| Abstract: | B. pertussis suspension was tested by De Voe et al. method (1970) and its modification with the solutions of a definite ionic composition and a lysozyme. The best results were obtained by the following modification elaborated by the authors. The microbes were grown on the casein-carbon agar for 36 hours and were washed with chilled 0.5 M NaCl. The suspension was washed 4 times with the same solution and then the precipitate was suspended in saccharose solution (0.5 M). In 2 hours the saccharose was replaced by a solution of salts with lysozyme. After a 2-hour incubation at 35 degrees C the substance was centrifugated for 20 minutes and the precipitate suspended in the tris-buffer at pH 7.8. The following changes were observed: after the washing and incubation with saccharose there was seen a strong stretching and separation of the cell wall (CW) from the cytoplasmic membrane (CPM); cells without the CW were rarely revealed; 2) after the lysozyme treatment there were many cells of spherical shape (phasic-contrast microscopy) without any CW, limited by the CPM only. Morphologically they were no different from the true protoplasts of the Gram-positive bacteria. The chemical analysis also confirmed a possibility of obtaining the true protoplasts of the Gram-negative bacteria. |
