Studies on the Toxins of Hemolytic Streptococci

Primary Report. On the Protein of Bacterial Bodies

Abstract

(1) A strain of hemolytic streptococci isolated from acute mastoiditis was cultivated in a large amount (71000 c.c.) of 0.2 per cent glucose bouillon. After incubation for 2 days the culture was centrifuged and the growth was collected. Extract I was prepared by the extraction of this growth with distilled water. By addition of an equal amount of quartz sand to the residue, mechanical grinding for a long time, and extraction with distilled water Extract II was prepared. These extracts reduced mice to fatal issues in a certain amount. When these extracts were intracutaneously injected into non-treated guinea pigs, rabbits and goat, the skin reactions such as reddening, swelling, necrosis and others occurred. Among these skin reactions, the necrosis could be only slightly aroused. On the other hand, the reddenings caused by both toxic original solutions and PF were very remarkable, particularly on the skin of rabbits. In this instance the sensibility of the skin reactions of animals decreased in the order of rabbit, guinea pig and goat.
(2) The minimum lethal dose of toxic original solution for mice was 4.04mgm. (D. W.) in Extract I and 2.12 to 5.3mgm. (D. W.) in Extract II. Although the minimum erythem doses (the doses which produce the reddening of 10mm. in diameter) are variable according to the individuality of animals and are difficult to determine exactly, the dose of Extract I for guinea pigs was estimated as 0.04 mgm. (D. W.) and that of Extract II as 0.023mgm. (D. W.), the dose of Extract I for rabbits as 0.01 rngm. (D. W.) and that of Extract II as 0.006mgm. (D. W.), the dose of both Extract I and II for goat as 0.05 mgm. (D. W.).
(3) When the extracts above described were added with hydrochloric acid, the precipitate of protein occurred in its isoelectric point. This precipitate was dissolved at pH 7.0 by the addition of weak alkali and water, purified by the further repeating of this process, and tentatively designated as protein fraction (PF).
(4) The supernatant fluid obtained by removal of PF was concentrated in vacuo, dialysed and reconcentrated after the removal. of residual PF, and precipitated with acetone. The precipitate was again dissolved in water and reprecipitated by actone. The substance which was obtained by further repeating this treatment was tentatively designated as carbohydrate fraction (CF).
(5) The solution of PF indicates almost all protein reactions. The nitrogen content of PF from Extract I was 11.8 per cent and that of PF from Extract II was 12.3 per cent. The content of phosphor in PF of Extract I was 0.259 per cent. The ash is contained to 3.8 per cent in PF of Extract I and to 5.4 per cent in PF of Extract II.
(6) The intraperitoneal injection of PF solution reduces animals to fatal issues under the symptoms of congestions of intestines, spleen, suprarenal capsules and in a small number of animals also of lungs. Diarrhoea and other symptoms were also observed. Although the minimum lethal doses are variable according to the individualities of the animals, and are difficult to determine exactly, the dose of PF of both extracts was about 5mgm. for mice weighing 10gm.
(7) The intracutaneous injections of PF into guinea pigs, rabbits and a goat produce skin reactions accompanied with reddenings and swellings 24 hours after injections, but produce no necrosis. Although the minimum erythem doses vary according to animals and are difficult to determine exactly, the dose of PF of both Extract I and II for guinea pigs was 0.025mgm., that for rabbits 0.005mgm., and that for goat was 0.25mgm. The sensibility of skin reaction is variable according to the species of animal, and is to be arranged in the order of rabbit, guinea pig and goat for both PF and toxic original solutions.