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. 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 cell culture contamination has long been the dream of many researchers, but it is an impractical, if not impossible, dream. Contamination cannot be eliminated, but it can be managed to reduce both its frequency of occurrence and the seriousness of its consequences. The goal of this guide 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.

 

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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 Refs. 1-3].

Media and Components
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 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.

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 organics 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 pipets 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.

Storage Vessels
Media stored in glass or plastic bottles that have previously contained solutions of heavy metals or organics, 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 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 pipets, storage bottles and instruments.

Fluorescent Lights
An important but often overlooked source of chemical contamination results from the exposure of media containing HEPES (an organic buffer commonly used to supplement bicarbonate based buffers), riboflavin or tryptophan to normal fluorescent lighting. These media components can be photoactivated giving rise to 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.

Endotoxins
Endotoxins, the lipopolysaccaride-containing 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 be a significant source of experimental variability [Reviewed in Ref. 6]. [See also Corning Technical Guide:  Endotoxins and Cell Culture]  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, levels in serum have been significantly reduced by some manufacturers by handling the raw products under more 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.

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 an 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 [For a comprehensive review see Refs. 7 and 8] 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; and those that are more difficult to detect, and as a result potentially more serious culture problems, - viruses, Protozoa, insects, mycoplasmas and other cell lines. 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, they can usually be readily detected in a culture, either by direct microscopic observation or by the effects they have on the culture (pH shifts, turbidity, and cell destruction), within two days of it becoming contaminated. 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's 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, 7a]. Since cytopathic viruses usually destroy the cultures they infect, they tend to be self-limiting. Thus when cultures self-destruct for no apparent reason and no evidence of common biological contaminants can be found, cryptic viruses are often blamed. 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 precautions 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, Epstein-Barr, 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.   See also these sites for information on laboratory safety.

Protozoa
Both parasitic and free-living single celled Protozoa, such as amoebae, have occasionally been identified as cell culture contaminants. Usually of soil origin, amoebae can form spores and are readily isolated from the air, and 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, amoebae 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 multi-legged 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.

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 and two major culture testing companies, at least 11-15% of the cell cultures in the United States are currently infected by mycoplasmas (Table 3), and since many of these cultures were from laboratories that test routinely for mycoplasma, the actual rates are probably higher [11-13] in the many laboratories that don't test at all. In Europe, mycoplasma contamination levels were recently 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 appear 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. Thus the validity of any research done using these unknowingly infected cultures is questionable at best. (See References 11, 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: these simple, bacteria-like microbes are the smallest self-replicating organism known (0.3-0.8µm in diameter), lack a cell wall, and 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 (10⁷-10⁸ colony forming units/mL are common) often without any visible signs of contamination - no turbidity, pH changes or even cytopathic effects. Even careful microscopic observation of live cell cultures cannot detect their presence (See Figure 1). 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 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].

Figure 1.   Can you identify this cell line and tell if it is contaminated by mycoplasmas? (Photomicrograph courtesy of Bionique Testing Laboratories, Inc.)

 

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 cross contaminated by cells of other species [7]. In 1967 isoenzyme analysis was used to show that 20 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 in fact 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).

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, and 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 (See Figure 1). 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, 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.

 

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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 large volumes of liquids (greater than 500mL 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 must 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 animal-derived biologicals are not heat or radiation sterilizable and require membrane filtration to remove biological contaminants. Products filter sterilized in your laboratory should always be tested for sterility before use (Discussed in detail later); commercially obtained sterile products are tested by the manufacturer before being sold. While filtration through 0.2µm 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 and that (in the early 1970's) sera was one of 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 0.1µm or smaller to remove mycoplasmas. This approach has been very successful at reducing the problem of mycoplasma in sera and other animal-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 4-28µm 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 mycoplasma 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 pipet 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 mycoplasmas [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 yeast-containing 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 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 travelled 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 side walls 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 (See Figure 2); 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 labelled WI-38 from a liquid nitrogen freezer thinking it contained the well known diploid human cell line. Once in culture it was immediately discovered to be a plant cell line derived from an equally well known 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 contaminations, 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 as lucky.

