CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



289 



B. proteus were organisms used) on 

 plain agar slants. 



(3) Suspend the growth of the organism 

 in a few drops or 1.0 cc. of sterile 

 plain bouillon or sterile normal saline 

 solution. 



(4) Treat the emulsion thus obtained in 

 one of the following ways: 



(a) Dilute 0.5 cc. of the emulsion with 

 5.0 cc. of bouillon, and heat for 

 1 hour at 60°C. in each of the cases 

 listed below. 



(b) Dilute as above, but heat at 70°C. 

 for 1 hour. 



(c) Boil the emulsion for 5 minutes 

 and then centrifuge to separate the 

 clear fluid extract from the bac- 

 terial bodies. 



(d) Add 0.5 cc. of the emulsion which 

 had been heated to 60°C. for 1 hour 

 to two tubes of bouillon. Place in 

 the ice box for one week. Centri- 

 fuge one tube and use the clear 

 liquid and also the centrifuged 

 suspension. 



(5) Add from 0.1 to 0.5 cc. of one of the 

 emulsions or extracts to a tube of 

 bouillon. 



Sterilization: Sterilization of bacterial ex- 

 tracts are given in step (4). Sterilization 

 of bouillon not specified. 



Use: To study nutrition of Bacillus influ- 

 enzae. Author reported that growth 

 appeared earlier and was heavier in bac- 

 terial extract bouillon than in blood 

 bouillon. Using Bacillus proteus extract 

 growth of Bacillus influenzae occurred 

 only in 2 or 3 generations. 



Reference: Thjotta (1921 p. 765). 



944. Kitchens' Basal Sugar Free Agar 

 Solution 

 Constituents : 



1. Sugar free bouillon 1000.0 cc. 



2. Agar (0.1%) 1.0 g. 



Preparation: 



(1) Dissolve 0.1% washed or purified agar 

 in sugar free bouillon. 



(2) Tube. 



(3) Add 1.0% of the desired added nu- 

 trients. These may be added before 

 sterilization or made up in concen- 

 trated solutions, sterilized and then 

 added to sterile (2). 



Sterilization: Method not given. 



Use: Cultivation of anaerobes. The tubes 

 are heated until a control tube containing 

 1.0 cc. of 1.0% methylene blue shows con- 

 siderable reduction, then cooled to 45°C. 

 until inoculated. 



Added nutrients: 1.0% of any desired car- 

 bohydrate, alcohol, etc. may be used. 



Variants : Andrade and phenol red may be 

 used as indicators. 



Reference: Kitchens (1922 p. 36). 



945. Hegner and Becker's Blood Agar 

 Solution 



Constituents: 



1. Water 270.0 cc. 



2. NaCl (0.85%) 2.3 g. 



3. Nutrient agar (2.0%) 30.0 cc. 



4. Blood, rabbit 

 Preparation : 



(1) Add 30.0 cc. of ordinary 2.0% nu- 

 trient agar (pH 7.6) to 270.0 cc. of 

 0.85% saline solution (pH 7.6). 



(2) Tube in 10.0 cc. quantities. 



(3) Add 20 drops of blood directly from 

 a rabbit's ear to each tube of sterile, 

 melted and cooled tube of (2). 



(4) Incubate 24 hours to test sterility. 

 Sterilization: Sterilize (2) at 120°C. in the 



autoclave. 

 Use: Cultivation of Emhodomonas in- 



testinalis. This medium is a modified 



Noguchi's serum medium described by 



Wenyon. 

 Reference: Hegner and Becker (1922 p. 17). 



946. Thjotta and Avery's Blood Cell Bouillon 



Constituents : 



1. Bouillon. 



2. Red blood cells. 

 Preparation : 



(1) Prepare plain bouillon. 



(2) Adjust the reaction of (1) to pH 

 = 7.8. 



(3) Wash the red cells from 20.0 cc, 

 sterile defibrinated blood three times, 

 each time with 50.0 cc. sterile salt 

 solution. The cells are removed by 

 centrifugation. 



(4) Make up the cells in sterile distilled 

 water to the original volume of blood. 



(5) Dilute (4) with (2) in varying propor- 

 tions from 1:10 to 1:100,000. 



