[Production of protoplasts and membranes in Actinomyces hygroscopus]

Protoplasts of Actinomyces hygroscopicus were obtained in a hypertonic medium containing 10.3% sucrose. Lysozyme was employed as a lysing agent at a concentration of 1 mg/ml. The cells were subjected to lysis during 3-4 hours at 37 degree C, the concentration of biomass being 0.05 g of dry mycelium per 1 ml of hypertonic solution. Production of protoplasts was controlled by phase-contrast microscopy; the amount of the protoplast was determined by optical density at lambda=530 nm. The highest amount of the protoplasts was obtained during growth of the culture on a medium containing 0.5% glycine. The membrane fraction was isolated after lysis of the protoplasts in a hypotonic medium, treatment with DNase and RNase, and centrifugation at 22,000 g in the cold. Production of the membranes was controlled by electron microscopy. The yield of the membranes was 17-19% of the dry mycelium weight. They contained about 35% of lipids, 50% of protein, and 5% of RNA.