The information presented above is designed to provide you with an increased awareness and understanding of the nature of biological and chemical contamination, and it serious consequences. The remaining sections will cover some basic ideas, techniques and strategies for actively detecting and combatting cell culture contamination in your own laboratory.

 

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How can Cell Culture Contamination be Controlled?

Cell cultures can be managed to reduce both the frequency and seriousness of culture-related problems, especially contamination. Lack of basic culture management procedures, especially in larger laboratories, frequently leads to long term problems, making contamination more likely for everyone. One solution is to actively manage your cultures to reduce problems and if necessary set up a program for others in your laboratory to use [27, 28]. This program should be designed to meet the needs of your specific working conditions and be based on the nature of your past cell culture problems; it can be very simple and informal, or more structured if required.

The first step in managing cultures is to determine the extent and nature of the culture losses in your lab. Everyone in the laboratory should keep an accurate record for a month or more of all problems, no matter how minor or insignificant, that result in the loss of any cultures. These problems may not only be contamination related but can also be from other causes such as incubator or equipment failures. Next, review the problems as a group to determine both their nature, seriousness and frequency. The group's findings may be surprising: what were thought to be individual and minor random occurrences of contamination often turn out to have a pattern and be more extensive than any individual realized. This problem sharing is often a painful process, but remember the goal is not to place blame but to appreciate the extent and nature of the problems confronting the laboratory. A critical part of this process is understanding the seriousness and actual costs of culture loss; placing a dollar value on these losses is often required before the full extent of the losses can be appreciated. It is very important for everyone in the laboratory to know the answers to the following questions:

1. How much time, money and effort has been invested in your cultures and experiments?
2. What are the consequences of their loss?
3. How expensive or difficult will it be to replace them?

Once the nature and consequences of the problems in the laboratory are better understood, the need for a management system, if necessary, can be determined. Basic problem solving tools [2] can be used to help identify the source of problems; changes to minimize or prevent the problems from reoccurring can then be implemented.

The following suggestions, concepts and strategies, combined with basic management techniques, can be used to reduce and control contamination (Table 6). These may require modification to fit your own needs and situation.

Use good aseptic techniques
Aseptic technique is designed to provide a barrier between microorganisms in the environment, and your cultures and sterile supplies, yet permit you to work with them. There are many successful techniques for achieving and maintaining aseptic cell cultures, ultimately your technique is 'good' if it routinely protects both you and your cultures from contamination. Teaching aseptic technique is beyond the scope of this guide; the goal here is to review some of its basic tenets and present some suggestions for improving it. The reader is referred to Freshney [3] for a basic introduction to this very important area.

The first step in developing sound, rational aseptic techniques is a solid understanding of both the nature and potential sources of biological contamination. This is reviewed in the beginning of this guide and covered in many of the references.

The second step, based on the nature of your work, is to determine the level of risk or danger to yourself and other laboratory personnel and then design your culture techniques accordingly. This is especially true when working with cultures known to be virally contaminated or derived from human and other primate sources. Ensure that all laboratory personnel have been trained in the safe handling and disposal of any potentially hazardous cultures and materials; refer to your facility's safety office for any necessary assistance or guidance [9].

Next, based on the potential costs and consequences if the cultures are lost, determine how rigorous your technique must be, and what degree of redundancy if any, is required. Very valuable or irreplaceable cultures can be carried by two or more workers using media from different sources and separate incubators to reduce the chance of their simultaneous loss [27, 28]. Evaluate whether workers need to be gloved, gowned and masked to reduce the potential for contamination.

The nature of your working environment and any problems it may present must also be considered in choosing appropriate aseptic techniques. Certified laminar flow hoods and safety cabinets are recommended for use whenever possible. Some of the techniques taught in introductory microbiology classes for use on the open bench, such as flaming, while popular, may not be appropriate or necessary in laminar flow hoods [16]. Hood manufacturers recommend against the use of Bunsen burners and other sources of flames in hoods; they disrupt the moving curtain of filtered air and the resulting turbulence can increase the probability of contamination by microbial laden aerosols and particles generated during routine culture manipulations.

