Testing for Bacteria and Fungi in Cell Cultures

Cook Book Volume 12

Fundamental Techniques in Cell Culture Laboratory Handbook-2nd Edition

Flow Scheme for Bacteria and Fungi Testing

Aim

In cases of gross contamination the naked eye may identify the presence of bacteria and fungi. However, in order to detect low-level infections the method above is recommended.

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Materials

  • Aerobic nutrient broth e.g. Soyabean Casein Digest (Tryptone Soya Broth,TSB) (15ml aliquots)
  • Anaerobic nutrient broth Fluid Thioglycollate Medium (20ml aliquots) (TGM)
  • Positive control organisms e.g.
    • Bacillus subtilis NCTC*
    • Candida albicans NCTC*
    • Clostridium sporogenes NCTC*

*Available from the National Collection of Type Cultures (NCTC)

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Equipment

  • Personal protective equipment (latex medical gloves, laboratory coat, safety glasses)
  • Waterbath set to 37°C
  • Microbiological safety cabinet at appropriate containment level
  • Incubator set at 32°C
  • Incubator set at 22°C

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Procedure

  1. Culture cell line in the absence of antibiotics for 2 passages prior to testing.
  2. Bring attached cells into suspension with the use of a cell scraper. Suspension cell lines may be tested directly.
  3. Inoculate 2 x aerobic broths and 2 x anaerobic broths with 1.5ml test sample.
  4. For each of the positive control organisms inoculate 2 x aerobic broths and 2 x anaerobic broths (e.g. 0.1ml control at 100 cfu per broth).
  5. Leave 2 aerobic and 2 anaerobic broths un-inoculated as negative controls.
  6. Incubate broths as follows:
    • Incubate one broth of each pair at 32°C the other at 22°C for 14 days
  7. Examine Test and Control broths for turbidity after 14 days.

Criteria for a Valid Result
All positive control broths show evidence of bacteria and fungi within 14 days of incubation and the negative control broths show no evidence of bacteria and fungi.

Criteria for a Positive Result
Test broths containing bacteria or fungi show turbidity.

Criteria for a Negative Result
Test broths should be clear and show no evidence of turbidity.

Notes

  1. Control organisms (Bacillus subtilis, Clostridium sporogenes and Candida albicans) are available from the National Collection of Type Cultures (NCTC), UK.
  2. This test procedure should be carried out in a microbiology laboratory away from the cell culture laboratory.

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