METHOD OF CULTIVATING STREPTOCOCCUS HEMOLYTICUS

1,157,947. Streptococcus hemolyticus of high anti-tumour activity. CHUGAI SEIYAKU K.K. Oct. 30, 1967 [Oct. 31, 1966], No.49275/67. Heading C6F. A process for obtaining cells of hemolytic Streptococci having high anti-tumour activity involves cultivating hemolytic Streptococci having anti-tumour activity, particularly Streptococcus hemolyticus ATCC No. 21060, in a culture medium containing a yeast extract, and separating bacterial cells from the cultivated broth. The yeast extracted may be obtained by dissolving a commerically available yeast extract or auto-lysate in water, neutralizing and heating the solution, and then taking away the insoluble fraction by filtering. The culture medium of pH 7À0-7À4 typically contains between 3 and 6 grams per decilitre of yeast extract and such other constituents as peptone, fish meat extract, beef extract, horse or whale flesh extract, ribonucleic acid and/or its ribonuclease core, sodium chloride, sodium hydrogen phosphate and sodium hydrogen carbonate. The hemolytic Streptococci may be preliminarily cultivated in a yeast extract medium or beef infusion medium at 37‹ C. for 24 hours. The main cultivation is then carried out at 37‹ C. for periods ranging from 10 to 30 hours (according to circumstances). The separated cells are suspended in a medium containing penicillin and heated at 30-38‹ C. for 10-30 minutes and then at 40-50‹ C. for 20-40 minutes to eliminate their virulence and hemolysin-producing activity. A suitable suspension medium (Bernheimer's Basal Medium) is composed of (a) 6 ml. of a solution consisting of 675 mg. of maltose and an aqueous solution containing 20 grams 1 decilitre of potassium dihydrogen phosphate, adjusted to pH 6À9-7À0 with sodium hydroxide, (b) 12 ml. of an aqueous solution containing 2 grams 1 decilitre of magnesium sulphate heptahydrate and (c) 66 ml. of distilled water.