The following suggestions are recommended to reduce the probability of contamination:

- Make it more difficult for microorganisms to gain entry by using sealed culture vessels whenever possible, especially for long term cultures. The sides of multiple well plates can be sealed with labelling tape, 35mm and 60mm dishes can be placed inside 150mm dishes, 100mm dishes placed in plastic boxes, and the caps on flasks sealed. Vented cap flasks (See Figure 3) use hydrophobic filter membranes to allow gas exchange but prevent the passage of microorganisms or liquids.

- Avoid pouring media from cell culture flasks or sterile bottles by using 50mL pipets to transfer larger volumes. A drop of medium remaining on the vessel's threads after pouring can form a liquid bridge when the cap is replaced providing a means of entry for bacteria, yeasts and molds. If pouring cannot be avoided, carefully remove any traces of media from the neck of the vessel with a sterile gauze pad.

- Always carry unsealed cultures in trays or boxes to minimize contact with airborne contaminants.

- Don't use the hood as a storage area. Storing unnecessary boxes, bottles, cans etc. in the hood, besides adding to the bioburden, disrupts the air flow patterns.

- Never mouth pipet. Besides the risk of injury to laboratory personnel, mouth pipetting has been implicated as the likely source of human mycoplasma species (M. orale and M. salivarium) often found in cell cultures [15].

- Use clean long sleeve lab coats or other protective clothing to protect against shedding contaminants from skin or clothes. Their use should be restricted to the cell culture area to avoid exposure to dirt and dust from other areas.

- Work with only one cell line at a time in the hood, and always use separate bottles of media, solutions, etc. for each to avoid possible cross-contamination. Use disinfectant to wipe down the hood's work surfaces between cell lines.

- Use antibiotic-free media for all routine culture work; this is a very important concept and will be discussed in detail below.

- Whenever possible package sterile solutions, such as trypsin, L-glutamine and antibiotics, in small volumes (i.e. stored in 15ml tubes) to reduce the number of times they must be used and thus reduce the probability of contamination.

Reduce opportunities for accidents
Accidents usually involve people, and reducing them must take into consideration both human nature and stress. Based on personal experience, accidents are far more likely on Friday afternoons, the day before a vacation begins, with new employees, and when people are stressed, overworked or rushed. The following suggestions can help reduce the confusion and misunderstanding that cause many accidents to happen in the laboratory.

- Be very careful when labelling solutions, cultures etc. Always clearly indicate if solutions or other supplies have been sterilized. Reduce misunderstandings in crowded or busy labs by using a color coding system: assign each worker their own color for labelling tape and marking pen inks.

- Be very careful with the use and choice of acronyms. Everyone in the laboratory should understand and agree to their meaning.

-Whenever possible use standardized recordkeeping forms; this simplifies their use and makes it more likely that good records will be kept.

- Use written protocols and formulation sheets when preparing media and solutions, listing the reagents used, lot numbers, weights and volumes, pH and any special treatments that were done. These will both reduce the potential for errors as well as provide a valuable aid in tracking down the cause of problems.

Clean up the work area and surrounding environment
Reducing the amount of airborne particulates and aerosols in the laboratory, especially around the incubator and the laminar flow hood, will reduce the amount of contamination. Routinely wipe floors and work surfaces to keep down dust. Incubators, especially those that maintain high humidity levels, require periodic cleaning and disinfecting. An often overlooked but important source of contaminants are the water baths used to thaw sera and warm media. Dirty water baths not only coat bottles with a layer of heavily contaminated water right before they are placed under the hood, but the water dripping from bottles generates heavily contaminated aerosols which can end up on lab coats and hands. Water baths should be emptied and cleaned on a regular basis, well before odor or visible turbidity develops. Pipet disposal trays and buckets, and other waste containers provide a source of potentially heavily contaminated materials in close proximity to the laminar flow hood and are a potential mycoplasma source [26]. Waste containers should be emptied daily and the wastes disposed of safely; autoclaving of any wastes that have been in contact with cells is recommended. The cooling coils on refrigerators and freezers are a major source of microbial laden airborne particulates that are often overlooked in otherwise very clean laboratories. These should be vacuumed at least yearly; besides removing a significant source of contamination, regular vacuuming will extend the life of the cooling units and allow them to run more efficiently. Some laboratories may also need to consider a pest management program to reduce the presence of mice, ants, cockroaches and other multi-legged creatures that can be sources of contamination. Potted plants, although attractive, can provide a home to these creatures and should not be kept in the culture vicinity. Care must be taken when using pesticides as part of a pest management program to prevent accidentally chemically contaminating the cultures in the laboratory.

