Contamination and Biosafety

Animal Cell Culture Guide Contamination and Biosafety

Table of Contents


Contamination of cells in culture can arise from many sources including other cell lines, reagents, supplies such as pipettes and culture vessels, equipment such as tissue culture hoods and incubators, and laboratory personnel. While the potential for contamination is constant, the risk can be reduced or eliminated by proper precautions: using only reagents of known quality and sterility, quarantining new cell lines until they are tested to be free from contamination, performing routine maintenance and cleaning of all equipment, and properly training cell culture personnel.

Check for Microbial Contamination

When most bacterial contamination occurs, it usually occurs within a few days and is typically obvious to the naked eye. Distinct changes to the medium such as turbidity, presence of particles visible in suspension, and a rapid decline in pH (yellow color, indicating acidity) are all indicators of bacterial contamination. Fastidious bacteria species that grow very slowly can be difficult to detect. 

Fungal contaminants may or may not cause a change in the pH of the medium and can be distinguished from bacteria by checking for the presence of filamentous structures in the suspension. Yeast cells are larger than bacteria, but may not appreciably change the pH of the medium, and will appear as separate round or ovoid particles. Microbacterial media which can be used to test for bacterial and fungal contamination include blood agar, thioglycollate broth, tryptic soy broth, BHI broth, Sabouraud broth, YM broth, and nutrient broth with 2% yeast extract.23 However, some microbial contamination is not apparent. For example, the use of antibiotics can suppress bacterial growth and thus mask contamination. Some viral infections do not alter the morphology of the cells, and detection of mycoplasma contamination requires specific assays.

Mycoplasma Contamination

IP-Brightness Contrast

Hoechst 33342 staining of an uncontaminated cell culture.

Vero - Neg control

Hoechst 33342 staining of a contaminated cell culture.

Cell lines are screened for mycoplasma contamination by direct (agarose and broth culture) and indirect (Hoechst) methods.24,25 For example, the fluorochrome Hoechst DNA stain will bind to the DNA of mycoplasma and the organisms can be detected easily when examined using a microscope equipped with appropriate fluorescence optics. The direct culture method requiring both broth and agar will permit isolation of cultivable strains as apparent by appearance of characteristic mycoplasma colonies on the agar medium. 

Both direct and indirect methods for detection of mycoplasma are used at ATCC several times while a cell line is expanded for the preparation of the token, seed and distribution stocks. 

Most cell culture laboratories have incorporated PCR-based mycoplasma testing, using kits such as ATCC’s Unversal Mycoplasma Detection Kit (ATCC® 30-1012K™) into their routine cell culture operations. Cell cultures can be submitted to the ATCC Mycoplasma Testing Service. See the ATCC Services section of the website for details.

Treating for Microbial Contamination

Eliminating contamination from a cell line is time consuming and does not always work. Discarding the culture and starting over is preferred. However, if the cells are unique and irreplaceable, one should first identify the contaminant and select a suitable antibiotic for treatment. It is best to test the contaminating microbe for its antibiotic sensitivity prior to treatment; this allows for a shorter treatment time and limits exposure of the cell line to potentially damaging reagents. 

The cells are cultured for 1 to 2 weeks in the presence of the antibiotic, and then cultured without antibiotic for 1 to 2 months. At this point, the line should be retested with a very sensitive test method to make sure that the culture is clean. Periodic retesting should be employed to make sure that the contaminant does not reappear. Since antibiotics may be toxic to cells, a selected population that no longer exhibits qualities of the parental line may result. It may be necessary to examine the cured culture to determine if it is sufficiently similar to the original line.

Cellular Cross-Contamination

Cross-contamination of one cell line with another can sometimes lead to the replacement of the original cell with the contaminant, particularly when the contaminant grows faster than the original line. HeLa cells are perhaps the most famous example of a cross-contaminating cell line overtaking and then masquerading as the original. 

In the 1950s and 1960s, many continuous lines were unknowingly cross-contaminated with other cell lines including HeLa cells. In the 1970s and 1980s, as many as one in three cell lines deposited in cell repositories were imposters.26 This cross-contamination was only uncovered with the development of suitable genetic markers beginning in 1967.27 Indeed, several “unique” cell lines in ATCC’s collection turned out to be HeLa cells upon further study. Despite the confirmation of their HeLa cell origin, cytogenetic analysis suggests that there are differences among these HeLa-derived cell lines. Several of them possess unique properties. However, these cell lines should not be used as functional models of their claimed tissues of origin. 

More recently, ATCC and other cell repositories have used DNA polymorphisms in addition to enzyme polymorphisms, HLA typing, and karyotyping to confirm the identity of their cell lines. One of the most reliable methods to study DNA polymorphisms is the profiling of short tandem repeats (STR) by PCR amplification followed by capillary electrophoresis.28 STR profiles for all ATCC human cell lines are available on the website in the catalog descriptions. (See: Figure 4)

STR profiles for two unrelated human cell lines

Figure 4. STR profiles for two unrelated human cell lines. Top: KU812E (ATCC® CRL-2100™). Bottom: MRC-5 (ATCC® CCL-171™). Amplicons were generated using Promega’s PowerPlex® platform, separated by capillary electrophoresis and analyzed using GeneMapper® software from Life Technologies.

Test cell cultures on a regular basis to ensure the absence of contamination from both microorganisms as well as from other cell lines. If contamination is found, discard the culture and start fresh with a new stock.


The need for precautions when experimenting with cells in culture depends upon the source and nature of the biological material, the experimental procedure, and the laboratory/containment conditions. Since every situation is different, the risks need to be identified and appropriate precautions need to be taken before any work begins. 

More information on risk assessment and precautions can be found in the Center for Disease Control (CDC) publication Biosafety in Microbiological and Biomedical Laboratories, (BMBL) 5th Edition.29 The text of this publication is available in its entirety online. Information on agent risk assessment and a description of the four biosafety levels can be found in this publication. 

ATCC assigns a biosafety level (BSL) to each cell line for purposes of packaging for safe shipment. When a cell line is known to contain an etiologic agent, ATCC classification is at least comparable to the BSL assigned to the agent by the CDC and in some cases the ATCC designation is more restrictive. ATCC follows federal biosafety guidelines and takes several factors into consideration when assessing potential hazard.

Biosafety Level 1

  • Cell lines with animal origin not included under Biosafety Level 2 

Biosafety Level 2

  • Cell lines that harbor mycoplasma or any other BSL 2 agent (See: NOTE)
  • Cell lines exposed to or transformed by a primate oncogenic virus
  • Primate cell lines that contain viruses
  • Cell lines carrying a part of certain viral genomes, even if whole virus is not released from the cell30


Some patent cell lines at ATCC are known to be contaminated with mycoplasma and are noted as such in the catalog.

As the recipient of a cell line, take into account not only the nature of the material but also the manipulations employed during its handling when assessing the potential laboratory risk. For example, procedures involving large volumes of cell lines that contain HIV or that include manipulation of HIV in high concentration should be conducted under BSL 3 conditions.29

It is not possible to screen cell lines for the presence of every agent. For added precaution, ATCC handles all cell lines under BSL 2 practices, even those classified as BSL 1. It is prudent to treat all mammalian cell lines as potentially hazardous.