660 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(8) Place in a mortar and knead it with 

 alcohol (amount not specified). 



(9) Dry in a vacuum desiccator. 



(10) Prepare a 2.0% agar solution in 

 0.5% HCl. 



(11) To 90.0 CO. of (10) add 4 to 5.0 g. of 

 (9) and exactly 10.0 cc. of a 10.0% 

 Na2C03 solution. 



(12) Mix thoroly. 



(13) Boil for several minutes. 



(14) Pour immediately into Petri dishes. 



(15) Allow the Petri dishes to remain 

 uncovered until the agar has 

 solidified. 



(16) It is recommended to dry the agar 

 plates in the incubator 1 to 2 hours 

 before use. 



Sterilization: Not specified. 

 Use: Isolation of cholera vibrio. 

 Reference: Teruuchi and Hida (1912 

 p. 573). 



2052. Norris' Trypsinized Caseinogen Agar 



Constituents : 



1. Water 50,000.0 cc. 



2. Na2C03 commercial an- 

 hydrous 200.0 g. 



3. Caseinogen 10.0 lbs. 



4. Pancreatic extract 2,000.0 cc. 



5. NaCl 125.0 g. 



6. CaCU 6.25 g. 



7. Agar 2,000.0 g. 



Preparation : 



(1) Boil 25 liters of water in a large tub 

 of about 70 liters capacity. 



(2) Add 2. 



(3) Gradually shake in 3, and bring the 

 whole to the boiling point. 



(4) Add 25 liters of cold water. 



(5) Reaction should be alkaline to 

 litmus. 



(6) If temperature is not more than 

 48°C. add 2 liters of pancreatic ex- 

 tract (prepared according to Cole, 

 Lancet, July 1, 1916). 



(7) Estimate amino acids by Sorensen's 

 method. 



(8) Digest (6) for 3.5 to 4 hours at 37°C. 



(9) Then boil and filter thru muslin. 



(10) Determine amount of amino-acids 

 present again by Sorensen's method. 

 This should show an increase equiva- 

 lent to about 3.0 cc. N/10 NaOH 

 per 10.0 cc. broth. 



(11) Add 2.5 g. NaCl, 0.125 g. of CaCls to 

 each liter of the broth. 



(12) Sterilize at 115°C. for one hour. 



(13) Filter and place 100.0 cc. together 

 with 4.0 g. desiccated agar (prepared 

 according to Cunningham, Ind. 

 J. Med. Res. April, 1919) into each 

 round whiskey bottle. 



SteriUzation: Sterilize (13) at 120° for 

 two hours. 



Use: Substitute for meat in growth of 

 organisms used for vaccines, B. typhosus, 

 B. para A and B, and B. cholerae. The 

 above quantities yield about 450 bottles. 



Variants: Harvey prepared the medium as 

 follows : 



(1) Add gradually 100.0 g. commercial 

 casein to 500.0 cc. boiling water con- 

 taining 0.8% anhydrous sodium 

 carbonate. 



Note: Or double the quantity of 

 washing soda. 



(2) Raise the temperature to boiling 

 point. 



(3) Add 500.0 cc. cold water. 



(4) Make the reaction faintly alkaline 

 to litmus. 



(5) Allow to cool to 45°C. 



(6) Add 400.0 cc. pancreatic extract to 

 the mixture. 



(7) Keep 4 hours at 37°C. 



(8) Raise the temperature to boiling 

 point. 



(9) Pour the digest mixture on to a wet, 

 thick clean cloth. 



(10) Collect the fluid which drains thru 

 the cloth together with that obtained 

 by squeezing the cloth. 



(11) Filter the fluid collected thru well- 

 wetted, thick filter paper. 



(12) Bring the volume up to 1000.0 cc. 

 by the addition of water. 



(13) Add 2.5 g. sodium chloride and 

 0.125 g. calcium chloride. 



(14) Adjust the reaction. 



(15) Steam 30 minutes. 



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

 thick filter paper. 



(17) Sterilize in autoclave. 



(18) Add to 30.0 cc. sterilized casein 

 digest in large test tubes 4.0 g. 

 desiccated agar. 



(19) Sterilize in autoclave. 



