322 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(3) Place for 3 to 5 days in the incubator, 

 shaking occasionally. 



(4) Each day test the mixture for trypto- 

 phane, by adding a few drops of 

 acetic acid to a sample and then 

 adding bromine water. 



(5) When the tryptophane content has 

 reached the maximum and the 

 tjTOsin has separated out as white 

 clumps, heat the fluid for a short 

 time at 80 °C. 



(6) Filter and neutralize with a few cc. 

 of HCl. 



(7) Heat moderately in a vacuum if 

 possible and evaporate to a syrupy 

 thickness. (Possibly may be fil- 

 tered again.) 



(8) Place in a mortar and knead it with 

 alcohol (amount not specified). 



(9) Dry in a vacuum desiccator. 



(10) This yields a light yellow colored 

 powder, which dissolves quite easily 

 and clear in water. The yield is 

 nearly quantitative. 



(11) Add 0.5% NaCl and 1.0% NajCO, to 

 a 5.0% watery solution of (9). 



Sterilization: Not specified. 



Use: Enrichment of cholera vibrio, toxin 

 production by Proteus vulgaris and gen- 

 eral culture medium. 



Variants : 



(a) Berthelot prepared a pancreatic ca- 

 sein digest, and dissolved 20.0 g. of 

 it in a liter of water. The medium 

 was sterilized with ether, method not 

 given, and used to produce toxin by 

 Proteus vulgaris. 



(b) Harvey prepared a "tryptamine" 

 medium as follows: 



(1) Prepare a suspension in a well 

 stoppered bottle: casein 1; dis- 

 tilled water 10. 



(2) Shake well to break up clumps. 



(3) Adjust the reaction if necessary 

 with the help of cresol red. 



Note: The optimum reaction 

 for the tryptic digestion of casein 

 is about pH = 8.1 at which point 

 cresol red indicator solution gives 

 a reddish violet color and phenol- 

 phthalein remains colorless. 



(4) Add per liter: Pancreatic extract 

 60.0 cc, toluol 5.0 cc. 



(5) Shake to mix. 



(6) Digest at 39 °C. for 10 days, with 

 daily shaking and addition of more 

 toluol if necessary. 



(7) Add per liter, 100.0 cc. 7.5 per cent 

 hydrochloric acid. 



(8) Steam 20 minutes. 



(9) Filter thru well-wetted, thick filter 

 paper. 



(10) Make the reaction nearly neutral 

 to litmus with 5% sodium hy- 

 droxide. 



(11) Preserve as stock tryptic broth or 

 stock "tryptamine." 



(12) Dilute for use— "tryptamine" 1: 

 water 2 = tryptamine bouillon. 



(c) Harvey prepared a similar medium as 

 follows: 



(1) Add very gradually 200.0 g. casein 

 to 1000.0 cc. boiling water contain- 

 ing 20.0 g. anhydrous sodium 

 carbonate. 



(2) Allow to cool to 45°C. 



(3) Add pancreatin 3.0 g. or pan- 

 creatic extract 50.0 cc, chloroform 

 15.0 cc 



(4) Place in incubator 5 days, shaking 

 vigorously each day to break up 

 clumps. 



(5) Add again pancreatin 3.0 g. or 

 pancreatic extract 50.0 cc. 



(6) Place in incubator again for 10 

 days. 



(7) Add 400.0 cc. N/1 hydrochloric 

 acid. 



Note: Or 400.0 cc. pure con- 

 centrated hydrochloric acid di- 

 luted with 350.0 cc water. 



(8) Steam 30 minutes. 



(9) Filter, while hot, thru well-wetted, 

 thick filter paper. 



(10) Add 120.0 cc. N/1 sodium hy- 

 droxide to the filtrate. 



(11) Adjust reaction. 



(12) Dilute for use 1/3 with 0.5 per cent 

 sodium chloride. 



(13) Sterilize in the autoclave or 

 steamer. 



References: Teruuchi and Hida (1912 

 p. 572), Berthelot (1914 p. 916), Harvey 

 (1921-22 p. 116). 



1126. Zipfel's Trypsinized Casein Solution 



Constituents: 

 1. Distilled water 1000.0 cc. 



