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CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



of the medium to produce toxin. Heat- 

 ing only to 70°C. and filtering thru a 

 Chamberland filter gave good results. 

 Other investigators utilized a similar me- 

 dium for the production of tetanus toxin. 

 Variants : 



(a) Thoinot and Masselin used the 

 stomachs of 5 hogs, but did not 

 specify the use of 200.0 g. They also 

 used 500.0 g. beef instead of veal. 



(b) BezanQon digested the stomach for 

 24 hours instead of 12 to 24, infused 

 beef instead of veal, heated the mix- 

 ture at 115° for 15 minutes instead of 

 heating at 70 °C. in step (11) above 

 and sterilized in the autoclave at 

 115° for 15 minutes. 



(c) Besson used 250.0 g. hogs stomach 

 instead of 200.0 g. and added 7.0 cc. 

 of normal soda solution to the me- 

 dium after it had been neutralized to 

 litmus. 



(d) Wilcox studied the production of 

 tetanus toxin using one of the follow- 

 ing combinations, prepared according 

 to Martin's process: 



(1) (a) 200 g. minced stomach in 



1000.0 cc. water. 



(b) 400 g. minced stomach in 



1000.0 cc. water. 



(c) 200 g. minced stomach in 



1500.0 cc. water. 



(d) 300 g. minced stomach in 



1000.0 cc. water. 



(2) Veal infusions prepared from one of 

 the following: 



(a) 500 g. veal to 1000.0 cc. water. 



(b) 500 g. veal to 500.0 cc. water. 



(3) Add 1.0% glucose if desired. 

 Author reported that 1 part (1) (a) 

 to 1 part (2) (a) gave highest toxin 

 production with organism studied. 



(e) Dopter and Sacquepee used beef in- 

 stead of veal, added 7.0 cc. of normal 

 soda to the medium after it had been 

 neutralized to litmus and autoclaved 

 at 117°C. for 15 minutes instead of 

 heating at 70°C. as in step (11) above. 



(f) Harvey neutralized the medium to 

 litmus added 7.0 cc. of normal NaOH 

 per liter, then filtered thru well- 

 wetted thick filter paper, steamed 30 

 minutes, filtered again thru well- 

 wetted thick filter paper while hot, 

 distributed and sterilized. 



(g) Klimmer used the stomachs of 5 hogs, 

 but did not specify the use of 200.0 g. 

 He carried on the digestion for 24 

 hours and then sterilized. Equal 

 volumes of the sterile digest 

 (1000.0 cc.) and the infusion from 

 500.0 g. of veal to 1000.0 cc. of water 

 were mixed, boiled and neutralized. 

 The reaction was made slightly alka- 

 line, or adjusted to any desired pH 

 value, filtered, tubed and sterilized 

 in the usual manner, 

 (h) Park, Williams and Krumwiede in- 

 fused the veal and 1000.0 cc. of water 

 for 18 to 24 hours at 35°C., heated at 

 45 to 48°C. for one hour, then boiled 

 briskly for 30 minutes and strained 

 thru cheese cloth. This infusion at 

 70 °C. was mixed with an equal 

 volume of stomach digest at 70°C., 

 prepared according to Martin's 

 method, and heated to boiling. The 

 reaction was adjusted to -f-0.5 to 

 phenolphthalein at room tempera- 

 ture. The medium was sterilized at 

 15 pounds pressure for 30 minutes 

 after the reaction was readjusted 

 and filtered thru cotton and paper. 

 They prepared a similar medium with 

 a reaction of +1.0 to phenolphthalein 

 and added 1.0% glucose. This me- 

 dium was used for the production of 

 tetanus toxin. 

 References: Martin (1898 p. 35), Thoinot 

 and Masselin (1902 p. 23), BezanQon 

 (1920 p. Ill), Besson (1920 p. 29), Wilcox 

 (1921 p. 414), Dopter and Sacquepee (1921 

 p. 119), Harvey (1921-22 p. 99), Klimmer 

 (1923 p. 200), Park, Williams and Krum- 

 wiede (1924 p. 133). 



1007. Besredka and Jupille's Egg Stomach 

 Digest Solution 



Constituents: 



1. Distilled water. 



2. Beef or veal 500.0 to 750.0 g. 



3. Egg. 



4. Stomach (hog). 

 Preparation : 



(1) Beat the white of eggs with 10 

 volumes of distilled water, adding 

 the water little by little. 



(2) To clarify run thru a sieve covered 

 with a thin layer of absorbent cotton. 



