Class II Facility
Introduction
What causes biological contamination?
Poor Aseptic technique is the number 1 cause of cell culture contamination
Biological contaminants can be divided into two subgroups depending on the ease of detecting them in cultures, with the easiest being most bacteria and fungi. Those that are more difficult to detect, and thus present potentially more serious problems, include Mycoplasmas, viruses, and cross-contamination by other mammalian cells.
Bacteria and fungi
Bacteria and fungi, including moulds and yeasts, are ubiquitous in the environment and are able to quickly contaminate cell cultures. Their small size and fast growth rates make these microbes the most commonly encountered cell culture contaminants. In the absence of antibiotics, bacteria can usually be detected in a culture within a few days of contamination, either by microscopic observation or by their direct effects on the culture (pH shifts, turbidity, and cell death). Yeasts generally cause the growth medium to become very cloudy or turbid, whereas molds will produce branched mycelium, which eventually appear as furry clumps floating in the medium.
Mycoplasmas
Mycoplasmas are certainly the most serious and widespread of all the biological contaminants, due to their low detection rates and their effect on mammalian cells. Although mycoplasmas are technically bacteria, they possess certain characteristics that make them unique. They are much smaller than most bacteria (0.15 to 0.3 μm), so they can grow to very high densities without any visible signs. They also lack a cell wall, and that, combined with their small size, means that they can sometimes slip through the pores of filter membranes used in sterilization. Since the most common antibiotics target bacterial cell walls, mycoplasmas are resistant.
Mycoplasmas are extremely detrimental to any cell culture: they affect the host cells’ metabolism and morphology, cause chromosomal aberrations and damage, and can provoke cytopathic responses, rendering any data from contaminated cultures unreliable. In Europe, mycoplasma contamination levels have been found to be extremely high—between 25 percent and 40 percent—and reported rates in Japan have been as high as 80 percent. Statistics show that laboratories that routinely test for mycoplasma contamination have much lower incidence; once detected, contamination can be contained and eliminated. Testing for mycoplasma should be performed at least once per month, and the Department of Bioengineering runs these for the users.
Viruses
Like mycoplasmas, viruses do not provide visual cues to their presence; they do not change the pH of the culture medium or result in turbidity. Since viruses use their host for replication, drugs used to block viruses can also be highly toxic for the cells being cultured. Viruses that cause damage to the host cell do, however, tend to be self-limiting, so the major concern for viral contamination is their potential for infecting laboratory personnel. Those working with human or other primate cells must use extra safety precautions.
Other mammalian cell types
Cross-contamination of a cell culture with other cell types is a serious problem that has only recently been considered alarming. An estimated 15 percent to 20 percent of cell lines currently in use are misidentified. Such contamination clearly poses a problem for the quality of research produced, and the use of cultures containing the wrong cell types can lead to retraction of published results.
Sources of biological contaminants in the lab
In order to reduce the frequency of biological contamination, it is important to understand how biological contaminants can enter culture dishes. In most laboratories, the greatest sources of microbes are those that accompany laboratory personnel. These are circulated as airborne particles and aerosols during normal lab work. Talking, sneezing, and coughing can generate significant amounts of aerosols. Clothing can also harbor and transport a range of microorganisms from outside the lab, so it is crucial to wear lab coats when working in the cell culture lab. Even simply moving around the lab can create air movement, so the room must be cleaned often to reduce dust particles.
Certain laboratory equipment, such as pipetting devices, vortexers, or centrifuges without biocontainment vessels, can generate large amounts of microbial-laden particulates and aerosols. Frequently used laboratory equipment, including water baths, refrigerators, microscopes, and cold storage rooms, are also reservoirs for microbes and fungi. Improperly cleaned and maintained incubators can serve as an acceptable home for fungi and bacteria. Overcrowding of materials in the autoclave during sterilization can also result in incomplete elimination of microbes.
Culture media, bovine sera, reagents, and plasticware can also be major sources of biological contaminants. While commercial testing methods are much improved over those of earlier decades, it is paramount to use materials that are certified for cell culture use. Cross-contamination can occur when working with multiple cell lines at the same time. Each cell type should have its own solutions and supplies and should be manipulated separately from other cells. Unintentional use of nonsterile supplies, media, or solutions during routine cell culture procedures is the major source of microbial spread.
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