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A photograph of bacteria used to help identify contamination in cell culture.
Bacteria (cocci) @ 320x magnification. In this example, the cells are the larger refractile bodies, while the bacteria appear as very small dark “dots” in the spaces between the cells.
A photo of bacteria rods used to help identify contamination in cell culture.
Bacteria (rods) @ high conc. between cells
A photo of small cocci used to help identify contamination in cell culture.
Bacteria (cocci) between cells
Description: There are three principle forms of bacteria, round-shaped (cocci), rod-shaped (bacilli), and spiral-shaped (spirilla). An average rod-shaped bacterium measures about 1 um in diameter and 4 um in length. However, bacteria vary considerably in size ranging from 0.5 to 1.0 um in diameter to 10 to 20 um in length in some of the longer spiral forms. Bacteria are not easily distinguishable under low power microscopy (~100X), but some indications of contamination are notable. The space between cells will appear uniformly granular and may shimmer from the movement of the bacteria. Also, flasks and cell culture reagents may become cloudy and develop a slight film on the surface of the growth vessel or reagent bottle that dissipates when the vessel is moved. High power microscopy (~400X) allows the resolution of individual bacterium which typically reveals cocci or bacilli contamination in the cell culture environment. Bacterial contamination is sometimes confused with precipitates of media constituents such as serum proteins or cellular debris. However, a regular particulate morphology and motility distinguish bacteria.Characteristics in mammalian cell cultures: Bacterial contamination will usually cause sudden changes in pH. If a culture is contaminated with aerobic bacteria, then the medium will become acidic and appear yellow. Most cases of bacterial contamination in the cell culture laboratory are caused by aerobes. However, if the bacteria are anaerobic, the contamination will cause the medium to become basic and will appear pink. Bacteria often produce toxins that disrupt cell function and ultimately destroy cell cultures. If at all possible, we recommend discarding contaminated cultures immediately. If caught early, it is possible to eradicate bacterial contamination with antibiotics. However, bacterial contamination is not easily treatable once the bacteria begin log phase of growth.

Typical routes of infection in cultures: Possible sources of contamination are innumerable, but the most likely source of bacterial contamination in the cell culture working environment is the water bath. We recommend that the water bath be emptied and cleaned every month. We recommend that the bath be filled with sterile ddH2O and 0.05% benzalconium chloride to inhibit microbial growth. Also, the humid environment of the CO2 incubator provides ideal growth conditions for many strains of bacteria. We recommend that the incubator be cleaned out once a month with a chemical disinfectant such as 70% ethanol and/or 1% benzalconium chloride. Also, it is beneficial to autoclave shelves and water pans. Water for humidification should be as recommended above for water baths. Other routes of infection to be aware of include: contact with non-sterile surfaces when performing cell culture manipulations, spillage on materials and the work surface, splash-back from pipetting or pouring cell suspensions and reagents, microscopic aerosols and infestation by insects, mites or dust into supplies.

Antibiotics: The most commonly used antibiotics in cell culture are penicillin/streptomycin (pen/strep), gentamicin, kanamycin, and ampicillin. All four are effective against both gram-positive and gram-negative bacteria (pen/strep only when used together). Gentamicin and kanamycin are also marginally effective against some strains of mycoplasma. Typically, pen/strep are used together and gentamicin and kanamycin can be used together. Avoid using cocktails of many antibiotics because all are, to some extent, slightly toxic in media and too many used together can result in a cumulative toxic effect on cells. A very useful list of antibiotics, the organisms they are effective against, and recommended working concentrations compiled by the Sigma-Aldrich Company can be found as a pdf document (129Kb, 3 pages).

Antibiotic List