554 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



heating at 15 pounds pressure for 15 

 minutes. 



Use: Differentiation of colon-typhoid-dy- 

 sentery group. 



Reference: Bailey and Lacy (1927 p. 185). 



1778. Holt, Harris and Teague's Eosine 



Methylene Blue Agar 

 Constituents : 



1. Water 1000.0 cc. 



2. Agar 15.0 g. 



3. Peptone (Witte's) 10.0 g. 



4. NaCl 5.0 g. 



5. Beef extract (Liebig's) 5.0 g. 



6. Sucrose 5.0 g, 



7. Lactose 5.0 g. 



8. Eosin (2.0% yellowish) 20.0 cc. 



9. Methylene blue (0.5%) 20.0 cc. 



Preparation : 



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



(2) Clear with egg white. 



(3) Flask. 



(4) Adjust sterile (3) to -fO.8. 



(5) To melted (4) add 6 and 7 and heat 

 for 10 minutes in the Arnold. 



(6) To every 60.0 cc. of the medium add 

 1.0 cc. of 2.0% yellowish eosin in 

 distilled water and then 1.0 cc. of 

 0.5% methylene blue in distilled 

 water 



(7) Mix thoroughly, and pour in sterile 

 Petri dishes. 



(8) Dry the surface in usual way and 

 inoculate. 



Sterilization: Sterilize in Arnold on three 

 successive days. 



Use: Isolation of Bacillus typhosus from 

 stools. Author reported that typhoid 

 colonies were colorless and transparent. 

 Colon colonies were black and did not 

 transmit light. Sugar-free infusion may 

 be substituted for Liebig's meat extract. 



References: Holt-Harris and Teague (1916 

 p. 597), Stitt (1923 p. 49), Park, Williams 

 and Krumwiede (1924 p. 128). 



1778a. Krumwiede, Pratt and McWilliams 

 Brilliant Green Agar 



Constituents : 



1. Water 1000.0 cc. 



2. Agar 15.0 g. 



3. Salt (NaCl) 5.0 g. 



4. Peptone (Witte) 10.0 g. 



5. Beef extract (Lie- 

 big's) 3.0 g. 



6. Lactose 10. g. 



7. Glucose 1.0 g. 



8. Brilliant green (0.1% 



solution) 2.0 or 3.0 cc. 



9. Andrade Indicator. . . 10.0 cc. 

 Preparation : 



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

 autoclave. 



(2) Adjust so that the reaction is slightly 

 alkaline to litmus. 



(3) Distribute in 100.0 cc. lots. 



(4) Before use readjust so that the 

 medium be set to Andrade Indicator. 

 The reaction to phenolphthalein 

 (hot titration) is 0.6 to 0.7% acid. 



(5) Add Andrade indicator (amount not 

 given). 



(6) Add 1.0% lactose and 0.1% glucose to 

 sterile (5), using sterile 25.0% 

 solutions. 



(7) Add appropriate amounts of 0.1% 

 solution of brilliant green (0.2 cc. or 

 0.3 cc. of a 0.1% solution per 100.0 

 cc. agar). 



(8) Pour in sterile Petri dishes. 



(9) Allow plates to stand open until 

 surface is dry. Inoculate as with 

 Endo plates. 



Sterilization: Sterilize (3) in the autoclave. 



Use: Isolation of typhoid bacilli. Author 

 reported that fecal types were restrained. 

 Typhoid colonies were large and pre- 

 sented a snow flaky appearance with 

 black background by light passing 

 obliquely thru agar. Medium gave 36.0% 

 positive increase over Endo agar. 



Variants : 



(a) Krumwiede, Kohn, Kuttner and 

 Schumm prepared the medium as 

 follows: 



(1) Dissolve 15.0 g. agar in 500.0 cc. 

 of water by heating over gas 

 stove. 



(2) Dissolve 3.0 g. of Liebig's extract 

 of beef, 10.0 g. peptone and 5.0 g. 

 NaCl in 500.0 cc. of 1 by heating 

 over gas stove. 



(3) After complete solution mix (1) 

 and (2). 



(4) Adjust to 0.6 or 0.7% acid to 

 phenolphthalein (neutral to And- 

 rade indicator). 



(5) Prepare Andrade indicator by 

 adding 16.0 cc. of a N/1 NaOH 



