70 



sugar. The least growth was in the 5 per cent alcohol. Evidently 

 the acid which was formed inhibited growth, although it did not 

 immediately kill all of the organisms. This was determined by mak- 

 ing 6 cultures from the 5 per cent alcohol on the twenty-third day (2 

 carrot, 2 potato, and 2 coconut cultures— 1 loop for each). The 

 oro-anism erew in all the.se tubes, but its development was slow. It 

 was not visible in any of them on the fourth day. The yellow growth 

 appeared in 5 of these tubes on the sixth day and in the sixth tube a 

 day or two later. A fact which shows the remarkably slow diffusion 

 of the acid is that the fluid in the closed end of the tubes (2i per cent 

 alcohol) remained alkaline while that in the open end became acid. 

 On tho thirtieth day, in 1 tube, the fluid in the bowl was " distinctly 

 acid to the blue paper and also to the pale red paper;" in the other it 

 was "'strongly acid to both red and blue papers." Nevertheless, 

 when the contents of these 2 tubes was poured out into a clean test 

 tube and thoroughly mixed it was no longer acid to either paper, but 

 had become slightly alkaline; i. e., not enough acid was produced in' 

 the open end of the tube to neutralize the sodium hydrate in the 25 

 c. c. of fluid (25 c. c. of ^ NaOH per liter). This fluid was then reduced 

 one-half by boiling, but no acid vapors appeared in the steam. 



(3) The experiments with glycerol and maltose were repeated to see 

 whether the faint clouding which finally appeared in the closed end, 

 in the experiments of 1897, should be attributed to facultative anae- 

 robism or only to some accident. The stock used was a 1:2 slightly 

 alkaline non-peptonized sugar-free beef bouillon (No. 450).^ 



To this was added 2 per cent of Schering's twice distilled c. p. 

 glycerin in the one case and 2 per cent of Merck's c. p. maltose in 

 the other. The experiments were carried through in duplicate. Hav- 

 ing been on the shelf 15 days since the last sterilization, the tubes 

 were resteamed for 20 minutes but no air bubbles appeared. Each 

 tube was then inoculated with one 3 mm. loop from a cloudy broth 

 culture 3 days old. The observations were continued 23 days. 



Beaiilt. — The tubes of glycerin bouillon clouded in the bowl and outer 

 three-fourths of the U on the second day, but remained entirely clear in 

 the closed end during the whole time. The glycerin gave no increased 

 clouding, i. e., not more than the simple bouillon. The line of demar- 

 cation in the U remained sharp. The fluid was slightly alkaline to 

 litmus when inoculated and was neutral to feebly alkaline at the close 

 of the experiment. 



The maltose bouillon was feebly clouded in the bowl and outer three- 

 fourths of the U on the second day. The line of demarcation in the 

 U was sharp on the third day. On the seventh day the bowl and outer 

 three-fourths of the U were uniformly and well clouded. This cloud- 

 ing was decidedly more than in the corresponding tubes of gh^cerin 



' Freed from muscle sugar by B. coli and clarified with white of egg. 