Sterility Testing
The best strategy for reducing contamination is to be proactive by routinely monitoring supplies, media and solutions, work areas and, most importantly, cell cultures for contaminants before they are used in critical applications and experiments. The key to developing a realistic contamination monitoring program is to keep it as simple as possible so that people use it, yet ensure that it can get the job done. Unfortunately there are no easy solutions: no single microbiological medium can detect all types of biological contaminants, and practical testing methods often miss low levels of contaminants. The process of detection is made even more difficult by the presence of antibiotics. The techniques and concepts presented below offer some practical approaches for monitoring contamination that can be readily adapted to meet the needs of most cell culture laboratories.

All autoclaves and dry heat ovens used to sterilize glassware, solutions and other supplies must be regularly maintained, and personnel properly trained in their loading and operation. Thermometers and chart recorders should be tested and calibrated periodically to ensure their accuracy. Inexpensive (when compared to the cost of a single autoclave failure) autoclave thermometers, spore test strips and capsules, or other testing devices can be placed inside autoclaves or into bottles of solutions or other packaged supplies during every run, or as necessary, to ensure proper loading and operation.

Samples of all in-house filter-sterilized solutions should be tested for sterility each time they are prepared and the solutions not used until testing is complete. Standard microbiological testing methods for bacteria, yeasts and fungi usually require placing samples for testing into several different broths (trypticase soy, thioglycolate and sabouraud broths) and semi solid media (brain-heart infusion, blood agar), and incubating them at both 30° and 37°C for at least two weeks [29].

Cell culture media, especially media that are outdated or no longer used in the lab (as long as they do not contain any antibiotics) can provide a very rich, readily available and useful substitute for standard microbiological media. A small amount of serum (3 to 5% - again outdated or unwanted sera can be used) should be added to promote growth. The medium can be dispensed in 10mL amounts into sterile 16mm by 125mm glass screw cap tubes or clear 15mL plastic centrifuge tubes and be stored at 4°C until needed. The sterility of filtered solutions, and cultures or products suspected of being contaminated, can be routinely and easily checked by placing a small samples into two tubes and incubating them at 30° and 37°C for at least two weeks.

This sterility test media substitute is also very useful for evaluating the amount or source of particulate contamination in an area, near a piece of equipment or by a technique. Hoods, and especially incubators, are frequently blamed by laboratory personnel as the source of their contamination problems as in: "my cultures keep getting contaminated because something is wrong with the hood" (or incubator). Until these areas are screened and eliminated as the source of the problem, the real problem, often simply aseptic technique, can not be dealt with effectively. These suspected problem areas can be screened by dispensing the test medium into 96 well culture plates or 100mm culture dishes (use agar-gelled media for the dishes). The vessels are then opened (with unopened vessels as controls) for 30 to 60 minutes at several locations within the test site prior to being sealed and incubated. Cultures can be initially checked for contamination after two to three days although slow growing contaminants may take two weeks or longer to appear. The rate of contamination (number of colonies or contaminated wells/vessel or unit area/unit time) can then be calculated and analyzed. Besides giving an accurate level of the bioburden in that area, microscopic observation of the contaminants in the liquid test media also allows their morphological comparison with the microorganisms found causing problems in the cell cultures. Past experience with this approach has shown it is a very useful tool when teaching aseptic technique as it clearly demonstrates that the air in a room, or even inside a humidified incubator is usually not a major source of contamination in a well maintained laboratory. It is also a useful tool in tracking down mysterious contamination outbreaks.

