CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



655 



(9) Make a biuret and tryptophane 

 test, when both are +, the digest 

 is yellowish green and contains 

 very little undigested debris. 



(10) Transfer to large bottles and 

 steam for 10 minutes to stop 

 digestion. 



(11) Strain thru cotton or preferably 

 store over night in ice chest and 

 decant after 24 hours. 



(12) Warm (11) to 70°C. and neutralize 

 with 2N Na2C03 to litmus. 



(13) Filter into a flask. 



(14) Add 5 and 6 to (13). 



(15) Adjust to desired reaction using 

 litmus or preferably to a definite 

 H-ion concentration (pH = 7.0 

 to 7.5). 



(16) Clear (15) by adding 5-10.0% of 

 the decanted beef serum. Steam 

 45 to 60 minutes. 



(17) Remove (16) from steamer and 

 allow the clot to form as a compact 

 mass. Decant or better centri- 

 fuge the medium to remove it. 



(18) Sterilize at 100° for 30 minutes 

 on 2 successive days. 



Reference: Stickel and Meyer (1918 

 pp. 79, 81). 



2037a. Harvey's Basal Peptic Digest Agar 



Constituents: 



1. Distilled water. 



2. Veal, fat free. 



3. Minced stomach. 



4. HCl. 



5. Agar. 

 Preparation : 



(1) Clean and wash a number of pigs' 

 stomachs. 



(2) Mince (1) finely. 



(3) Add 10.0 cc. of strong hydrochloric 



acid to 1000 cc. water at 50°C. 



(4) Add 200 g. (2) to (3) and keep at 



50°C. for 20 hours. 



(5) Raise the temperature to the boiling 

 point. 



(6) Pour the mi.xture on a thick clean 

 cloth. Collect the fluid and that ob- 

 tained by squeezing the cloth and 

 its contents. 



(7) Heat the fluid to 80°C. and make 

 faintly alkaline to litmus while at 

 80°C. 



(8) Filter thru well-wetted thick filter 

 paper while hot. 



(9) Steam 30 minutes. 



(10) Filter again thru well-wetted thick 

 filter paper. 



(11) Mince finely, fat free veal, and add 

 500 g. to 1000 cc. water. 



(12) Incubate for 18 hours at 37°C. 



(13) Pour the mi.xture on a thick clean 

 cloth. 



(14) Collect the fluid, and that obtained 

 by squeezing the cloth and its 

 contents. 



(15) Mix (14) with an equal volume of 

 pepsin digest solution (10). 



(16) Heat to 70°C. 



(17) Make the reaction neutral to litmus. 



(18) Add 7 cc. of normal NaOH per liter. 



(19) Filter thru well-wetted thick filter 

 paper. 



(20) Add sufficient agar to give a solid 

 medium. 



(21) Steam 30 minutes. 



(22) Filter while hot thru well-wetted 

 thick filter paper. 



Sterilization : Method of sterilization of the 



agar base not given. 

 Added nutrients : The author used the agar 



base in the following manner: 



(A) Defibrinated blood. 



(1) Mix one part defibrinated blood 

 with three parts of distilled water. 



(2) Add 0.5 cc. of 10% NaOH to 100 cc. 

 of (1). 



(3) Sterilize (2) in the autoclave at 

 112°C. 



(4) Mix one part (3) with two parts 

 sterile agar, base at 80°C. under 

 aseptic conditions. 



(5) Slant. 



(B) Formalinized Serum. 



(1) Add 1 cc. formalin to 500 cc. horse 

 serum. 



(2) Add 1% ammonia solution to (1) 

 to neutralize to litmus. 



(3) Mix one part (2) with two parts dis- 

 tilled water. 



(4) Sterilize at 110°C. for 15 minutes. 



(5) Mix one part (4) with three parts 

 sterile agar base. 



Use: Isolation and cultivation meningo- 

 coccus. 

 Reference: Harvey (1921-22, p. 74, 81). 



