Testing for Bacteria and Fungi

Aim In cases of gross contamination the naked eye may identify the presence of bacteria and fungi. However, it is necessary to detect low-level infections by incubation of cell cultures and/or their products in microbiological broth. Equally these sterility tests can be used to confirm the absence of bacteria and fungi from the preparation which is important when preparing cell banks or cell culture products.

Materials

• Soybean Casein Digest (Tryptone Soya Broth,TSB) (15ml aliquots) (Prod. No. S1674 ) TSB Powder (Prod. No. T8907 )
• Fluid Thioglycollate Medium (20ml aliquots) (TGM) (Prod. No. F4797 )
• Bacillus subtilis NCTC*
• Candida albicans NCTC*
• Clostridium sporogenes NCTC*

Equipment

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

Click here for Figure 12. Flow Scheme for Bacteria and Fungi Testing

Procedure

Culture cell line in the absence of antibiotics for 2 passages prior to testing. Bring attached cells into suspension with the use of a cell scraper. Suspension cell lines may be tested directly. Inoculate 2 x Thioglycollate Medium (TGM) (Prod.No. F4797 ) and 2 x Tryptone Soya broth (TSB) (Prod.No. T8907 ) with 1.5ml test sample. Inoculate 2 (TGM) and 2 (TSB) with 0.1ml C.albicans (containing 100 colony forming units, cfu). Inoculate 2 (TGM) and 2 (TSB) with 0.1ml B. subtilis (containing 100cfu). Inoculate 1 TGM with 0.1ml C. sporogenes (containing 100cfu). Leave 2 (TGM) and 2 (TSB) un-inoculated as negative controls. Incubate broths as follows:

• For TSB, incubate one broth of each pair at 32ºC the other at 22ºC for 14 days
• For TGM, incubate one broth of each pair at 32ºC the other at 22ºC for 14 days
• For the TGM inoculated with C.sporogenes incubate at 32ºC for 14 days

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

The positive controls should be handled in a laboratory remote from the main tissue culture laboratory. Control organisms (Bacillus subtilis , Clostridium sporogenes and Candida albicans ) are also available from the National Collection of Type Cultures (NCTC), UK*. This test procedure should be carried out in a microbiology laboratory away from the cell culture laboratory.