CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



317 



Preparation : 



(1) Mince finely an average sized ox 

 heart. 



(2) Add to 400.0 cc. tap water. 



(3) Heat slowly to 75°C. 



(4) Allow to cool to 45°C. 



(5) Add trypsin solution to 1 per cent. 



Note: e.g., Liq. trypsin Co. (A 

 (and H)). 



(6) Place in incubator 21 hours. 



(7) Test for peptone by the Biuret test. 



Note: Add 1.0 cc. 5 per cent 

 copper sulphate to 5.0 cc. trypsin 

 digest, followed by 5.0 cc. N/1 po- 

 tassium hydro.xide. Note the color 

 change. If the color is pink, pep- 

 tonization is complete; if bluish 

 purple incomplete. 



(8) Make faintly acid to litmus with 

 4 per cent acetic acid. 



(9) Boil 15 minutes. 



(10) Allow the solid matter to settle. 



Note: Or simply strain thru cloth. 



(11) Pour off the supernatant fluid. 



(12) Add 1.0 g. sodium chloride and 0.5 g. 

 calcium chloride. 



(13) Make faintly alkaline to litmus. 



(14) Steam 45 minutes. 



(15) Bring up to original volume. 



(16) Adjust the reaction. 



(17) Steam 30 minutes. 



(18) Clarify by the addition of egg 

 albumin. 



(19) Tube. 



Sterilization: Sterilize in the autoclave or 

 steamer. 



Use: General culture medium. Author re- 

 ported good growth of meningococci and 

 similar organisms when pea extract or 

 serum was added. 



Variants: The author gives the following 

 variants : 



(a)|Add 25.0% of sterile rabbit or horse 

 serum. 



(b) Add 5.0% of a sterile pea extract pre- 

 pared by steaming a mi.xture of 50.0 g. 

 pea flour and 100.0 g. NaCl in 

 1000.0 cc. distilled water for 30 

 minutes, filtering thru paper and 

 sterilizing. 



(c) The medium was prepared as fol- 

 lows from horse heart, omitting the 

 NaCl and CaCh. 



(1) Mince finely fat-free horse heart. 



(2) Add 500.0 g. to 1000.0 cc. water. 



(3) Make faintly alkaline to litmus. 



(4) Heat slowly to 75°C. 



(5) Allow to cool to 45°C. 



(6) Add trypsin solution to 1 per cent 

 and 35.0 cc. chloroform. 



(7) Place in a loose stoppered bottle. 



(8) Keep 10 days at 37°C. with daily 

 shaking. 



Note: The reaction must be 

 frequently tested and made faintly 

 alkaline to litmus. 



(9) Add, at the end of this time, 

 trypsin solution again to 1 per 

 cent. 



(10) Keep a further period of 10 days 

 at 37°C. without shaking: 



Note: The reaction must be 

 frequently tested and made faintly 

 alkaline to litmus. 



(11) Make faintly acid to litmus with 

 4 per cent acetic acid. 



(12) Boil 15 minutes. 



(13) Allow the solid matter to settle 

 by placing in the ice chest over 

 night. 



(14) Pour off the supernatant fluid or 

 filter thru well-wetted, thick, filter 

 paper. 



(15) Make the reaction 1.2 per cent 

 acid to phenolphthalein. 



(16) Steam 45 minutes. 



(17) Filter. 



(18) Sterilize in the steamer or auto- 

 clave. 



Reference: Harvey (1921-22 p. 114). 



1118. Distaso's Trypsinized Serum Solution 



Constituents : 



1. Distilled water 500.0 cc. 



2. Serum (beef of sheep) 500.0 cc. 



Preparation : 



(1) Mix equal parts of water and sheep or 

 beef serum. 



(2) Digest sterile (1) for 24 hours at 60°C. 

 with a pancreatic extract from a hog 

 in the presence of chloroform. Acti- 

 vate the extract with an extract of 

 the upper portions of the small in- 

 testines. 



(3) Filter on paper. 



(4) Tube. 



Sterilization: Sterilize (1) at 120° for 15 

 minutes. Sterilize (4) method not given. 