Detecting mycoplasma in cultures
No monitoring program is complete unless it can effectively detect contaminated cultures, especially those infected by mycoplasma. Unfortunately mycoplasma detection is not simple, and because of this, and a lack of awareness, many cell culture users (as many as 50%, see survey results in Table 7) simply don't bother to test. As a result mycoplasma contamination rates in the United States are estimated to be at least 15%. Because of these outrageously high levels of contamination and the proven ease with which mycoplasmas can be spread from contaminated cultures [26], it is very important to quarantine all cultures coming into the laboratory until they have been tested for mycoplasma. This is especially true of gifts of cell lines from other labs, often these 'gifts' end up infecting your cultures.

There are two basic testing methods for mycoplasma, direct culture in media and indirect tests that measure specific characteristics of mycoplasmas [16]. Direct culture is the most effective and sensitive method for detecting mycoplasma, but it is also the most difficult and time consuming. It requires several carefully tested liquid and semisolid media and controlled environmental conditions [See Ref. 30 for detailed protocols], and must be run with live mycoplasma controls. Additionally, although direct culture is the most sensitive method, it is the slowest (requiring up to 28 days) and it cannot reliably detect some fastidious strains of mycoplasma, making it less than 100% effective. Budget permitting, direct culture testing is best contracted to an outside testing facility for two reasons: first, given the ease with which mycoplasma can spread in the laboratory, bringing live mycoplasma into a cell culture facility for the required controls is not recommended; second to do it well, direct testing requires a serious effort and commitment of resources better spent in doing cell culture. These tests are commercially available at a reasonable cost from several cell culture testing companies.

There are a wide variety of indirect test methods available, including DNA probes, DNA fluorochrome staining, autoradiography, ELISA, immunofluorescence and specific biochemical assays. These tests are faster than direct culture, all are commercial available in kit form, and they can detect the fastidious, difficult to cultivate strains that are occasionally missed by direct culture. However they lack the sensitivity of direct culture, requiring much higher levels of contamination for detection. As a result, they have more frequent false negatives than direct culture methods, potentially leaving researchers who rely solely on a single indirect test with a false sense of security [Reviewed in Refs. 11, 18].

The most widely used and recommended indirect test is DNA fluorochrome staining [see 31 for detailed protocols]. This easy and relatively fast procedure stains all DNA using a fluorescent dye. When stained and fixed cells are examined under a UV microscope equipped with the proper filter package, DNA fluoresces brightly (See Figure 4). Not only will this test detect mycoplasma but as an added benefit it will also detect any other microbial contaminants. This staining method can be combined with an indicator cell line to increase its sensitivity. Interpreting results is not always easy, especially with hybridoma cultures; suitable positive and negative control slides should always be used to help interpret staining results. These positive and negative mycoplasma control slides are commercially available; since they have already been fixed, they are safe to use in the laboratory.

The best overall testing approach is a combination of both methods: direct culture can provide very high sensitivity while DNA fluorochrome staining can detect any fastidious mycoplasma that the direct culture misses. This combined approach is required by both the FDA and USDA for cell culture derived products, such as monoclonal antibodies, vaccines and drugs, and the cells required to produce them. If resources do not permit the combined approach, then the DNA fluorochrome staining procedure using an indicator cell line, combined with one other indirect test method should provide a minimum level of security.

Detecting other biological contaminants in cultures
The traditional microbiological media described earlier for testing the sterility of solutions can be adapted for testing cultures for bacteria, yeasts and fungi [29]. However, although not designed for this purpose, the direct culture tests and the indirect DNA fluorochrome test for mycoplasma will also detect most bacteria, yeasts and fungi, including intracellular forms, reducing the need for the traditional tests. Special culture procedures are also available for detecting suspected protozoan contaminants in culture [Details in Ref. 32].

