F3 - Detection of mycoplasmas by DNA staining and fluorescent antibody methodology

Abstract

This chapter focuses on detection of mycoplasmas by DNA staining and fluorescent antibody methodology. The diagnostic value of a fluorescent preparation is related to the specific affinity of the stain. In the case of DNA-binding fluorochromes (DNAF), when a mycoplasma is stained it appears as a small fluorescent body because it contains DNA. Mycoplasmas also stain with specific fluoresceinated antibodies. It is possible to apply both of the stains, a DNAF and a fluorescent antibody, to the same preparation and observe that a particular small body binds both stains, thus providing diagnostic power unavailable with either stain by itself. The reagents are applied directly to the cell culture sample to be tested for mycoplasma or, alternatively, to a cell culture sample which had been inoculated into a known mycoplasma-free indicator cell culture. Most often cell culture contaminant mycoplasmas are present in high concentrations and produce infections that are readily detectable by use of these methods.