662 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(3) Add 5.0 cc. pancreatic extract, 5.0 cc. 

 enterokinase solution, and 1.5 cc. 

 chloroform. 



(4) Shake to mix. 



(5) Incubate 8 days with daily shaking 

 the first three days. 



(6) Remove the flask without disturbing 

 the sediment. 



(7) Pipette off for use, with sterile pre- 



cautions, as much clear supernatant 

 fluid as possible. 



(8) Filter the remainder thru sterilized 

 filter paper. 



(9) Mix one part sterile (9) with four 

 parts sterile trypsinized caseinogen 

 agar (see Harvey's variant of me- 

 dium 2052) at 45°C. 



Sterilization: Method of sterilization of 



agar not given. 

 Use: Cultivation of B. influenzae. 

 Reference: Harvey (1921-22 p. 117). 



2057. Harvey's Blood Tryptamine Agar 



Constituents : 



1. Distilled water 3000.0 cc. 



2. Casein 100.0 g. 



3. Agar 60.0 g. 



4. Blood, defibrinated 150.0 cc. 



Preparation : 



(1) Prepare a suspension of one part 

 casein in 10 parts water in a well 

 stoppered bottle. 



(2) Shake well to break up clumps. 



(3) Adjust the reaction if necessary 

 with the help of cresol red. 



Note: The optimum reaction for 

 the tryptic digestion of casein is 

 about pH = 8.1 at which point 

 cresol red indicator solution gives a 

 reddish violet color and phenol- 

 phthalein remains colorless. 



(4) Add per liter 60.0 cc. pancreatic 

 extract and 5.0 cc. toluol. 



(5) Shake to mix. 



(6) Digest at 39°C. 10 days, with daily 

 shaking and addition of more toluol 

 if necessary. 



(7) Add per liter, 100.0 cc. 7.5% hydro- 

 chloric acid. 



(8) Steam 20 minutes. 



(9) Filter thru well-wetted, thick, filter 

 paper. 



(10) Make the reaction nearly neutral to 

 litmus with 5.0% sodium hydro.xide. 



(11) Mix one part (10) and two parts 

 water. 



(12) Make distinctly acid to litmus by 

 the cautious addition of strong 

 hydrochloric acid. 



(13) Add agar 2.0%,. 



(14) Steam 150 minutes. 



(15) Filter, while hot, thru well-wetted, 

 thick, filter paper by placing filter 

 funnel, stand, and receptacle for 

 filtrate in the steamer and steaming 

 till filtration is completed. 



(16) Add per liter of melted agar at 65 °C., 

 50.0 cc. defibrinated sheep blood and 

 the beaten up whites of 2 eggs. 



(17) Steam 50 minutes. 



(18) Strain the mixture thru a fine wire 

 sieve and squeeze the fluid out of 

 the clot. 



(19) Filter thru glass wool, taking care 

 to keep the mixture hot during the 

 process. 



(20) Adjust the reaction. 

 (21)^ Distribute in test tubes. 



Sterilization: Sterilize 15 minutes at 100°C. 



on three successive days. 

 Use: General culture medium. 

 Reference: Harvey (1921-22 p. 74). 



2058. Teague and Deibert's Serum Tryp- 

 sinized Casein Agar 



Constituents: 



1. Water 100.0 cc. 



2. Casein (10.0%) 10.0 g. 



3. Trypsin 



4. Serum (Rabbit or sheep) 



5. Agar solution 

 Preparation : 



(1) Prepare a 10.0% casein solution and 

 digest with trypsin at 37 °C. for 

 8 days. Add chloroform to prevent 

 bacterial growth 



(2) Boil and filter thru cotton. 



(3) Obtain sheep or rabbit serum from 

 freshly drawn defibrinated blood. 



(4) Heat (3) for 15 minutes at 55°C. 



(5) Add various amounts of sterile (3), to 

 an agar solution containing 20.0 g. 

 agar, and 5.0 g. NaCl per liter. 



Sterilization: Sterilize (2) in the autoclave. 



Sterilization of agar, or final sterilization, 



not specified. 

 Use: To study growth requirements of 



Unna-Ducrey bacillus. Authors reported 