There are several other important quality control tests that should be used to both identify and characterize the cell cultures used in your research. Besides the serious and widespread problem of cross contamination by other cell lines described earlier, cells are also continually evolving in culture: important characteristics can be lost, mutations can occur, or chromosomes can undergo rearrangements or changes in number. Monitoring these changes is important because altered cell cultures can have a significant impact on the reproducibility of your research [Reviewed in Ref. 33]. The following characterization methods are recommended for monitoring cell cultures; refer to the cited references for details. Most laboratories should incorporate at least one of these methods as part of their monitoring program:

- Karyotyping, a relatively simple method used to determine the modal chromosome number and presence of any unique marker chromosomes [34].

- Electrophoresis and isoenzyme analysis to generate a protein 'fingerprint' that can be used to determine species or for future comparisons [33].

- Immunological or biochemical techniques to detect markers that are unique to the tissue, cell line or the species from which it is derived [33].

- DNA fingerprinting, a relatively new technique but one that is becoming increasingly useful, can be used to detect both intra- and interspecies contamination [35].

The results from these characterization tests can serve as an important baseline against which any future changes can be compared.

Recommendations for a testing program
The cell culture testing program you choose should be the best you can afford as it is the cornerstone of your research. An inadequate program (or worse no program at all) provides a false sense of security and can eventually lead to failure compromising the validity of your research. The following steps are recommended for setting up a sound yet practical culture monitoring program:

- Test all current in-house cell lines using the methods described above to ensure they are free from mycoplasma and other microbial contaminants, and to check their identity. Then incorporate these tested cultures into your cell repository and rely only on them for all future experiments.

- Quarantine and then test all incoming cell lines and any cultures currently stored in your cell respository that were not tested when they were frozen.

- Test all cell lines that are in continuous use at least every three to four months and any time they behave suspicious. Better yet, save time, money and effort by periodically discarding these cultures and replacing them with cultures from your tested cell repository. (This strategy will be discussed in detail later in the section on using a cell repository.)

- New lots of sera should be evaluated before widespread use.   The simplest test method is to use the new serum in an indicator cell culture for several weeks and then test the culture for mycoplasma contamination using DNA staining or other suitable test.

Detecting chemical contaminants
Determining that a chemical contaminant is the cause of a cell culture problem is usually much more difficult than with biological contaminants because it is so hard to detect. Often the first signs that something is wrong are widespread alterations in the growth, behavior or morphology of the cultures in the laboratory; however, it may take weeks before these changes are noticed. Once noticed, the cause is frequently misconstrued to be of biological origin; only after extensive and unsuccessful testing for the usual microbial suspects does attention focus on the possibility it might be a chemical contaminant.

Begin the problem solving process by identifying all changes that have occurred in the lab in the weeks prior to the problem being noticed, especially in equipment, solutions, media and supplies, that may be related to the problem. Good record keeping is essential for this process to be successful. Bring together laboratory personnel to brainstorm for all of the possible causes and then select the best possibilities for evaluation. Simple comparison experiments can then be done to eliminate each possibility as the source of the problem; media, solutions, sera and other products to use as controls in the testing can be obtained from other labs or sources. The best way to avoid chemical contamination is to test all new lots of reagents, media and especially sera, and test the water purity at least yearly using the most sensitive culture assay available.

Strategic use of a frozen cell repository
A cryogenic cell repository is commonly used in laboratories to reduce the need to carry large numbers of cultures and to provide replacements for cultures lost to contamination or accidents. Freezing cultures also stops biological time for them, preventing them from acquiring the altered characteristics that can normally occur in actively growing cells as a result of environmental or age related changes. However a cell repository is only a reliable resource if the cultures it contains have been properly tested, labelled and stored [Reviewed in Ref. 36]. [See also the Corning Technical Guide:  General Guide for Cryogenically Storing Animal Cell Cultures.]

