Maintaining clean primary cultures: simple yet vital tips

Maintaining clean primary cultures: simple yet vital tips

The term “contamination” can send shivers down the spine of any tissue culture lab personnel! When the extent of contamination is searched across literature, the following observations emerge: 2012 published research in the Cell journal by Nikfarjam and Farzaneh showed that on an average, Mycoplasma is present in 5 to 30% of the world’s cell lines! The obligate parasites-viruses are usually specific for their hosts. To cite an example of how viruses can affect cultures is the work published in PLoS ONE by Uphoff and colleagues (2015). 3.3% of 577 human cell lines tested positive for murine leukaemia viruses (MLV). Of these, active viruses could be produced from 17 of these cell lines as shown by PCR!

Contamination leads to loss of cells and hence, possible products not to mention the loss of time, energy, reagents and money! One of the major issues in the lab is undetected contamination! The time-lapse between the incidence of contamination and its detection can finally affect the factors listed including reputation!

As far as Mycoplasma is concerned, the lab personnel themselves are a major source of contamination. 6.2% of the M. salivarium cases are due to talking in the lab. The number of these organisms in vital parts is given-106 organisms/cm2 on the scalp; 107 organisms in 1 ml of saliva and 105 organisms/cm2 on the forehead (Nikfarjam and Farzaneh, 2012). This makes the use of clean lab coats, masks and gloves an absolute must. Talking is a strict no-no and it is better to avoid working in the lab with a cold or allergy.

Another important aspect of maintaining clean cultures is avoiding accidents. According to John Ryan (Corning), a vial labelled “WI-38” was used as a diploid cell line by a lab worker. Post-culture, it was revealed to be Wisconsin 38-a tobacco cell line that was also labelled as WI-38. Though the situation was rectified before the problem escalated, it becomes very important to be completely aware of labels and conduct experiments with complete awareness.

The maintenance of clean cultures can involve the following key tips:

  1. Correct use of acronyms as labels. Each worker should be aware of the acronym used.
  2. Good record keeping; the protocols for media or reagents should be written down along with the pH, volume, lot and any treatment done. If sterilized, it should be labelled to avoid confusion.
  3. A separate colour coding system can be used in a busy lab with different colours for each worker.
  4. In a lab, cooling coils of freezers and refrigerators require regular vacuuming. This can remove potential contamination.
  5. Cell lines in the lab that are used continuously should be screened every 3-4 months. The microbiological culture method where cell cultures are added to media and observed for bacterial or mycoplasma contamination is standard. Staining DNA using fluorochromes or testing using PCR or fluorescence in situ hybridization (FISH). Other techniques include analyzing the chromosomes, profiling short tandem repeats (STR) or isoenzyme analysis. One or more these techniques can be used regularly.
  6. New lots of chemicals, media and sera should be checked using the most precise assay.
  7. The purity of water in the lab should be checked at least once a year.

When correct aseptic techniques are followed with good record- keeping and lab practices, contamination can be minimized so that primary cultures can be continuously generated and used to study the intricate mechanisms of human biology.

References:

Nikfarjam, L., & Farzaneh, P. Prevention and detection of Mycoplasma contamination in cell culture. Cell journal13(4), 203–212 (2012).

Uphoff, C. C., Lange, S., Denkmann, S. A., Garritsen. H, S, P. and Drexler, H. G. Prevalence and Characterization of Murine Leukemia Virus Contamination in Human Cell Lines. PLoS ONE 10(4):e0125622 (2015).

A Guide to Understanding and Managing Cell Culture Contamination-Corning.

John Ryan, Understanding and Managing Cell Culture Contamination.  Technical Bulletin- Corning Life sciences.

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