Vital Staining of Mycoplasma and L-Forms with Chlorazol Black E

Vital staining ofMycoplasma colonies was attempted because other dye visualization techniques kill the organisms and preclude reisolation for further studies. The lipophilic amphoteric dye Chlorazol Black E (CBE) was the most successful of 14 vital dyes tested onMycoplasma hominis, M. pharyngis, M. fermentans, M. arthritidis, M. salivarium, M. pneumoniae , and L-forms ofStaphylococcus aureus when used in 1:1,000 (w/v) saline dilution as the sterile suspension medium for inoculation of Hayflick's medium under both aerobic and microaerophilic (Fortner method) conditions. Colonies of all species stain homogeneously in the periphery and center portion, the latter being more refractive under positive phase contrast. All stained colonies were successfully subcultured. The most striking and promising result of the use of CBE as a tool for physiological study ofMycoplasma was a very significant increase in diameter of all colonies except those ofM. pneumoniae grown with CBE: 1.5 × forM. hominis and 5 × for L-formS. aureus . This size increase inM. hominis is proportional to the concentration down to a 1:50,000 dilution only under microaerophilic conditions. Whether this increase in colony size is due to an increased number of cells, to larger cells, or to the adsorption of CBE on the lipid membrane is unknown at present.