Equally important, a cell repository can also be used strategically to convert continuously carried cultures into a series of short term cultures, thereby greatly reducing both the amount of quality control testing required and potential problems from cryptic contaminants. When cultures are continuously carried for long periods in the laboratory they should be tested for contaminants at least every three to four months (more often for critical applications). If they are not tested regularly, then when a cryptic contaminant, such as a mycoplasma or another cell line, is finally uncovered, it is impossible to determine how long it has been in the culture and how much research has been invalidated by its presence. In addition, if the contaminant is mycoplasma, it is likely that it may have spread to other cultures. However regular testing, although very important to ensure the integrity of your cultures, can require considerable effort, especially in laboratories using multiple cell lines. Rather than test cultures several times a year, it is easier to simply discard them every three months replacing them from the repository with cultures that have been previously tested to ensure their integrity.

Tested stocks should be set up in the cell repository for each culture that is routinely used in your laboratory. The cultures should be grown for at least two weeks in antibiotic-free media, then thoroughly tested to check their viability, ensure they are free of contamination, and confirm their identity and presence of any important characteristics. Testing should be done both immediately before and after freezing; however if you don't mind assuming some added risk, testing can be left until after freezing. The freezer stock should always be prepared from pooled cultures and contain enough vials, assuming a consumption rate of five vials per year (or higher based on your experience), to last the planned lifetime of any research projects involving them. A better alternative may be to first develop a seed stock (10-20 vials is usually sufficient, depending on your envisioned needs), and then from that develop a working stock (approximately 20 vials). When the original working stock is depleted, it is replaced by using a vial from the seed stock to develop a new working stock. Assuming a consumption rate of five vials per year, each working stock will be good for four years, with the seed stock lasting for 40 to 80 years, long enough to finish most research projects. This approach reduces the amount of routine testing (usually only newly introduced cultures will require testing) to practical levels and, equally important, destroys any undiscovered biological contaminants that may have gained access to the cultures, limiting both their damage to the integrity of the research and their spread to other cultures.

Strategic use of antibiotics
Antibiotics are a useful tool in cell culture when used intelligently but a very dangerous one when overused or used incorrectly. Experienced cell culture users have recommended for many years that antibiotics never be used routinely in culture media [3, 7, 17, 18, 26, 27]. Barile found in one study that 72% of cultures grown continuously in antibiotics were contaminated by mycoplasma, but only 7% grown without antibiotics, a ten-fold difference [37]. Similar results are common: workers who routinely and continuously use antibiotics in their media tend to have higher contamination problems, including mycoplasma, than workers who don't. Over reliance on antibiotics leads to poor aseptic technique. It also leads to increased antibiotic resistance among common culture contaminants: in an ongoing study [12] of the antibiotic sensitivity of culture-derived mycoplasmas, 80% were resistant to gentamycin, 98% to erythromycin, and 73% to kanamycin, all commonly used antibiotics widely claimed to be effective against mycoplasmas. Mycoplasmas also showed resistance to the antibiotics recommended and sold specifically for cleaning up mycoplasma infected cultures: 15% were resistant to ciprofloxacin, 28% to lincomycin, and 21% to tylosin.

Why does the routine use of antibiotics lead to higher rates of mycoplasma contamination? Everyone generates and sheds a relatively constant flow of particles, consisting of fibers, aerosols and droplets, as they work in the laboratory. These particles can have a mixture of bacteria, yeast, fungi, and even mycoplasmas bound to them. If one of these contamination laden particles enters an antibiotic-free culture, the chances are that at least one of the contaminants will produce a highly visible infection within 24 to 48 hours. As a result the contaminant is quickly detected and the culture discarded. It is very unlikely that particles shed by laboratory personnel would ever consist of just difficult to detect contaminants, such as mycoplasmas, that could enter cultures and not cause visible signs of contamination. However if the culture contains antibiotics, there is a chance that the antibiotics will prevent the growth of the usually more easily detected contaminants but allow mycoplasma or other cryptic contaminants to grow undetected. As a result, instead of being discarded, the cryptically infected culture remains in use, is utilized in experiments, and becomes a potential source of serious contamination for the other cultures in the laboratory.

