CULTURE MEDIA FOR CULTIVATION OF MICROORGANISMS 



627 



(6) Incubate to test sterility. 



Sterilization: Not specified. 



Use: Isolation of Treponema pallidum 

 Inoculate by forcing small bits of pal- 

 lidum-containing tissue far into the butt 

 of the tube. Other investigators culti- 

 vated a variety of organisms on similar 

 media. 



Variants : 



(a) Zinsser, Hopkins and Gilbert pre- 

 pared a similar medium as follows 

 for the cultivation of large quantities 

 of Treponema pallidum. 



(1) Prepare serum agar. 



(2) Melt (1) and pour to the height of 

 about one inch into the bottom of 

 a 200.0 cc. flask. 



(3) Drop in sterile bits of tissue. 



(4) Inoculate. 



(5) When the agar has solidified, cover 

 with a mixture of either salt solu- 

 tion and heated ascitic fluid, or 

 slightly acid broth and ascitic fluid. 

 Fill to the neck of the flask. 



(6) Add a few bits of sterile tissue. 



(7) Cover with liquid paraffin oil. 

 Authors reported that large quanti- 

 ties of Treponema pallidum may be 

 found within periods of from two 

 to four weeks. It is not necessary 

 to add the agar. The tissues may 

 be heated in the autoclave. 



(b) Fle.xner, Noguchi and Amoss culti- 

 vated organisms from poliomyelitic 

 tissue on a medium prepared as 

 follows: 



(1) Prepare 1.0% nutrient agar and 

 bouillon. 



(2) Introduce a fragment of kidney into 

 an Erlenmeyer or Florence flask of 

 100.0 cc. capacity. The inoculum 

 is placed on the kidney. 



(3) Mix equal volumes of agar and 

 ascitic fluid. (.\gar to be melted 

 and cooled at 40°C.) 



(4) Pour 15.0 cc. of (3) over the kidney 

 (giving a solid layer 1 cm. deep). 



(5) When solidified add 50.0 cc. of an 

 equal mixture of sterile ascitic fluid 

 and bouillon. 



(6) Cover with a layer of sterile para- 

 ffin to yield a layer about 1 cm. 

 deep. 



(7) Incubate at 37°C, 



(c) Rosenow and Towne cultivated pole- 



morphic streptococci (causing polio- 

 myelitis) on a medium prepared as 

 follows : 



(1) Prepare 1.5% nutrient agar. 



(2) Mix two parts (1) with one part 

 ascitic fluid. 



(3) Method of sterilization not given. 



(4) Distribute in test tubes of 0.8 cc. 

 in diameter to a depth of 13 centi- 

 meters. 



(5) Add to each tube a piece of fresh 

 sterile rabbit kidney. 



(6) Add a layer of sterile mineral oil. 



(d) Loewe and Strauss cultivated organ- 

 isms causing epidemic encephalitis 

 on a medium prepared as follows: 



(1) Mix one part sterile 2.0% nutrient 

 agar with four or five parts ascitic 

 fluid. Ascitic fluid should be ster- 

 ile, contain no bile or fibrin, and 

 have a high specific gravity. 



(2) Place kidney into tube and cover 

 with (1). 



(3) Inoculate. 



(4) Pour on or cover (2) with auto- 

 claved petrolatum and incubate. 



(e) Stitt cultivated treponemata on a 

 medium prepared as follows : 



(1) Prepare a 2.0% nutrient agar. 



(2) Adjust (1) so that it is slightly 

 alkaline. 



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

 ascitic or hydrocele fluid. 



(4) Fill 2 by 20 cm. test tubes by the 

 addition of 15.0 cc. of (3). 



(5) Place a fragment of fresh sterile 

 rabbit kidney or testicle in each 

 tube. The tissue should be in the 

 bottom of the tube. 



(6) After solidification add sterile para- 

 ffin oil so that it covers the solid 

 medium to a depth of 3 cm. 



(7) Inoculate in the bottom of the tube 

 by means of a capillary pipette. 



References: Noguchi (1912 p. 91), Flexner, 

 Noguchi and Amoss (1915 p. 92), Zinsser, 

 Hopkins and Gilbert (1915 p. 215), Rose- 

 now and Towne (1917 p. 177), Loewe and 

 Strauss (1920 p. 253), Stitt (1923 p. 53). 



1964. Gozony's Kidney Agar 



Constituents: 



1. Water 500.0 cc. 



2. Bouillon 500.0 cc. 



3. Peptone 20.0 g. 



