22 



C, and finally at 100° C; squeezed fluid through a clean towel, and steamed again 



for 1 hour. Filtered through S. & S. paper, flasked, and sterilized formmg stock 204. 



Fermentation tubes of this broth (25 c. c.) yielded about 1^ c. c. of gas with Bacillus 



doacse, showing the presence of muscle sugar. 1,700 c. c. of 204-f-255 gr. gelatin 



(Coignet Pere et Cie gelatine extra) . Gelatin soaked in broth 1^ hours, then heated 



in steamer to 100° C, cooled, added whites of 6 eggs (previously neutralized with 



c. p. HCl) to clarify, steamed, filtered, flasked, and steamed, added 17 gr. "Witte's 



peptonum siccum, steamed and filtered; 1,500 c. c. of acid fluid remained; when 



N 

 titrated with caustic soda, 3.4 c. c. -ttt NaOH were required to exactly neutralize 10 



c. c, using phenolphthalein as indicator; i. e., the acidity was +34. Reduced the 



2N 

 acidity to +25 by adding 500 c. c. of distilled water. Then added 25 c. c. -y soda, 



and steamed, filtered, flasked, and sterilized. The gelatin was steamed as short a 

 time as possible on each occasion. This stock contained about 13 per cent of gelatin. 



(2) Malic acid gelatin. — 680 gr. finely minced, lean beef; 1,500 c. c. distilled water. 

 Mixed and put into the ice box for 24 hours. Boiled three-fourths hour, filtered, 

 cooled to 70° C, added 3 gr. NaCl, 12 gr. Witte's pept. sic, 100 gr. of brown German 

 gelatin, and when the gelatin was dissolved the whites of 6 eggs, which were incor- 

 porated by repeated pourings. Steamed 40 minutes at 100° C, filtered through 

 flannel, and titrated with caustic soda and phenolphthalein — acidity +45. Measured 

 out 300 c. c. into each of three flasks and exactly neutralized each to phenolphtha- 



2N 

 lein by addition of 6.7 c. c— y-NaOH. To one flask was then added 7.8 c. c, to 



another 10.4 c. c, and to the third 11.7 c. c. of a mahc acid water, 1 c. c. of which 

 exactly balanced 13.75 c. c.-ttt NaOH; i. e., enough acid so that the gelatins should 



be approximately +36, +48, and +54 of Fuller's scale. These three acid gela- 

 tins were then filtered, tubed, and sterilized by steaming for a few minutes on each 

 of three consecutive days. These stocks contained 8 per cent of gelatin. 



(3) Stock £44c.— 2,000 c. c. distilled water, 1,000 gr. lean minced beef, 300 gr. 



gelatin (one-half white French, one-half brown German), 20 gr. Witte's peptonum 



siccum. The beef was soaked in 1,200 c. c. distilled water for 1 hour, then brought 



slowly up to 65° C. on a water bath with constant stirring, then steamed 1 hour and 



filtered. The gelatin was soaked for a few hours in 1,000 c. c. of distilled water and 



then added to the hot beef broth, along with the peptone. Steamed 45 minutes, 



filtered, added 50 c. c. water to make up the 2,000 c. c. Fluid acid to litmus. Titrated 



N 

 with caustic soda, using phenolphthalein as an indicator. 2.85 c. c. — NaOH was 



required to exactly neutralize 5 c. c. of gelatin, i. e., the acidity was -i-57. 14 c. c. of 



2N 



-j-NaOH was then added to 500 c. c. of the medium to make stock 244c, which was 



then flasked and sterilized. This stock, which contained 15 per cent of gelatin, was 

 not perfectly solid at 24° C. A fermentation tube of the beef broth (25 c. c.) used 

 for this gelatin yielded several c. c. of gas upon inoculating with Bacillus cloacx indi- 

 cating the presence of muscle sugar. 



To test the effect of varying grades of alkalinity, stock 2tW:C was 

 compared with three other portions of the same gehitin to which dif- 

 ferent amounts of caustic soda were added, viz. (1) with a gelatin con- 



N 

 taining, per liter, -iOc. c. less of -3- NaOH (stock 244a); (2) Avith one 



containing, per liter, 20 c. c. less of the normal soda solution (244b); 

 and, finally, (3) with one containing, per liter, 25 c. c. more of the nor- 

 mal soda solution (244d). These gelatins I common]}' designate, fol- 



