will determine the extent of damage it creates in a laboratory or research project.
Bacteria, Molds, and Yeasts Bacteria, molds and yeasts are found virtually everywhere and are able to quickly colonize and flourish in the rich and relatively undefended environment provided by cell cultures. Because of their size and fast growth rates, these microbes are the most commonly encountered cell culture contaminants. In the absence of antibiotics, microbes can usually be readily detected in a culture within a few days of becoming contaminated, either by direct microscopic observation. (See Figures 1 and 2.) or by the effects they have on the

a
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Figures 3a and 3b. Photomicrographs of a winter flounder (Pseudopleuronectes americanus) fibroblast-like cell culture. Figure 3a shows an apparently healthy early passage culture; Figure 3b shows the same culture approximately 24 hours later. Electron microscopy showed virus-like particles in these cells. Multiple attempts to establish cell lines from this species were unsuccessful and showed cytopathic effects that appeared to be caused by an unknown virus.
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culture (pH shifts, turbidity, and cell destruction). However, when antibiotics are routinely used in culture, resistant organisms may develop into slow growing, low level infections that are very difficult to detect by direct visual observation. Similar detection problems can occur with naturally slow growing organisms or very small or intracellular bacteria that are difficult to see during routine microscopic culture observation. These cryptic contaminants may persist indefinitely in cultures causing subtle but significant alterations in their behavior. By the time these cryptic contaminants are discovered, many experiments and cultures may have been compromised.
Viruses
Due to their extremely small size, viruses are the most difficult cell culture contaminants to detect in culture, requiring methods that are impractical for most research laboratories. Their small size also makes them very difficult to remove from media, sera, and other solutions of biological origin. However, most viruses have stringent requirements for their original host species’ cellular machinery (may also be tissue specific) which greatly limits their ability to infect cell cultures from other species. Thus, although viruses may be more common in cell cultures than many researchers realize, they are usually not a serious problem unless they have cytopathic or other adverse effects on the cultures. (Reviewed in Ref. 7, 40.) Since cytopathic viruses usually destroy the cultures they infect, they tend to be self-limiting. Thus, when cultures selfdestruct for no apparent reason and no evidence of common biological contaminants can be found, cryptic viruses are often blamed. (See Figures 3a and 3b.) They are perfect culprits, unseen and undetectable; guilty without direct evidence. This is unfortunate, since the real cause of this culture destruction may be something else, possibly mycoplasma or a chemical contaminant, and as a result will go undetected to become a more serious problem.
A major concern of using virally infected cell cultures is not their effects on the cultures but rather the potential health hazards they pose for laboratory personnel. Special safety pre-

cautions should always be used when working with tissues or cells from humans or other primates to avoid possible transmission of viral infection (HIV, hepatitis B, EpsteinBarr, simian herpes B virus, among others) from the cell cultures to laboratory personnel (9). Contact your safety office for additional assistance if in doubt as to appropriate procedures for working with potentially hazardous tissues, cultures or viruses.
Protozoa
Both parasitic and free-living, single-celled protozoa, such as amoebas, have occasionally been identified as cell culture contaminants. Usually of soil origin, amoebas can form spores and are readily isolated from the air, occasionally from tissues, as well as throat and nose swabs of laboratory personnel. They can cause cytopathic effects resembling viral damage and completely destroy a culture within ten days. Because of their slow growth and morphological similarities to cultured cells, amoebas are somewhat difficult to detect in culture, unless already suspected as contaminants (7). Fortunately, reported cases of this class of contaminants are rare, but it is important to be alert to the possibility of their occurrence.
Invertebrates
Insects and arachnids commonly found in laboratory areas, especially flies, ants, cockroaches and mites, can both be culture contaminants as well as important sources of microbial contamination. Warm rooms are common sites of infestation. By wandering in and out of culture vessels and sterile supplies as they search for food or shelter, they can randomly spread a variety of microbial contaminants. Occasionally they are detected by the trail of “foot prints” (microbial colonies) they leave behind on agar plates, but usually they don’t leave any visible signs of their visit other than random microbial contamination. Mites can be a serious problem in plant cell culture facilities, especially those doing large scale plant propagation. Although bacteria, molds and yeast may sometimes appear to ‘jump’ from culture to culture, these multilegged contaminants really can. While not nearly as common as other culture contaminants, it is important to be alert to the presence of these invertebrates in culture areas.

