Contamination

Contamination

The presence of microorganisms can inhibit cell growth, kill cells, and lead to inconsistent results. Contamination of cell cultures can occur with both cell culture novices and experts.

Potential contamination routes are numerous. For example, cultures can be infected through poor handling, from contaminated media, reagents, and equipment (e.g., pipets), and from microorganisms present in incubators, refrigerators, and laminar flow hoods, as well as on the skin of the worker and in cultures coming from other laboratories.

Bacteria, yeasts, fungi, molds, mycoplasmas, and other cell cultures are common contaminants in animal cell culture. To safeguard against accidental cell culture loss by contamination, we recommend freezing aliquots of cultured cells to re-establish the culture if necessary (see Freezing and viability staining of cells).

The characteristic features of microbial contamination are presented in the table Characteristic features of microbial contamination. The presence of an infectious agent sometimes can be detected by turbidity and a sharp change in the pH of the medium (usually indicated by a change in the color of the medium), and/or cell culture death. However, for some infections, no turbidity is observed and adverse effects on the cells are not easily observed.

Cell cultures should be routinely evaluated for contamination. Mycoplasmal infections are one of the more common and difficult-to-detect infections; their detection and eradication are described in further detail below.

Characteristic features of microbial contamination
Characteristic Bacteria Yeast Fungi 
Change in pH pH drop with
most infections
pH change with
heavy infections
pH changes
sometimes
Cloudy medium:
Under microscope
(100–400x)
Shimmering in spaces
between cells; rods or
cocci may be observed
Round or ovoid
particles that bud off
smaller particles
Thin filamentous mycelia;
sometimes clumps of spores

Mycoplasmas are small, slow-growing prokaryotes that lack a cell wall and commonly infect cell cultures. They are generally unaffected by the antibiotics commonly used against bacteria and fungi. Furthermore, as mycoplasma do not overgrow cell cultures and typically do not cause turbidity, they can go undetected for long periods of time and can easily spread to other cell cultures. The negative effects of mycoplasmal contamination include inhibition of metabolism and growth, as well as interference with nucleic acid synthesis and cell antigenicity. Acute infection causes total deterioration of the cell culture, sometimes with a few apparently resistant colonies that may, in fact, also be chronically infected. There are two main approaches to detect mycoplasma — Hoechst 33258 staining (1, 3) and mycoplasma-specific DNA probes. Alternatively, a PCR-based, mycoplasma-testing service is offered by the ATCC or other organizations on a fee-for-service basis.
The best action to take with a culture containing chronic mycoplasmal infection is to discard it by either autoclaving or incineration. Only if the cell culture is absolutely irreplaceable should eradication be attempted. This process should be performed by experienced personnel in an isolated hood that is not used for cell culture, preferably in a separate room. Elimination of mycoplasma is commonly achieved by treatment with various commercially available antibiotics such as a quinolone derivative (Mycoplasma Removal Agent), ciprofolxacin (Ciprobay), enrofloxacin (Baytril), and a combination of tiamulin and minocycline (BM-Cyclin). Treatment procedures and appropriate antibiotic concentrations can be found in the suppliers’ instructions and in references 1 and 3.
Cross-contamination of one cell culture with fast-growing cells from another culture (such as HeLa) presents a serious risk. To avoid cross-contamination, only use cell lines from a reputable cell bank; only work with one cell line at a time in the hood; use different pipets, bottles of reagents, and bottles of media for different cell lines; and check cells regularly for the correct morphological and growth characteristics
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