Antibiotics should never be used as a substitute for good aseptic technique, however they can be used strategically to reduce the loss of critical experiments and cultures. The key is to use them only for short term applications: for the first week or two of primary cultures, during the initial production stages of hybridomas, for experiments in general where the cultures will be terminated in the end. Whatever their use, the antibiotics ultimately chosen should be proven effective, noncytotoxic and stable [37].

Curing contaminated cultures
Autoclaving is the preferred method for dealing with contaminated cultures - it always works and is guaranteed to keep the infection from spreading to other cultures. However, occasionally contamination will be found in a valuable culture that cannot be replaced and attempts will be made to save it. This is a task that should not be undertaken lightly as it usually entails considerable effort and frequently turns out unsuccessful. In addition, cultures can lose important characteristics as a result of the clean up procedure. If the contaminant is a fungus or yeast success is unlikely since amphotericin B (Fungizone) and nystatin will not kill these organisms, but only prevent their growth. Many bacterial culture contaminants come from human or animal sources and are likely to have developed resistance to most commonly used cell culture antibiotics.

Most clean up attempts however are usually made against mycoplasma infected cultures. Treating with antibiotics is the most widely used approach, but as discussed earlier, cell culture mycoplasma strains are often resistant to some of the antibiotics specifically recommended for cleaning up mycoplasma infected cultures. Furthermore, the more attempts made at cleaning up contaminated cultures with these antibiotics the more likely resistant mycoplasma strains will develop. Other approaches, usually combining the use of antibiotics with specific antisera or other chemical treatments, can be used as well [Reviewed in Refs. 11, 16, 37], however none of these methods are 100% successful and clean up should only be tried as a last resort. A word of caution, often these treatments reduce the level of contamination below that which can be detected by indirect methods such as DNA staining. As a result, clean up attempts often appear successful for the first month or more following treatment because the low level of surviving mycoplasmas can escape detection. But eventually the few remaining undetected mycoplasmas recover leading to more serious problems. Budget permitting, there is at least one commercially available mycoplasma clean up service for contaminated cultures, it is relatively expensive but usually successful [17].

A Final Warning

In the United States alone, losses due to cell culture contamination, especially by mycoplasma, are estimated to cost cell culture users millions of dollars annually; money that could otherwise be used for additional research. Unfortunately this serious problem does not appear to be getting any better. As shown by the survey results below (Table 7), contamination is a problem for most cell culture workers. At least 23% of respondents have experienced mycoplasma contamination of their cultures, but an additional 44% suspected mycoplasma contamination but were not sure. The reason for their uncertainty is clarified by the response to the next question: 50% of all respondents do not test for mycoplasma, as a result they are unaware of the status of their cultures. The answer to the last question points out one important reason for widespread contamination problems - over use of antibiotics; with 65% of respondents using antibiotics on a regular basis, the continued frequent occurrence of cryptic contaminants, especially mycoplasmas, are likely.

Because of the very serious nature of mycoplasma contamination and its widespread distribution, it is important to summarize the major sources of mycoplasma contamination and review the basic steps for preventing it from happening in your laboratory. The number one source of mycoplasma contamination is currently other infected cell lines; it is essential to quarantine all cultures brought into the laboratory until they have been screened for mycoplasma contamination, and to use only tested cultures in research. The second common source is the cell culturist; good aseptic technique combined with the strategic use of a tested cell repository and limited use of antibiotics will greatly reduce the opportunities for contamination via this route. The last important source of mycoplasma is sera and other biologicals that are sterilized by filtration; buy only from sources that have a proven track record and that use currently acceptable filtration (0.1µm or smaller) and testing procedures.

Cell culture contamination will never be totally eliminated, but through a better understanding of the nature of contamination and the implementation of some basic concepts it can be better controlled and its damage greatly reduced. The information in this guide has been compiled to provide you with the basic foundation (Table 8) upon which you can build a contamination management program designed to fit your own needs.

For additional assistance in these areas, please contact Corning, Inc. Technical Information Center at 800-222-7740.

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