CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



475 



(6) Mix one part chloroform with 200.0 

 to 250 parts (5). 



(7) Place (5) in incubator 48 hours with 

 occasional shaking. 



(8) Test sterility. 



(9) Add one part (8) to 20 parts (4) 

 with sterile precautions, without 

 shaking. 



(10) Mix with sterile precautions at 45 °C. 

 one part (9) with seven parts 

 sterile (1). 



(11) Distribute with sterile precautions 

 into test tubes. 



(12) Slope. 



(13) Stack the test tubes in the horizontal 

 position. 



(14) Test sterility bj' incubating 48 hours. 

 Sterilization: Method not given. 



Use: Cultivation of meningococci. The 

 author reported that the medium should 

 be clear and free from all traces of red 

 color. 



Variants : Levinthal (Harvey) used one 

 part (9) with 3 parts sterile agar. 



Reference: Harvey (1921-22 p. 75). 



1644. Harvey's Litmus Blood Peptone Agar 



Constituents : 



1. Distilled water 



2. Peptone 5.0 g. 



3. NaCl 2.5 g. 



4. Glucose 5.0 g. 



5. Litmus solution ■ 20.0 cc. 



6. Defibrinated blood 

 Preparation: 



(1) Lake defibrinated blood with the 

 smallest possible amount of sterile 

 distilled water. 



(2) Prepare a medium from 1, 2, 3, 4 and 5 



as indicated in medium 595, and 

 solidify with agar. 



(3) Add a sufficient amount of (1) to (2) 



to give a deep color to the medium. 

 Sterilization: See medium 595 for steriliza- 

 tion of the agar. 

 Use: Cultivation of meningococci. 

 Reference: Harvey (1921-22 p. 76). 



1645. Tochtermann's Serum Peptone Agar 



Constituents : 



1. Water 1000.0 cc. 



2. Agar 20.0 g. 



3. Peptone 10.0 g. 



4. NaCl 5.0 g. 



5. Glucose 3.0 to 5.0 g. 



Preparation: 



(1) Dissolve 2, 3, 4 and 5 in 1. 



(2) Filter. 



(3) Mix (2) with equal parts sheep blood 

 serum or 3 parts serum to 2 parts agar, 



(4) Filter. 



(5) Tube. 



Sterilization: Sterilize in the usual manner 



(method not given). 

 Use: Diagnosis of diphtheria. The author 



reported that diphtheria colonies after 



24 hours were white. 

 Variants : 



(a) Dieudonne gave the following method 

 of preparation: 



(1) Allow sheep blood to stand for 

 24 hours. 



(2) Pour off the serum. 



(3) Dissolve 10.0 g. peptone, 5.0 g. 

 NaCl and 20.0 g. agar in 1000.0 cc. 

 water. 



(4) Filter and add 3.0 to 5.0 g. glucose 

 to (3). 



(5) Add an equal volume (or 3 parts 

 serum to 2 parts agar) of (2) and 

 boil for 15 or 30 minutes. 



(6) Filter. 



(7) Distribute into test tubes and 

 sterilize in streaming steam for 1 to 

 I5 hours on each of 3 successive 

 days. 



(b) Kolle and Wasserman prepared the 

 medium as follows: 



(1) Dissolve 20.0 g. agar, 10.0 g. pep- 

 tone, 5.0 g. NaCl, and 3.0 to 5.0 g. 

 glucose in 1000.0 cc. water. 



(2) Mix (1) with equal parts sheep 

 serum (or in the ratio of 2:3). 



(3) Boil for 75 to 90 minutes. 



(4) Filter. 



(5) Tube. 



(c) Besson specified the use of Chapo- 

 teaut's or Witte's peptone, heated in 

 the autoclave at 115 to 120°C. for 

 30 minutes and sterilized the medium 

 at 115°C. 



(d) Harvey specified the use of calf serum, 

 boiled for 20 minutes and sterilized 

 for 50 minutes at 100°C. or 30 minutes 

 at 117°C. 



