526 Bulletin 167 



The details necessary for duplication of our media are given 

 below for the convenience of any one who may have occasion 

 to review the results. Unless otherwise noted, media were pre- 

 pared with a distilled water of high purity and adjusted against 

 phenolphthalein to a reaction of +10 Fuller's scale. Sterilization 

 was effected by exposing in the autoclave at 6 pounds pressure 

 for 15 minutes, except with milk, potato, gelatin, and carbohy- 

 drate and glycerin media which were steamed in the Arnold for 

 15 minutes on each of three successive days. 



Nutrient broth. — Ten grams of "Witte's Peptonum Siccum" 

 and 5 grams of "Liebig's Extract of Meat" were added to one 

 liter of distilled water and cooked in a double cooker until dis- 

 solved (about 30 minutes), restored to volume, titrated and ad- 

 justed in reaction to +10 Fuller's scale, cooked in double cooker 

 20 to 30 minutes, boiled 5 minutes over the open flame, titrated 

 and adjusted in reaction if necessary, filtered and sterilized. 



Agar medium. — -This medium was prepared by adding 1.5', 

 of Bausch and Lomb agar flour to the nutrient broth, with sub- 

 sequent autoclaving, adjusting of reaction to +10 Fuller's scale, 

 and clearing with either fresh white of egg or dried albumen. 



Gelatin medium. — Nutrient broth was stiffened with 12' i 

 of "Nelson's Photographic Gelatin No. 1" unless otherwise speci- 

 fied. The broth was heated, the gelatin added, steamed a few 

 minutes in the Arnold, adjusted to +10 Fuller's scale, cooled, 

 cleared with egg albumen, filtered, and sterilized by the inter- 

 mittent method. 



REJUVENATION AND STOCK 



The organisms were rejuvenated as recommended by trans- 

 ferring to nutrient broth, incubating 24 hours, transferring from 

 the young culture to a second tube of broth, incubating 24 

 hours again, and so on for three days. Plates were poured 

 from the third tube of broth to insure the purity of the 

 strain and transfers from the colonies were made to tubes of agar 

 and broth. The agar .and broth series was transferred monthlv 



