indirect DNA staining technique using the fluorochrome dye Hoechst 33258, enzyme-
linked immunosorbent assay (ELISA) or polymerase chain reaction (PCR).
With the microbiological culture technique, cells in suspension are inoculated into
liquid broth and then incubated under aerobic conditions at 37C for 14 days. A non-
inoculated flask of broth is used as a negative control. Aliquots of broth are taken
every 3 days and inoculated onto an agar plate, which is incubated anaerobically as
above. All plates are then examined under an inverted microscope at a magnification
of 300after 14 days of incubation. Positive cultures will show the typical myco-
plasma colony formation, which has an opaque granular central zone surrounded by
a translucent border, giving a ‘fried egg’ appearance (Fig. 2.3). It may be necessary
to set up positive controls in parallel, in which case plates and broth should
be inoculated with a known strain of mycoplasma such asMycoplasma oraleor
Mycoplasma pneumoniae.
The DNA binding method offers a rapid alternative for detecting mycoplasma
and works on the principle that Hoechst 33258 fluoresces under ultraviolet light
once bound to DNA. Thus, in contaminated cells, the fluorescence will be fairly
dispersed in the cytoplasm of the cells owing to the presence of mycoplasma. In
contrast, uncontaminated cells will show localised fluorescence in their nucleus only.
The Hoechst 33258 assay, although rapid, is relatively less sensitive when compared
with the culture technique described above. For this assay, an aliquot of the culture to
be tested is placed on a sterile coverslip in a 35-mm culture dish and incubated at
37 C in a cell culture incubator to allow cells to adhere. The coverslip is then fixed by
adding a fixative consisting of 1 part glacial acetic acid and 3 parts methanol,
prepared fresh on the day. A freshly prepared solution of Hoechst 33258 stain is
added to the fixed coverslip, incubated in the dark at room temperature to allow
the dye to bind to the DNA and then viewed under ultraviolet fluorescence at 1000.
All positive cultures will show fluorescence of mycoplasma DNA, which will appear
as small cocci or filaments in the cytoplasm of the contaminated cells (Fig. 2.4b,
see also colour section). Negative cultures will show only fluorescing nuclei ofFig. 2.3Photograph of mycoplasma, showing the characteristic opaque granular central
zone surrounded by a translucent border, giving a ‘fried egg’ appearance.47 2.4 Aseptic techniques and good cell culture practice