ZOOLOGY AND 150 TAN V, MICROSCOPY, ETC. 187 



sets to a dark brown coagulum. The eoagulum is then broken up with 

 a glass rod and added to twice its volume of absolute alcohol or methy- 

 lated spirit. A dilute solution of alkaline hasinatin is readily obtained 

 by this method in a few minutes. For complete extraction, the mixture 

 is allowed to stand overnight when, on filtering off the solution, a dark, 

 tarrj fluid, consisting of alkali hasniatin with some alkali and lipoid 

 substances, is obtained. The residue may be rubbed up with 66 p.c. 

 alcohol to extract the remainder of the hasmatin. The solution may be 

 neutralized with hydrochloric acid, when the hsematin is precipitated. 

 The precipitate is purified by successive washings with distilled water, 

 alcohol and ether. The hasmatin may also be precipitated by adding 

 twice its volume of ether to the solution, or by the use of calcium 

 chloride and ammonia, or with baryta mixture. These precipitates may 

 be washed with dilute hydrochloric acid, to remove the precipitated 

 carbonate, then with distilled water, alcohol and ether. 



Cultivation of Tubercle Bacilli from Sputum and Fasces. * 

 S. A. Petroff has elaborated a new and rapid method for the isolation 

 and cultivation of tubercle bacilli directly from sputum and fasces. In 

 each case the material is digested in a solution of sodium hydrate, and 

 cultures made subsequently on special media. The medium employed 

 consists of egg, beef or veal juice and gentian violet, and is prepared as 

 follows. Sterilize the shells of the eggs by immersion for ten minutes 

 in 70 p.c. alcohol, or by pouring hot water upon them ; break the eggs 

 into a sterile beaker, and after mixing well, filter through sterile gauze. 

 The meat juice is prepared by infusing 500 grm. of beef or veal in 

 500 c.cm. of a 15 p.c. solution of glycerin in water. Twenty-four hours 

 later the meat is squeezed in a sterile meat-press and collected in a 

 sterile beaker. One part by volume of meat juice is added to the eggs 

 and a sufficiency of 1 p.c. alcoholic gentian violet to make a dilution of 

 1 in 10,000. The medium is tubed in sterile test-tubes and inspissated 

 for three successive days ; on the first day at 85° C, on the second and 

 third at 75° C. For cultivations of the bovine type of bacillus omit the 

 glycerin and infuse the meat for 24 hours in water. 



For isolating the bacilli from sputum, equal parts of fresh sputum 

 (about 5 c.cm.) and 3 p.c. sodium hydrate are well shaken and left in 

 the incubator for twenty minutes, or till the sputum is fairly well digested. 

 The sputum is then neutralized to litmus, centrifugalized, and the 

 sediment inoculated into the above-described medium. By employing 

 this method 69 positive cultures were obtained from 69 specimens of 

 sputum from practically all stages of tuberculosis, six being negative by 

 direct microscopical examination. In many cases the growths were in 

 pure culture, the inhibitory effect of the gentian violet killing out all 

 extraneous organisms. 



The method advocated for isolating the bacilli from fasces consists 

 in collection in wide-mouthed jars, diluting with three volumes of water 

 and filtering to remove solid particles. The filtrate is then saturated 

 with sodium chloride, and the floating film resulting is mixed with an 

 equal volume of sodium hydrate. After digestion and neutralization 



* Journ. Exper. Med., xxi. (1915) pp. 38-42. 



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