308 WATER HYGIENE 
This was oxidized to acetyl-methyl-carbinol. 
H H Od8 
woot 
HOH OR 
When this was mixed with potassium hydroxide, in the presence of 
peptone, an eosin-like color was obtained after a period. Walpole (1910) 
found that under aerobic conditions Bacillus aerogenes gave a greater 
amount of acetyl-methyl-carbinol. The coloration was not secured when 
butylene glycol or acetyl-methyl-carbinol were mixed with potassium 
hyrdoxide. Harden (1905) later explained this by finding that the 
acetyl-methyl-carbinol was oxidized to diacetyl CHsCO—CO—CHs. 
This, in some way, acted with the pepton to give a pink color. Harden 
and Norris (1911) later secured the same pink color between diacety] 
and arginine, dicyanamide, creatin and gaunidine acetic. They state 
that the reaction depends upon the group NH==-C—-N=HR, R has not 
NHe 
been determined. 
Levine demonstrated that the Voges-Proskauer reaction had much 
sanitary significance and that it would distinguish between fecal and 
non-fecal strains of Bacillus colon. He states that the natural habitat 
of those strains which form the acetyl-methyl-carbinol is probably the 
soil. Bacillus colt from non-fecal origins would then be methyl red and 
V-P plus. 
Standard Methods gives the following procedure for the differen- 
tiation of fecal from non-fecal members of the B. coli group. 
American Public Health Association Method for the Differentiation 
of Fecal and Non-fecal B. coli. (1) At least ten cultures should be 
used. If possible these should be sub-cultured from plates made direct 
from the water since all the cultures obtained by plating from fermenta- 
tion tubes may be descendants of a single cell in the water. If cul- 
tures from water plates are not available those obtained from the plates 
made as prescribed under B may be used. 
(2) Inoculate each culture into dextrose potassium phosphate broth,* 
* The preparation of this medium has been described in the chapter on media. 
Since devismg this medium, these investigators (Clark and Lubs, 1917) have de- 
scribed the preparation of a substitute. The new medium is a synthetic solution 
without Witte’s peptone. Aspartic acid is used in its place, thus satisfying the 
nitrogen requirements and acting as a buffer. The preparation of this new medium 
is also given in the chapter on media. 
