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Contamination in Cultured Cells
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- 2. Introduction Cell cultureis the process of obtaining cells from A plant or animal and then growing them in an artificial environment. Contamination of cell culture is common problem in cell culture labs. Cell culture contaminants can be divided into two categories: Chemical contaminants Biological / microbial contaminants Contamination frequency can be reduced by Following good aseptic techniques. Understanding sources of contamination.
- 3. Source of Contamination Failure in sterilization procedure for glassware and pipettes Turbulence and particulates in the air. In room poorly maintained incubators and refrigerators. Types of Microbial Contamination Bacteria Molds Mycoplasma Yeast Viruses
- 4. Bacterial Contamination Large,ubiquitous group of unicellular microorganisms. Size - few micrometers in diameter Shape - variety of shapes Can grow along with yeast and molds. Visible microbial contamination It is most commonly found contaminants.
- 5. SIGNS- Infected culturesusually appear cloudy. May form thin film on surface. Sudden drop in pH of the culture medium. Visible under a low-power microscope as tiny, moving granules. Higher magnification, bacteria variety of shapes of bacteria.
- 6. Yeast Contamination Unicellulareukaryotic microorganism in kingdom of fungi. Size - few - 40 micrometers. Cause quick contamination. Easy to identify due to large size
- 7. SIGNS- Infected culturebecome turbid. Very little change in pH until contamination become heavy, then pH usually increase. Under microscope, yeast appears as oval or spherical particles that may show budding of smaller particles. Stimulated phase contrast image of cells in culture contaminated with yeast.
- 8. Mold Contamination Moldsare eukaryotes in the kingdom of fungi. Grow as multicellular filaments called hyphae. Spores can servive in extreme harsh condition, dormant stage. Activated when found suitable growth conditions.
- 9. SIGNS- Infected cultureremain clear until culture becomes heavily infected and media become turbid. pH remains stable during initial infection, then rapidly increase. Under a microscope , mycelia appears as thin filamentous, sometimes with a denser cluster of spores. With some toxic infection sine deterioration of the cells will be apparent.
- 10. Viral Contamination Virusesare microscopic infectious agents that uses the host cells machinery to reproduce. Detection is difficult due to extremely small size.(20-400nanometer) Virally infected cell cultures can cause serious health hazard to the laboratory personnel. Not particularly common SIGNS- No observable signs apart from adverse effects on culture. Can be detected by electronic microscopy, ELISA, or PCR with appropriate viral primers.
- 11. Mycoplasma Contamination Singlebacteria - lack a cell wall The smallest self replicating organism. Very difficult to detect until they achieve extremely high densities Cause the cell culture to deteriorate Some slow growing mycoplasma may persists in culture without causing cell death, but they can alter the behavior and metabolism of the host cells in the culture.
- 12. SIGNS- Decreased rateof cell proliferation, reduced saturation density, and agglutination in suspension cultures Detected by Fluorescent staining, ELISA, PCR, autoradiography or microbiological assays. Un-visible Microbial Contamination
- 13. Monitoring of contamination Check for contamination by eye and with a microscope at each handling of a culture. If it is suspected, but not obvious - Remove a sample from the culture and place it on a microscope slide. Check the culture with a microscope, preferably by phase contrast. If it is confirmed discard the pipettes, swab the hood or bench with 70% alcohol do not use the hood or bench until next day.
- 14. Record thenature of contamination. If the contamination is new and not widespread discard the culture, media feeding bottles, other components (trypsin) If contamination is new and widespread (i.e. in at least two different cultures) , discard all media, stock solution, trypsin, etc. If the contamination is widespread, multispecific and repeated, check the laboratory's sterilization procedure
- 15. Cross-contamination The transferof different cells- directly or indirectly into the cell culture is called as cross contamination. They have very short doubling times and high plating efficiencies. Although these properties makes such cell lines valuable experimental material, they also make them potentially hazardous for cross-infection other cell line. Such HeLa cell line.
- 16. To Avoid Cross-Contamination Obtain cell line from a reputable cell bank. Handle rapidly growing lines. Never use the same pipette for different cell lines. Never use the same bottle of medium, trypsin etc. for different cell lines. Do not put pipette back into a bottle of medium, trypsin, etc. Add medium and any other reagents to the flask first and then add cells last. Check the characteristics of the culture regularly and suspect any sudden change in morphology, growth rate , etc. Cross-contamination or its absence may be confirmed by DNA fingerprinting, karyotype,etc.
- 17. References Cell CultureContamination: Sources, Consequences, Prevention, and Elimination Carolyn Kay Lincoln and Michael G. Gabridget Understanding and Managing Cell Culture Contamination John Ryan, Ph.D.