Cell Culture: contamination

1. Ranked causes

   1. Bacterial contamination (turbid media, rapid pH shift to yellow)Media cloudy with a sudden color change within 24 h.
   2. Fungal contamination (visible hyphae or floating spore clusters)Fuzzy particulates visible under low magnification, media stays clear otherwise.
   3. Mycoplasma (no visible turbidity, but slow growth / viability decline)No visible contamination but chronic underperformance; mycoplasma test missing or overdue on the Lab Index line.
   4. Cross-contaminated or misidentified cell lineMorphology or marker profile does not match authenticated stock.
   5. Breach in aseptic technique at the biosafety cabinetNew contamination event coincides with cabinet airflow or filter service.

2. Recovery steps

   1. Quarantine and discard the affected flask immediately — do not open near other culturesPrevents cross-contamination of the rest of the incubator.
   2. Decontaminate the incubator shelf and biosafety cabinet; re-verify HEPA filter service dateAddresses the likely source before resuming culture.
   3. Thaw a fresh, early-passage vial from the verified cryostockRestarts from a known-clean population rather than trying to rescue a contaminated line.
   4. Test for mycoplasma on all other active lines in the same incubatorMycoplasma spreads silently between flasks sharing an incubator or media bottle.

3. Controls first

   • Did uninoculated media blanks / negative controls stay clear?If no: Reagent or incubator contamination — discard shared bottles and decontaminate before rescue attempts.
   • Is the media visibly turbid or has it shifted color rapidly?If yes: Likely bacterial — discard immediately, do not attempt to rescue.
   • Is the media visibly turbid or has it shifted color rapidly?If no: Consider mycoplasma testing if growth has been chronically poor without visible cause.

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