uncontaminated cells against a dark cytoplasmic background (Fig. 2.4a, see also
colour section). However, this technique is prone to errors, including false-negative
results. To avoid the latter, cells should be cultured in antibiotic-free medium for two
to three passages before being used. A positive control using a strain of mycoplasma
seeded onto a coverslip is essential. Such controls should be handled away from the
cell culture laboratory to avoid contaminating clean cultures of cells. It is also
important to ensure that the fluorescence detected is not due to the presence of
bacterial contamination or debris embedded into the plastics during manufacture.
The former normally appear larger than the fluorescing cocci or filaments of myco-
plasma. Debris, on the other hand, would show a non-uniform fluorescence owing to
the variation in size of the particles usually found in plastics.
ELISA detection of mycoplasma is now becoming more commonly used and can
be carried out using specifically designed kits following the manufacturer’s protocol
and reagents supplied. In this assay, 96-well plates are coated with the antibodies
against different mycoplasma species. Each plate is then incubated at 37C for 2 h
with the required antibody or antibodies before blocking with the appropriate
blocking solution and incubating with the test sample(s). A negative control, which
is simply media with sample buffer, and a positive control normally provided with the
kits, should also be included in each assay. A detection antibody is subsequently
added to the samples, incubated for a further 2 h at 37C before washing and
incubating with a streptavidin solution for 1 h at 37C. Each plate is then detected
for mycoplasma by adding the substrate solution and read on a plate reader at 405 nm
after a further 30 min incubation at room temperature. This method is apparently
suitable for detecting high levels of mycoplasma and could also be used to identify
several species in one assay.(a) (b)Fig. 2.4Hoechst 33258 staining of mycoplasma in cells. (a) A Hoechst-negative stain, with the dye staining
cellular DNA in the nucleus and thus showing nuclear fluorescence. (b) A Hoechst-positive stain, showing
staining of mycoplasma DNA in the cytoplasm of the cells. (See also colour plate.)48 Cell culture techniques