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Figures 4a and 4b. These scanning electron micrographs show 3T6 cells (ATCC # CCL-96) with (4b) and without (4a) mycoplasma infections. The level of contamination of these cells by the mycoplasma shown here is typical of contaminated cells. Examination of this contaminated culture by phase contrast microscopy did not show any evidence of contamination; nor did the medium show any turbidity.

Mycoplasmas
Mycoplasmas were first detected in cell cultures by Robinson and coworkers in 1956. They were attempting to study the effects of PPLO (pleuropneumonialike organisms — the original name for mycoplasma) on HeLa cells when they discovered that the control HeLa cultures were already contaminated by PPLO (10). In addition, they discovered that the other cell lines currently in use in their laboratory were also infected with mycoplasma, a common characteristic of mycoplasma contamination. Based on mycoplasma testing done by the FDA, ATCC, and two major cell culture testing companies, at least 11 to 15% of the cell cultures in the United States are currently infected by mycoplasmas (Table 3). Since many of these cultures were from laboratories that test routinely for mycoplasma, the actual rates are probably higher in the many laboratories that do not test at all (11-13). In Europe, mycoplasma contamination levels were found to be even higher: over 25% of 1949 cell cultures from the Netherlands and 37% of 327 cultures from former Czechoslovakia were positive (14). The Czechoslovakia study had an interesting, but typical finding: 100% of the cultures from labs without mycoplasma testing programs were contaminated, but only 2% of the cultures from labs that tested regularly. Other countries may be worse: 65% of the cultures in Argentina and 80% in Japan were reported to be contaminated by mycoplasma in other studies (11).
Unfortunately, mycoplasmas are not relatively benign culture contaminants but have the ability to alter their host culture’s cell function, growth, metabolism, morphology, attachment, membranes, virus propagation and yield, interferon

induction and yield, cause chromosomal aberrations and damage, and cytopathic effects including plaque formation (12). Thus, the validity of any research done using these unknowingly infected cultures is questionable at best. (See references 11, 12, and 15-18 for good overviews of this very serious mycoplasma contamination problem.)
What gives mycoplasmas this ability to readily infect so many cultures? Three basic characteristics: a) these simple, bacteria-like microbes are the smallest selfreplicating organism known (0.3 to 0.8 µm in diameter), b) they lack a cell wall, and c) they are fastidious in their growth requirements. Their small size and lack of a cell wall allow mycoplasmas to grow to very high densities in cell culture (107 to 109 colony forming units/mL are common) often without any visible signs of contamination — no turbidity, pH changes or even cytopathic effects. (See Figures 4a and 4b.) Even careful microscopic observation of live cell cultures cannot detect their presence. These same two characteristics also make mycoplasmas, like viruses, very difficult to completely remove from sera by membrane filtration. In addition, their fastidious growth requirements (unfortunately, easily provided for by cell cultures) make them very difficult to grow and detect using standard microbiological cultivation methods. Thus, these three simple characteristics, combined with their ability to alter virtually every cellular function and parameter, make mycoplasmas the most serious, widespread, and devastating culture contaminants.
Mycoplasmas have been described as the “crabgrass” of cell cultures, but this is too

Table 3. Mycoplasma Contamination of Cell Cultures
Number of Cultures Tested Food and Drug Administration (FDA) (1970’s to 1990’s) (11)
20,000 cultures tested
Bionique Testing Laboratories (several years prior to 1993) (41) 11,000 cultures tested
Microbiological Associates (1985 to 1993) (13) 2,863 cultures tested
American Type Culture Collection (ATCC) (1989 to 1994) (42) 5362

Number Positive over 3000 (15%)
1218 (11.1%) 370 (12.9%) 752 (14%)

