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DIPHTHERIA tOXINE. 73 



the addition of fragments of marble to the cultivation. 



oLLE, 1 differing from SPRONCK, has recommended the use of 

 quite fresh flesh. MARTIN 2 has obtained successful results by 

 the use of a mixture in equal parts of extract of beef, or, better, 

 veal, and of extract of pig's stomach. 



He gives the following recipes : 



(i.) Extract of Pig's Stomach. Five pigs' stomachs are finely minced or 

 crushed, and the whole (mucosa and muscidaris) mixed with 10 grms. of 

 pure hydrochloric acid (20 per cent.) and 1,000 grms. of water to each 200 

 grms., and allowed to stand for twenty-four hours at 50 C. The mixture 

 is then boiled and filtered through loosely-packed cotton wool, and the 

 filtrate mixed with 0'2 per cent, of acetic acid, neutralised while still hot, 

 filtered through paper, and heated to 120 C. in an autoclave. The last 

 process can also be replaced by repeated heating to 100 C., careful 

 removal of the albuminous scum and fat, and, finally, clarification by the 

 addition of calcium chloride and sodium phosphate (formation of a pre- 

 cipitate of calcium phosphate which simultaneously carries down the 

 albuminous turbidity). 



(ii.) Meat Extract. Perfectly fresh veal is allowed to stand for twenty 

 hours at 35 C., after which it is treated with 1,000 grms. of water for each 

 500 grms. of material, and again left for twenty hours at the same tem- 

 perature. It is then expressed, and the resulting liquid mixed with 

 5 grms. of sodium chloride and 20 grms. of peptone, neutralised, and, after 

 the addition of 7 c.c. of normal soda solution, sterilised at 120 C. 



A mixture of equal parts of the two extracts, heated to 70 C. and 

 sterilised by filtration through Chamberland filters, forms an excellent 

 medium which does not become acid and in which very active toxines are 

 speedily formed, O'l c.c. being required for a lethal dose after thirty hours, 

 and 0'002 c.c. after five to seven days. 



MADSEN's 3 method is to leave finely-minced lean veal, two to three days' 

 old, in contact with twice its weight of water for fifteen hours, after which 

 it is boiled and strained. The liquid is then mixed with 1 per cent, of 

 Witte's peptone and 1 per cent, of sodium chloride, rendered faintly 

 alkaline, boiled for forty-five minutes, filtered, and the filtrate transferred 

 to Erlenmeyer flasks holding a litre, and sterilised for fifteen minutes at 

 120 C. in autoclaves. He determines the reaction with litmus paper, and 

 also the "titer" of the bouillon i.e., the amount of normal soda solution 

 required to give a distinct pink coloration with phenol-phthalein as indi- 

 cator. The ratio of the two measurements is such that neutral solutions 

 showing a "titer" of over 20 c.c. are unmistakably acid to litmus, while 

 with 16 c.c. they are amphoteric, and below 10 c.c. are alkaline. Accord- 

 ing to his experience the age of the flesh has no material influence. The 

 addition of calcium carbonate recommended by SPRONCK (loc. cit. ) and VAN 

 TURENHOUT (loc. cit.) keeps the reaction permanently alkaline, but at the 

 same time does not materially promote the formation of toxine. 



1 Nicolle, "Preparation de la toxine diphte'rique," Ann. Past., x., 333, 

 1896. 



>2 Martin, "Production de la toxine diphte'r," ibid., xii., 26, 1898. 



3 Madsen, "Zur Biologic des Diphtheric bacillus," Zeit. f. Hyg., xxvi., 

 157, 1897. 



