THE ALCOHOL TEST IN KELATION TO MILK. 
15 
In order to show the effect of rennet of bacterial origin, the action 
of a pure culture of a rennet-forming organism was studied. Two 
flasks of sterile skim milk were inoculated with different amounts of a 
pure culture of a rennet-forming organism. These flasks were 
incubated at 37° C, and the bacterial increase was determined at 
definite intervals, together with the alcohol test. The results are 
shown in Table 11. From a study of the table it is evident that 
rennet-forming bacteria will cause a positive alcohol test, but there 
must be a large bacterial increase to produce rennet enough to cause 
a positive test. 
The acidity was also increased during the incubation, but we believe 
this acidity played a minor part in causing the positive alcohol test. 
Table 11. — Influence on the alcohol test of rennet produced in milk by the growth of a pure 
culture of a rennet-forming organism. 
Experi- 
ment 
No. 
Age of 
cul- 
ture. 
Bacteria 
per cubic 
centimeter. 
Acid- 
ity. 
Alcohol test. 
75 per 
cent. 
68 per 
cent. 
44 per 
cent. 
I 
II 
Hours. 

2 
4 
5 
6 
7 

2 
4 
34,000 
62,000 
4,700,000 
9,000,000 
21,000,000 
31,000,000 
147,000 
200, 000 
15,000,000 
1.98 
"""2*62' 
2.06 
2.10 
2.11 
1.94 
2."i6" 
+ L 
+ L 
+ L 
+ L 
+ L 
+L 
+ L 
+s 
1 See footnote under Table 1. 
DIFFERENTIATION BETWEEN ACIDITY AND RENNET ACTION. 
The fact that reducing the acidity did not cause a negative alcohol 
test, as mentioned above, led us to believe that it might be possible 
to differentiate between a positive alcohol test caused by acidity and 
one caused by rennet action. In order to determine whether this 
was true two flasks of sterile skim milk were prepared. One was 
inoculated with a pure culture of a lactic-acid-forming organism and 
the other with equal amounts of a pure culture of lactic-acid bacteria 
and rennet-forming bacteria. The two flasks were then incubated at 
37° C. As may be seen from Table 12, the milk containing the 
lactic-acid bacteria had an acidity of 2.23 after 3 hours' incubation 
and the test was positive with both 75 per cent and 68 per cent 
alcohol. When the acidity was reduced to 1.49 all the alcohol tests 
were negative. The milk containing a mixed culture of lactic-acid 
bacteria and rennet-forming bacteria after 3 hours' incubation had 
an acidity of 2.32 and the alcohol test was positive with 75 per cent 
and 68 per cent alcohol. In both cases the coagulation was in the 
form of large flakes. When the acidity was reduced to 1.70 the 