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benign a description for what are the most significant and widespread cell culture contaminants in the world. Unfortunately, even with the advances in detection methods (discussed in detail later) mycoplasma infection rates (Table 3) have not changed noticeably since they were first detected in cell cultures. Aggressive management against mycoplasma contamination must be the central focus for any cell culture laboratory contamination or quality control program (16).
Cross-Contamination by Other Cell Cultures
With the advent of improved karyotyping methods in the late 1950’s, it soon became apparent that some cell lines were crosscontaminated by cells of other species (7). In 1967, isoenzyme analysis was used to show that 20 commonly used human cell lines were intraspecies contaminated by HeLa cells (19,20). Contaminated is actually a misnomer since in fact 100% of the original cells had been replaced by the HeLa contaminant. Unfortunately, the scientific community was slow to respond to this very serious problem. Tests done at one research center on 246 cell lines over an 18 month period prior to 1976 showed that nearly 30% were incorrectly designated: 14% were the wrong species and 25% of the human cell lines were HeLa cells (21). A 1981 survey of cultures showed over 60 cell lines that were actually HeLa cells, 16 other human cell lines contaminated by non-HeLa human cell lines, and 12 cases of interspecies contamination (See Table 4). Nor is the problem limited to contamination by HeLa cells. The advent of DNA analysis

has shown that cells from a variety of sources have contaminated many other cell lines (42).
The seriousness of cross-contamination, while not as common as microbial contamination, cannot be overstated. The validity of experimental results from cultures having inter- or intraspecies contamination is, at the very least, questionable. Furthermore, their use can lead to the embarrassment of having to retract published results. Whenever the invading cell is better adapted to the culture conditions and thus faster growing than the original cells, it will almost always completely replace them. Because of the outward physical similarities of different cell lines and the wide morphological variations that can be caused by the culture environment, it is impossible to rely only on microscopic observation to screen for cross-contamination of cultures. Simple accidents are one of the most common means by which other cell lines gain entry into cultures and will be discussed separately in the next section.
Remember, the seriousness of any culture contaminant is usually directly proportional to the difficulty of detecting it; those that go undetected the longest have the most serious consequences. Cultures containing nonlethal (but not harmless), cryptic chemical or biological contaminants are sometimes used in research for months or even years before being uncovered; during this time the quality and validity of all research done with those cultures is compromised, as is the reputation of the researchers using them.

Table 4. Some HeLa Contaminated Cell Lines

Detroit 6 (CCL-3)

Conjunctiva (CCL-20.2)*

Minnesota-EE (CCL-4)

AV3 (CCL-21)*

L132 (CCL 5)*

HEp-2 (CCL-23)*

Intestine 407 (CCL-6)*

J-111 (CCL-24)

Chang Liver (CCL-13)

WISH (CCL-25)*

KB (CCL-17)*

Giardia Heart (CCL-27)

Detroit 98 (CCL-18)

Wilm’s Tumor (CCL-31)

NCTC 2544 (CCL-19)

FL (CCL- 62)*

CCL# is the ATCC catalog designation. All except CCL-20.2, CCL-31 and CCL-62 were shown to be HeLa by Gartler in 1968 (20). Those marked with an asterisk can be found in the Cell Biology Collection on the ATCC web site (www.atcc.org) where they are clearly marked as HeLa contaminants.

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Table 5. How Do Biological Contaminants Enter Cultures?
◗ Contact with nonsterile supplies, media, or solutions ◗ Particulate or aerosol fallout during culture manipulation, transportation, or incubation ◗ Swimming, crawling, or growing into culture vessels ◗ Accidents and mistakes

What Are the Sources of Biological Contaminants?
To reduce the frequency of biological contamination, it is important to know not only the nature and identity of the contaminants but also where they come from and how they gain entry into cultures. This section will detail some of the most common sources of biological contaminants (3).
Nonsterile Supplies, Media and Solutions
Unintentional use of nonsterile supplies, media or solutions during routine cell culture procedures is a major source of biological contaminants. These products may be contaminated as a result of improper sterilization or storage, or may become contaminated during use.
Glassware, including storage bottles and pipettes, is usually sterilized by autoclaving or dry heat sterilization. Serious contamination outbreaks are frequently traced to improper maintenance or operation of sterilization autoclaves and ovens. Packing too much into an autoclave or dry heat oven will cause uneven heating, resulting in pockets of nonsterile supplies. Using too short a sterilization cycle, especially for autoclaving volumes of liquids greater than 500 mL per vessel or solutions containing solids or viscous materials, such as agar or starches, is a common mistake. The size, mass, nature and volume of the materials to be sterilized must always be considered and the cycle time appropriately adjusted to achieve sterility (23). Then, once achieved, sterility must be maintained by properly storing the supplies and solutions in a dust- and insect-free area to prevent recontamination. Care must also be taken to avoid condensation on bottles of solutions stored in refrigerators and cold rooms. Of course,

