306 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



992. Mueller's Hydrolyzed Casein Heart 

 Solution 



Constituents: 



1. Water 1000.0 cc. 



2. NaCl (1.0%) 10.0 g. 



3. MgS04 (0.04%) 0.4 g. 



4. CaCl. (0.02%) 0.2 g. 



5. K2HPO4 (0.2%) 2.0 g. 



6. Glucose (0.2%) 2.0 g. 



7. Phenol red (0.02% solution). 80.0 cc. 



8. Heart, beef 1.0 lb. 



9. Casein. 

 Preparation : 



(1) Prepare beef heart infusion by heat- 

 ing 1.0 pound of beef heart in 

 500.0 cc. of tap water to boiling. 

 Strain and filter. 



(2) Decolorize (1) by boiling for 25 

 minutes with 10.0% "Norit," a com- 

 mercial grade of wood charcoal. 



(3) Filter thru paper. 



(4) Prepare casein hydrolysate by boil- 

 ing commercial casein 18 hours with a 

 mixture of 6 times its weight of water 

 and 3 times its weight of concen- 

 trated H2SO4. 



(5) Free from H2SO4 by the addition of 

 Ba(0H)2. Wash precipitate with 

 water and concentrate the filtrate 

 and washings. 



(6) The quantity of casein hydrolysate 

 used in the following preparations is 

 equivalent to 0.5 g. of the original 

 casein. 



(7) Dissolve 2, 3, 4, 5, 6 and 7 in 1000.0 cc. 

 of water. 



(8) Prepare the following solutions: 



[Decolorized infusion (1).. 25.0 cc. 



(a) i Glucose salt solution (7) . . 25.0 cc. 

 [Casein hydrolysate (6) 



to 0.5 g. 



[Water 25.0 cc. 



(b) ■{ Glucose salt solution (7).. 25.0 cc. 

 [Casein hydrolysate 0.5 g. 



(9) Adjust to pH = 7.8. 



(10) Filter if necessary and tube. 



Sterilization: Sterilize at 10 pounds pres- 

 sure for 10 minutes. 



Use: To study food requirements of strep- 

 tococci and pneumococci. Author re- 

 ported growth with casein hydrolysate 

 present. No growth in other solutions. 



Reference: Mueller (1922 p. 327). 



993. Robinson and Rettger's Extract 

 Hydrolyzed Casein Solution 



Constituents: 



1. Water 1000.0 cc. 



2. Casein 50.0 g. 



3. Meat extract (Liebig's) 



(0.5%) 5.0 g. 



Preparation : 



(1) Boil 50.0 g. of casein with 10.0% HCl 

 under a reflex condenser until the 

 solution no longer responds to the 

 Biuret test. 



(2) Evaporate on the water bath until 

 nearly all the HCl is removed. 



(3) Neutralize the remaining acid with 

 NaOH. This solution is of dark 

 brown color and is known as "Ca- 

 sein C." 



(4) Dissolve 50.0 g. of (3) in 1000.0 cc. 

 water. 



(5) Decolorize by filtering thru animal 

 charcoal. 



(6) Add 0.5% Liebig's meat extract. 



(7) Heat on a boiling water bath for 

 20 minutes. 



(8) Adjust the reaction to +0.6 to phenol- 

 phthalein, and filter. 



(9) Distribute in 20.0 cc. lots in 150.0 cc. 

 Erlenmeyer flasks. 



Sterilization: Sterilize at 12 to 14 pounds 



pressure for 15 minutes. 

 Use: Cultivation of B. diphthcriae and 



toxin production. 

 Reference: Robinson and Rettger (1917 



p. 363). 



994. Groer and Srnka's Hydrolyzed 



Placenta Solution 



Constituents: 



1. Water 4000.0 cc. 



2. Placenta 4000.0 g. 



Preparation : 



(1) Mix about 4 liters (3 placenta to 

 1 liter on an average) placenta with 

 the blood, cut into medium sized 

 pieces, with 4 liters of water. 



(2) Add about 10.0 cc. concentrated HCl 

 and mix well. 



(3) Boil for about 2 hours to a brown mix- 

 ture, adding water. 



(4) Strain and filter thru paper while 

 still warm. 



(5) Add 10.0% NaOH until phenol- 



