CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



617 



sterile beef bile and 1.0 cc. of a solu- 

 tion of 1.0 g. of malachite green I 

 (Hochst) in 60.0 cc. distilled water. 

 It is preferable to mix 20.0 g. of agar 

 with 0.05, 0.1, 0.15, 0.2, etc., cc. of a 

 2.0% malachite green solution and 

 pour into plates. Inoculate with 

 colon and typhoid bacilli and use 

 that dilution of malachite green 

 that inhibits the colon organism but 

 not the typhoid organism. Add a 

 corresponding amount of malachite 

 green and bile to the 100.0 cc. of 

 agar. 



Sterilizatiou : Method of sterilization of (8) 

 not given. 



Use: Cultivation of colon-typhoid group. 



Reference: Klimmer (1923 p. 212). 



1936. V. Drigalski and Conradi's Crystal 

 Violet Nutrose Agar 



Constituents : 



1. Water 2000.0 cc. 



2. Beef 3.0 lbs. 



3. Peptone (Witte) 20.0 g. 



4. Nutrose 20.0 g. 



5. NaCl 100 g- 



6. Agar 60.0 g. 



7. Litmus solution 



8. Lactose 30.0 g. 



9. Crystal violet (B. Hochst) 

 Preparation : 



(1) Allow 2 liters of water to stand with 

 3 pounds of finely chopped beef over 

 night. 



(2) Filter off the meat water and boil 

 one hour. 



(3) Filter. 



(4) Add 3, 4, 5 and boil one hour. 



(5) Filter. 



(6) Add 60.0 g. of fine agar, boil 3 hours 

 (or autoclave one hour) and make 

 slightly alkaline to litmus. 



(7) Filter and boil 3 minutes. 



(8) Boil 260.0 g. of litmus solution (pre- 

 pared according to Kubel and Tie- 

 mann, method or reference not 

 given) for 10 minutes and add 30.0 g. 

 c.p. lactose. 



(9) Boil (8) for 15 minutes. 



(10) Mix hot (9) and (7). 



(11) Again adjust to slight alkalinity. 



(12) Add 4.0 cc. of a hot, sterile 10.0% 

 water free soda solution. 



(13) Add 20.0 cc. of a freshly prepared 

 solution of 0.1 g. Crystal violet B 

 Hochst in 100.0 cc. sterile warm dis- 

 tilled water. 



(14) Pour into plates. 

 Sterilization: Not specified. 



Use: Differentiation of colon-typhoid 

 group. Authors reported that coli colo- 

 nies were red, deep red and wine red. 

 Bad. typhi colonies were small, of bluish 

 color and glossy. 



Variants: The following authors prepared 

 the medium as indicated: 



(a) Frost used a sugar free infusion broth 

 and specified that the medium be 

 sterilized for 20 minutes on 3 suc- 

 cessive days. 



(b) Harris (Heinemann). 



(1) Dissolve 20.0 g. nutrose and 40.0 g. 

 agar in 2000.0 cc. glucose free infu- 

 sion broth. 



(2) Neutralize (1) to phenolphthalein. 



(3) Autoclave at 120° for 5 minutes. 



(4) Clarify with the white of eggs and 

 filter. 



(5) Add 30.0 g. lactose, 260.0 cc. of 

 litmus solution and 20.0 cc. of a 

 0.1% aqueous solution of Crystal 

 violet. 



(6) Tube. 



(7) Sterilize in the Arnold, 

 (c) Wesbrook. 



(1) Add 680.0 g. finely chopped lean 

 beef to 1000.0 cc. water and place 

 in cold for 24 hours. 



(2) Express the juice and make up to 

 one liter. 



(3) Coagulate albumin either by vigor- 

 ous boiling 10 minutes or by heat- 

 ing at 120°C. in the autoclave. 



(4) Filter. 



(5) Dissolve 10.0 g. Witte's peptone, 

 10.0 g. nutrose and 5.0 g. NaCl in 

 (4). 



(6) Heat in autoclave at 120°C. for 

 30 minutes or boil vigorously for 

 15 minutes. 



(7) Render slightly alkaline to litmus 

 paper. 



(8) Filter. 



(9) Add 30.0 g. agar to (8). 



(10) Heat in autoclave at 120°C. for 

 30 minutes, or over flame until 

 agar is dissolved. 



