Mycoplasma contamination prevention and detection

Mycoplasma are everywhere. Members of this genus of bacteria are the smallest free-living organisms able to self-replicate. In the 1950s, when mycoplasma were first isolated from cell cultures, contamination was found in 57–92 percent of lab-grown cells. Because the organisms lack a cell wall, they are resistant to common antibiotics, such as streptomycin and penicillin, and they easily slip through filters. Out, Damned Mycoplasma by Kelly Rae Chi. The Scientist Magazine

Doing a quick google search for “mycoplasma cell culture” one can find lots of results explaining what they are, and so on. I’ve founded a good article by Kelly Rae Chi on “The Scientist” Magazine entitled “Out, Damned Mycoplasma” about it. They have talked to experts  about the ways to prevent, detect, and treat the contamination.

Prevention

To prevent mycoplasma contamination on a shared culture room where nobody seems to care about it, we:

  1. Keep a set of pipettes to be used on cell culture only
  2. Never share tissue hood, incubator, solutions or culture media with other colleagues, especially if they don’t belong to our laboratory
  3. Test cell lines periodically and thaw a new vial of cells every 3 months

To improve our prevention protocol we can:

  1. As suggested by the article on The Scientist magazine
    1. Quarantine and test new cell lines: we already test new cell lines, but we could maintain freshly thawed, line maintenance flasks and other people’s [when they ask for help on maintaining their cells on a myco-free incubator] on a separate tray to be tested before being used, as we can’t afford having a dedicated incubator for that.
    2. Don’t speak: Because people’s mouths host some of the major mycoplasma contaminants, such as M. oraleM. fermentans, and M. hominis, you should not talk, whistle, or sing at the clean bench.
    3. Bad practices whiteboard: Write down every bad practice one witness on the culture room to further discuss how to improve them in a weekly group meeting.

If the contamination is found within the routine tissue culture laboratory, cell culture work should be closed down immediately and all living cultures discarded. All surfaces (particularly hoods and incubators) must be thoroughly cleaned and disinfected and all partly used medium discarded before taking fresh cultures from the cell bank and retesting all the cells Nature Protocols 5, – 929 – 934 (2010)

As we don’t currently work with “precious cell lines”, and we have “myco-free” batches of cryopreserved lines, we don’t treat contaminated cell lines, we just purge them. Treating contaminated cells could take weeks and does not assure that they won’t be damaged in the end.

Detection

To perform mycoplasma detection by conventional PCR using a commercial kit (Minerva Biolabs, Venor GeM).

Procedure

Accordingly to Young (2010), “the cultures must have been grown without antibiotics for at least three subcultures or for 2 weeks (whichever is greater)”, so after our cultures reaches this condition is when we are testing the cells.

  1. Change medium of an exponentially growing culture at 50-60% of confluence
  2. 24 hours after changing the medium, we take a 100-µl sample.
  3. Incubate sample at 95ºC for 5 minutes
  4. Centrifuge to pellet cell debris
  5. Prepare a master mix for all reactions (n+0.5) as follows:
    Component              Volume (1 reaction)
    PCR grade water             15.3 µl
    10x reaction mix             2.5 µl
    Primer/nucleotide mix        2.5 µl
    Internal control DNA         2.5 µl
    Polymerase (5 U/μl)          0.2 μl
    Total volume                23.0 µl
  6. Prepare a PCR tube for the positive control (2 µl of the positive control), the negative control (2 µl of PCR grade water) and for each sample (2 µl of sample) being tested
  7. Dispense 23 µl of the master mix in each reaction tube
  8. Run the samples on the Progene thermocycler if using 0.5 ml tubes or on the Bio-Rad one if using PCR tubes/strips:
    Cycle   Temperature    Time
      1        94 °C      02:00 
     39        94 °C      00:30
               55 °C      00:30
               72 °C      00:30
    cool down to 4 °C to 8 °C
  9. Cast a 1.5 % standard agarose gel including DNA stain (maximal 5 mm thick, 5 mm comb).
  10. Load 25 µl of each sample mixed with 5 µl of bromophenol blue loading dye.
  11. Stop electrophoresis after 2 cm run distance (depending on the electrophoresis chamber used run for approx. 20 minutes at 100 V).
  12. Result reading:
    1. Internal control: 191 bp
    2. Mycoplasma spp.: 265-278 bp

VenorGem