640 



CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



(3) Mix sterile (2) with 85.0 cc. neutral 

 agar. 

 Sterilization: Sterilize (2) in streaming 



steam. 

 Use: Isolation of cholera vibrio. 

 Variants : 



(a) Costa gave the following method of 

 preparation : 



(1) Pulverize crystalline blood in a 

 mortar. 



(2) Add distilled water slowly to (1). 

 This forms sort of a sticky mixture. 



(3) Add 100.0 cc. of a normal KOH 

 solution to (2). 



(4) Heat in the autoclave for 30 

 minutes. 



(5) Filter. 



(6) Sterilize by heating in the auto- 

 clave at 100°C. for several hours. 



(7) Mix 3 volumes of (6) with 7 volumes 

 of sterile melted agar under aseptic 

 conditions. 



(8) Pour into sterile Petri dishes. 



(b) Kohlisch and Otto prepared the 

 medium as follows: 



(1) Rub 15.0 g. of Merck's horse haemo- 

 globin in a mortar. 



(2) Mix 15.0 cc. of N/1 NaOH with 

 (1), and add 15.0 cc. distilled 

 water. 



(3) Sterilize for one hour in the 

 steamer. 



(4) To 15.0 cc. of (3) add 850.0 cc. of 

 neutral agar. 



(5) Mix thoroly. 



(6) Pour in plates. 



(7) Dry the plates at room tempera- 

 tures for one hour. 



(c) Klimmer used the following method 

 in preparing the medium: 



(1) Dissolve 50.0 g. of Merck's hemo- 

 globin in 300.0 cc. of half normal 

 KOH by heating in a steamer for 

 an hour. 



(2) To hot (1) add 1700.0 cc. of melted 

 hot neutral agar. 



(3) Pour into plates. 

 References: Esch (1910 p. 559), Costa (1912 



p. 846), Kohlisch and Otto (1915 p. 438), 

 Klimmer (1923 p. 218). 



1999. Kabeshima's Alkaline Hemoglobin 

 Agar 



Constituents : 

 1. Nutrient agar (3.0%) 80.0 cc. 



2. Soda (18.0% solution) 10.0 cc. 



3. NaCl (0.85% solution) 10.0 cc. 



4. Hemoglobin extract (Pfeuffer) 3.0 g. 

 Preparation : 



(1) Place 80.0 cc. of melted neutral nu- 

 trient 3.0% agar in an Erlenmeyer 

 flask. 



(2) Add 10.0 cc. of 18.0% soda solution 

 and boil this mixture for about 10 

 minutes. 



(3) Cool to 50°C. 



(4) Dissolve 3.0 g. hemoglobin extract 

 (Pfeuffer) in 10.0 cc. of 0.85% NaCl 

 solution. 



(5) Mix (4) and (3) thoroly. 



(6) Pour into 7 Petri dishes and allow to 

 stand with covers removed until 

 solidification has taken place. 



(7) Place the dishes in an incubator with 

 cover removed for 20 to 30 minutes to 

 remove the water of condensation. 



Sterilization: Not specified. 



Use: Selective medium for cholera vibrio. 

 Author reported that medium was trans- 

 parent and dark brown. Some cholera- 

 like vibrio and other organisms were 

 inhibited on this medium. 



Variants: Klimmer prepared the medium 

 in the following manner: 



(1) Dissolve 3.5 g. of Pfeuffer's hemo- 

 globin extract in 10.0 cc. of physio- 

 logical salt solution. 



(2) Add 10.0 cc. of a 5.5% solution of 

 water free soda and 2.0 cc. of KOH 

 solution (strength not given) to (1). 



(3) Steam for 15 minutes. 



(4) Cool to 50°C. 



(5) Add (4) to 80.0 cc. of 3.0% melted 

 nutrient agar, neutral to litmus and 

 cooled to 80 or 90°C. 



(6) Mix thoroly. 



(7) Pour in plates. 



(8) Dry thoroly in the incubator. 

 References: Kabeshima (1913 p. 203), 



Klimmer (1923 p. 220). 



2000. Besson's Glycerol Hemoglobin Agar 

 (Tanner) 



Constituents: 



1. Glycerol agar. 



2. Hemoglobin. 

 Preparation : 



(1) Melt tubes of glycerol agar and cool 

 to 40°C. 



(2) Add 1.0 cc. of a solution of hemoglobin 



