PRESERVATION OF SURFACE-LIPIDS AND DETERMINATION OF ULTRASTRUCTURE OFMYCOBACTERIUM-KANSASII BY FREEZE-SUBSTITUTION

The cell wall architecture of a slowly growing mycobacterium, Mycobact erium kansasii, was examined by freeze-substitution following growth i n vitro. Freeze-substituted bacteria were marked by the presence of an electron-translucent space (or electron-transparent zone [ETZ] descri bed by previous workers [T. Yamamoto, M. Nishiura, N. Harada, and T. I maeda, Int. J. Lepr. 26:111-114, 1958]) surrounding the majority of ce lls. At least two morphotypes of mycobacteria were revealed by freeze- substitution In the first, a relatively thin (11 +/- 2.3 to 35 +/- 3.1 nm), uniform ETZ surrounded intact cells which contained cytoplasm fi lled with well-stained ribosomes and a DNA nucleoid distributed throug hout the cell. The second morphotype consisted of a small proportion o f organisms that were distorted in shape and were surrounded by a much thicker (59 +/- 2.6 to 198 +/- 2.5 nm) ETZ in areas of the cell which appeared to have retracted from the space it had originally occupied, leaving depressions in the ETZ. The lipid nature of the ETZ was demon strated because cells n ere devoid of an ETZ when organisms were freez e-substituted in the absence of osmium tetroxide in the substitution m edium or treated with neutral lipid solvents (acetone or ethanol) befo re freeze-substitution. Moreover, thin-layer chromatography of acetone or ethanol extracts obtained from solvent-treated cells identified a lipid component which corresponded to the M. kansasii-specific phenoli c glycolipid. In contrast, negligible amounts of glycolipids were dete cted in extracts obtained from control HEPES (N-2-hydroxyethylpiperazi ne-N'-2-ethane- sulfonic acid) buffer-treated cells, and these cells r etained an ETZ. These results demonstrate that species-specific phenol ic glycolipids are essential components in the architecture of the M. kansasii ETZ. Furthermore, we show that freeze-substitution is a relia ble technique for the retention and precise preservation of lipid-cont aining polymers in the mycobacterial cell wall.