good aseptic technique is also required to maintain the sterility of properly sterilized supplies and solutions once they are in use.
Plastic disposable cell culture vessels, pipettes, centrifuge tubes, etc. are usually sterilized by their manufacturer using a high intensity gamma or electron beam radiation source after they are sealed in their packaging. This is a very reliable process, however care must be taken when opening and resealing the packaging to avoid contaminating the products within.
Most media, sera and other animalderived biologicals are not heat sterilizable and require membrane filtration (Sometimes radiation is also used.) to remove biological contaminants. Products filter sterilized in your laboratory should always be tested for sterility before use (discussed in detail later); commercially produced sterile products are tested by the manufacturer before being sold. While filtration through 0.2 µm membranes is very effective in removing most biological contaminants, it cannot guarantee the complete removal of viruses and mycoplasmas, especially in sera (16,18,24). In an excellent review of the rates and sources of mycoplasma contamination (25), Barile and coworkers reported that 104 out of 395 lots (26%) of commercial fetal bovine sera tested were contaminated by mycoplasma. They concluded in the early 1970’s that animal sera were among the major sources of cell culture contamination by mycoplasma. Many sera manufacturers responded to this problem over the next decade by improving both filtration and testing procedures; they currently use serial filtration through at least three filter membranes rated at 0.1 µm or smaller to remove mycoplasmas. This approach has been very successful at reducing the problem of mycoplasma in sera and other animal-

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derived products (16). While these products are no longer a major source of mycoplasma contamination, they must still be considered as potential sources to be evaluated whenever mycoplasmas are detected in cultures.
Airborne Particles and Aerosols
In most laboratories, the greatest sources of microbial contamination are airborne particles and aerosols generated during culture manipulations. The microbial laden particles are relatively large (generally 4 to 28 µm in diameter) and settle at a rate of approximately one foot per minute in still air. As a result, the air in a sealed, draft-free room or laboratory (no people, open windows or doors, air handling units, air conditioners, etc.) is virtually free of biological contaminants. However as soon as people enter the room, particles that have settled out will be easily resuspended. In addition certain equipment and activities can generate large amounts of microbial laden particulates and aerosols: pipetting devices, vacuum pumps and aspirators, centrifuges, blenders, sonicators, and heat sources such as radiators, ovens, refrigerators and freezers. Animal care facilities and the animals they house are especially serious particle and aerosol generators, and should always be kept as far from the culture area as possible.
McGarrity used a cell culture that was intentionally infected with mycoplasma as a model to study how mycoplasmas are spread in a laminar flow hood during routine subculturing procedures (26). (This reference is especially recommended for a better understanding of how mycoplasma can be spread in a lab.) Following trypsinization of the infected culture in a laminar flow hood, live mycoplasma were isolated from the technician, the outside of the flask, a hemocytometer, the pipettor, and the outside of the pipette discard pan. Live mycoplasma could even be successfully recovered from the surface of the laminar flow hood four to six days later! A clean culture, that was subcultured once a week in the same hood following the work with the contaminated cells, tested positive for mycoplasma after only 6 weeks.

