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precipitate filling the lower portion of 

 the tube. 

 Reference: Flint (1896 p. 4). 



911. Grace and Highberger's Ascitic Fluid 

 Extract Broth 



Constituents: 



1. Water 1000.0 cc. 



2. Beef extract (Liebig's) 



(0.3%) 3.0 g. 



3. Peptone (Difco) (1.0%) .... 10.0 g. 



4. NaCl (0.5%) 5.0 g. 



5. Glucose (0.2%) 2.0 g. 



6. Ascitic fluid (5.0%) 50.0 cc. 



Preparation : 



(1) Dissolve 2, 3 and 4 in 1. 



(2) Add 0.2% glucose and 5.0%, sterile 

 ascitic fluid that has been made 

 alkaline by the addition of 2.5% of a 

 40.0% NaOH solution. 



(3) Adjust to pH = 6.4, 6.8, 7.2, 7.6 or 8.0. 



(4) Heat for 15 minutes at 15 pounds 

 pressure. 



(5) Filter. 



(6) Tube and sterilize. 

 Sterilization: Sterilize (1) in the autoclave 



by heating at 20 pounds pressure for 15 

 minutes. Sterilize the ascitic fluid by 

 autoclaving at 15 pounds pressure for 

 15 minutes. Final sterilization in the 

 autoclave. 



Use: To study the effect of reaction on 

 the growth of Streptococcus viridans. 

 Author reported that best growth was ob- 

 tained in the pH range 6.4 and 7.6. 

 Growth in pure culture nearly as good 

 in a 1.0% glucose broth omitting the 

 ascitic fluid. 



Reference: Grace and Highberger (1920 

 p. 451). 



912. Olszewski and Kohler's Bile Extract 

 Broth 



Constituents: 



1. Gall 500.0 cc. 



2. Peptone (Witte) 50.0 g. 



3. Meat extract (Liebig) 25.0 g. 



4. NaCl 50.0 g. 



5. Trypsin 10 g. 



6. Litmus solution 600.0 cc. 



7. Lactose 75.0 g. 



Preparation: 



(1) Boil 50.0 g. Witte's peptone, 25.0 g. 

 Liebig's meat extract and 25.0 g. 



NaCl with 500.0 cc. of filtered and 

 sterile gall. 



(2) Neutralize with N/1 soda solution to 

 litmus and then add 3.5 cc. of the 

 normal soda solution. 



(3) Cool to 40°C. and add 1.0 g. trypsin 

 (Grubler's), 5.0 cc. chloroform and 

 1.0 cc. toluol. 



(4) Place in a glass stoppered flask, and 

 place in an incubator at 37°C. for 36 

 hours, shaking occasionally. 



(5) Filter thru a double filter paper, 

 moistened with gall. 



(6) Dissolve 75.0 g. lactose and 25.0 g. 

 NaCl in 600.0 cc. of Kubel and Tie- 

 mann's litmus solution (from Kahl- 

 baum's litmus). 



(7) Boil 15 minutes, filter and neutralize 

 with normal soda solution. 



Sterilization: Sterilize the filtrate from (5) 

 method not given. Sterilize (7), method 

 not given. 



Use: Indol production in water analysis. 

 Before use mix 5 parts of sterile (5) with 

 6 parts of sterile (7) and distribute in 

 10.0, 5.0, 2.5, and 1.0 cc. lots in such sized 

 containers so that 100.0, 25.0 and 10.0 cc. 

 of water under investigation may be 

 added to the flasks. 



Reference: Olszewski and Kohler (1923 

 p. 312). 



913. Rosenow's Brain Bacto Broth (Haden) 



Constituents : 



1. Distilled water 1000.0 cc. 



2. Nutrient broth (Bacto) 8.0 g. 



3. NaCl 8.0 g. 



4. Glucose (c.p.) 2.0 g. 



5. Andrade Indicator 10.0 cc. 



6. Calf brain. 



7. Marble. 

 Preparation : 



(1) Dissolve 2 and 3 in 1 by heating. 



(2) Cool and add 4 and 5. 



(3) Tube in 6 by 0.75 inch test tubes so 

 that the depth is at least 3.5 to 

 4.0 inches. 



(4) Add 3 pieces of crushed calf brain 

 about 1.0 cmm. square and two or 

 three pieces crushed marbles to each 

 tube. 



Sterilization: Autoclave for 20 minutes at 

 15 pounds pressure. 



