Protocol for Microbial Decontamination

Protocol for Microbial Decontamination

1.        Collect the contaminated medium carefully. If possible, the organism should be tested for sensitivity to a range of individual antibiotics. If not, autoclave the medium or add hypochlorite.

2.        Washthe cells in DBSS (Hanks BSS without bicarbonate, with Penicillin, Streptomycin, Amphotericin B and Kanamycin or Gentamycin). For monolayers, rinse the culture 3 times with DBSS, trypsinize and then wash the cells twice more in DBSS by centrifugation and re-suspension. For suspension cultures, wash the culture five times (in DBSS) by centrifugation and re-suspension.

3.        Reseed a fresh flask at the lowest reasonable seeding density, depending on cell type.

4.        Add high antibiotic medium and change the culture every 2 days.

5.        Subculture in a high antibiotic medium. Repeat Steps 1 to 4for three subcultures.

6.        Remove the antibiotics, and culture the cells without them for a further three subcultures.

7.        Recheck the cultures (phase contrast microscopy, Hoechst staining).

8.        Culture the cells for a further two months without antibiotics, and check to make sure that all contamination has been eliminated.

Mycoplasmas are simple bacteria that lack a cell wall, and they are considered the smallest self-replicating organism. Because of their extremely small size (typically less than one micrometer), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection. Chronic mycoplasma infections might manifest themselves with decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures; (Fig. 5) however, the only assured way of detecting mycoplasma contamination is by testing the cultures periodically using fluorescent staining (e.g., Hoechst 33258), ELISA, PCR, immunostaining, autoradiography, or microbiological assays