Cultures were washed several times with high concentrations of antibiotics by rinsing monolayer cultures or by resuspending non-adherent cells by centrifugation, and then the cultures were passaged three times with antibiotic-added medium and three times with non-antibiotic-added medium, and tested for contamination after each passaging.

Operation method

Program 19.4 Decontamination of Microorganisms

Principle

Cultures were washed several times with high concentrations of antibiotics by rinsing monolayer cultures or by resuspending non-adherent cells by centrifugation, and then the cultures were passaged three times with antibiotic-added medium and three times with non-antibiotic-added medium, and tested for contamination after each passaging.

Materials and Instruments

DBSS
Highly concentrated antibiotic culture solution
Material for passaging cultures Phase contrast microscope with 40× or 60× objective lens Material for mycoplasma staining

Move

1. Carefully collect the contaminated culture fluid and, if possible, test the organism for susceptibility to a range of antibiotics, failing which the culture fluid should be autoclaved or hypochlorite added.

2. rinse the cells with DBSS (each rinse reduces the contaminant concentration by two logarithmic orders of magnitude unless the contaminant adheres to the cells).

(a) For monolayer cultures, rinse the culture three times with DBSS, then trypsinize, and wash the cells more than twice with DBSS by centrifugation and resuspension.

(b) For suspension cultures, wash the culture five times with DBSS by centrifugation and resuspension.

3. Inoculate the cells in new culture flasks at the lowest possible and appropriate cell concentration.

4. Add medium containing high concentrations of antibiotics and change every two days.

5. Perform passaging culture with high concentration antibiotic medium.

6. Repeat steps 1 to 4 three times.

7. Remove the antibiotic and passage culture these cells three more times in the absence of antibiotic.

8. Examine the culture by phase contrast microscopy and Hoechst staining.

9. The cells are cultured for another two months without antibiotics and examined to make sure that all contamination has been eliminated.

Common Problems

The general principle is that contaminated cultures should be discarded without attempting to remove the contamination, except in the case of cell lines where it is vital that they be retained before removing the contamination is considered. In any case, it is difficult to achieve complete elimination of contamination. Particularly in the case of yeast contamination, attempts to remove the contamination may result in the production of more recalcitrant antibiotic-resistant cells.

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