296 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



and 2000.0 cc. of bouillon with a re- 

 action of +0.6 to phenolphthalein. 



(c) Besson mixed ^, 3 or J the volume of 

 sterile ascitic fluid with sterile 

 bouillon. 



(d) Kligler and Robertson cultivated 

 Spirochaeta obermeieri in a medium 

 prepared as follows: 



(1) Prepare a 10.0% peptone broth 

 (exact method not given). 



(2) Add 1.0% of (1) to undiluted ascitic 

 fluid. 



(3) Adjust to pH = 7.2. 



(4) Distribute in 3.0 to 4.0 cc. lots into 

 test tubes approximately 1.0 cm. 

 in diameter. 



(5) Inoculate with 1 drop of infected 

 blood, or if subcultures are being 

 made, add a drop of fresh rabbit 

 blood and rotate the tube gently. 

 (This furnishes the necessary 

 fibrin.) 



(6) Cover with a layer of oil about 

 1.5 cm. high. 



They reported that the spirochetes 



grew well at 37°C. but degeneration 



changes tended to set in early. 



After the culture was well started, 



growth proceeded well at room 



temperature. 



References: Veillon (1898 p. 24), Elser and 



Huntoon (1909 p. 382), Besson (1920 p. 31), 



Kligler and Robertson (1922 p. 315). 



966. Kahn's Casein Digest Ascitic Fluid 

 Bouillon 



Constituents : 



1. Bouillon 1000.0 cc. 



2. Casein (2.0%) 20.0 cc. 



3. Ascitic fluid 200.0 cc. 



4. CaCOs 

 Preparation : 



(1) Preparation of bouillon not given. 



(2) Mix 1 part ascitic fluid and 5 parts 

 (1) adjusted to +0.3. 



(3) Add CaCOs (amount not given). 

 (This is Lyall's broth.) 



(4) Add 2.0% casein digest as prepared 

 by Kahn (see 648). 



(5) Tube. 



(6) Seal the tubes with a vaseline cap by 

 boiling the medium and vaseline. 



Sterilization: Not specified. 

 Use: To determine hemolysis by spore 

 forming anaerobes. The medium was 



inoculated thru the liquid vaseline and 

 cooled quickly, incubated for 24 hours 

 and 0.1, 0.2, 0.4 and 0.5 cc. of the culture 

 transferred to 1.0 cc. of a 5.0% suspension 

 of washed fresh sheep erythrocytes. Add 

 sufficient physiological salt solution to 

 each tube to bring the volume to 20.0 cc. 

 and incubate at 37°C. for one hour. 

 Reference: Kahn (1922 p. 198). 



967. Lyall's Carbonate Ascitic Fluid 

 Bouillon 



Constituents : 



1. Bouillon 1000.0 cc. 



2. Ascitic fluid 200.0 cc. 



3. CaCOa 

 Preparation : 



(1) Preparation of bouillon not given. 



(2) Mix one part ascitic fluid and 5 parts 

 of (1). 



(3) Add CaCOs (amount not given). 

 Sterilization: Not specified. 



Use: To determine hemolysis by strepto- 

 cocci. Grow the organism in the medium 

 for 18 hours. Add a definite amount of 

 the 18 hour broth culture to 1.0 cc. of a 

 5.0% solution of washed red blood cells 

 of a sheep. Incubate in a water bath for 

 1 hour at 37.5°C. Author reported that 

 three degrees of hemolysis are possible (1) 

 complete solution of erythrocytes, (2) 

 change of undissolved cell from a bright 

 red to a dark brown due to a transforma- 

 tion of oxyhemoglobin to methemo- 

 globin, but cells are not dissolved, (3) no 

 hemolysis or change in color of the cells. 



Reference: Lyall (1914 p. 497). 



968. Roddy's Bile Bouillon 



Constituents: 



1. Bouillon 1000.0 cc. 



2. Ox bile 1000.0 cc. 



Preparation: (1) Mix equal parts of ox bile 



and bouillon. 



Sterilization: Sterilize in the steam steril- 

 izer for 20 minutes on each of 3 successive 

 days. 



Use: Culture medium for parasitic and 

 saprophytic organisms. 



Reference: Roddy (1917 p. 42). 



969. Mayer's Mucin Bouillon 



Constituents: 



1. Bouillon. 



2. Mucin (Merck). 