It is easy to understand from this study how the entry of a single mycoplasma infected culture into a laboratory can quickly lead to the infection of all the other cultures in the laboratory. This explains the frequent finding that if one culture in a laboratory is mycoplasma contaminated then usually most if not all of the other cultures will be as well. Currently, the major source of mycoplasma contamination is infected cultures acquired from other research laboratories or commercial suppliers.
Another major source of particulates and aerosols are laboratory personnel. Street clothes and dirty lab coats are dust magnets. Placing a dust-laden sleeve into a laminar flow hood generates a cloud of dust particles that can easily fall into and contaminate cultures during routine processing. Talking and sneezing can generate significant amounts of aerosols that have been shown to contain mycoplasma (26). Mouth pipetting is both a source of mycoplasma contamination and a hazard to personnel and must not be permitted under any circumstances. Dry, flaky skin is another source of contamination laden particles; this common condition is aggravated by the frequent hand washing required in the laboratory; even the lotions designed to moisten dry skin have occasionally been found to be contaminated. Some laboratory personnel shed yeastcontaining particles for several days following bread making or beer brewing at home. Attempts by these individuals at cell culturing during this period have routinely ended in failure due to yeast contamination.
Incubators, especially those maintained at high humidity levels, can be a significant source of biological contamination in the laboratory. Dirty water reservoirs, and shelves or culture vessels soiled by spilled media, allow the growth of spore-generating fungi. The fans used in many incubators to circulate the air and prevent temperature stratification can then spread these spores and other particulates. Some incubators humidify incoming gases by bubbling them through the water reservoirs at the bottom of the incubator; the

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Figures 5a and 5b. Photomicrographs of contaminants growing on the outside surfaces of culture vessels. Eventually, these organisms may grow into the culture.

aerosols generated by this will quickly spread any contaminants in the water.
While laminar hoods and incubators are the major sites where biological contamination occurs, transporting cultures between these two sites also provides opportunities for contamination. Most cell culture laboratories try very hard to keep their incubators and laminar flow areas clean, but sometimes they overlook the potential sources of contamination found in less clean laboratory areas transversed going from one location to the other. Rooms containing open windows, air conditioners, microbiology and molecular biology work areas, and the other major particle generators discussed above, add to the potential hazards of moving cultures around the laboratory. This problem increases both with the distance traveled and when the culture vessels are unsealed.
Swimming, Growing, and Crawling into Cultures
Unsealed culture plates and dishes, as well as flasks with loose caps to allow gas exchange, provide another common way for contaminants to enter cultures. It is very easy for the space between the top and bottom sidewalls of a dish, or a flask and its cap to become wet by capillary action with medium or condensation. This thin film of liquid then provides a liquid bridge or highway for microorganisms to either swim or grow into the culture vessel.
Even without any detectable film, fungi, as well as other microorganisms, can grow on the outside of culture vessels (Figures 5a and 5b); eventually their hyphae grow right up the side wall of the dish or past the cap into the neck of the flask. This is more often observed in long term cultures (a month or more) maintained in the same unsealed culture vessel. Small insects and other invertebrates can also make temporary visits into unsealed cultures, especially dishes and plates, leaving behind (unless they fall in and drown) only the contaminants carried on their feet.

Accidents
Accidents are often overlooked as a significant source of cell culture problems. An accident is defined as “an undesirable or unfortunate happening, unintentionally caused and usually resulting in harm, injury, damage or loss” (Webster’s Encyclopedic Unabridged Dictionary, 1989). Cell culture-related accidents are one of the leading causes of cross-contamination by other cell cultures. The following actual cases demonstrate how relatively simple accidents can result in serious cross-contamination problems:
◗ A technician retrieved a vial labeled WI-38 from a liquid nitrogen freezer thinking it contained the widely used diploid human cell line. Once in culture, it was immediately discovered to be a plant cell line derived from a common strain of tobacco called Wisconsin 38, also designated WI-38.
◗ Two separate research laboratories, both attempting to develop cell lines from primary cultures, shared a walk-in incubator. One lab used the acronyms HL-1, HL-2, etc. to identify the primary cultures they derived from human lung. The other lab worked with cultures derived from human liver, but they too (unknowingly) used the identical coding system. It wasn’t long before a culture mix up occurred between the two laboratories.
Fortunately, both of the above accidental cross-contamination cases, although serious, were caught before they caused catastrophic problems. But how many times have similar accidents occurred and not been caught? Based on continuing reports in the literature (7,8,19-22) many researchers have not been lucky enough to identify their mistakes.
The information presented above is designed to provide you with an increased awareness and understanding of the nature of biological and chemical contamination, and its serious consequences. The remaining sections will cover some basic ideas, techniques and strategies for actively detecting and combating cell culture contamination in your own laboratory.

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Understanding and Managing Cell Culture Contamination