Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal.

Mycoplasma pneumoniae was grown on Formvar- and carbon-coated electron microscope grids and treated with the nonionic detergent Triton X-100 to gently remove the membrane and cytoplasm. The detergent mixture was composed of 0.5% Triton X-100 in SSR-2 broth base. After this treatment, the grids were rinsed in a mixture of 0.1 M KCl, 5 mM MgCl2, and 6 mM potassium phosphate buffer (pH 7.05) and negatively stained with uranyl acetate. The Triton X-100-resistant remains of M. pneumoniae after gentle removal of the membrane and cytoplasm consisted of fibrous structures oriented similarly to the undisrupted cells. The thin fibers displayed a negative staining quality and diameter analogous to that of rabbit muscle F-actin. The fibrous moieties ended in rodlike condensations which appeared striated in negatively stained and shadowed preparations. These striations were regular, and the majority of rod structures had lengths of 220 to 300 nm and widths of 50 to 80 nm. Specific antibody to rabbit muscle actin, produced in guinea pigs, was used in indirect immunofluorescence of the M. pneumoniae colonies. Fluorescence was detected, with concentrations at the colony center and at the tips of filamentous cells.