16 
unhydrolyzed casein at intervals with 1 per cent acetic acid, then 
making total nitrogen determinations in the precipitates as recom- 
mended by Walters. 1 Ammonia was determined in the filtrates by 
distilling with an excess of magnesia. In order to have a check on 
autohydrolysis, which is known to take place in solutions of casein, 
a number of the flasks were set aside without the addition of infu- 
sions and bacterial action prevented by the addition of 0.2 cubic 
centimeter of toluol. The casein was precipitated from these at inter- 
vals by adding 20 cubic centimeters of 1 per cent acetic acid and the 
nitrogen determined as above. The data showing autohydrolysis 
will be submitted first. 
Autohydrolysis of casein in solution. 
[Average of 2 samples.] 
Period of incubation. 
N present. 
N precipi- 
tated. 
N hydro- 
lyzed. 
Per cent of 
Nhydro- 
lyzed. 
lhour 
3 days 
9 days 
Mg. 
94.3 
94.3 
94.3 
Mg. 
90.4 
89.6 
87.6 
Mg. 
3.9 
4.7 
6.4 
4.13 
4.96 
6.79 
The above data show that in one hour's time 4.13 per cent of the 
nitrogen underwent autohydrolysis, and this was increased upon 
standing for 9 days to 6.79 per cent. 
The effects of bacterial action on the ammonification and hydrol- 
ysis of casein are shown as follows : 
Some results of bacterial action on casein. 
[Average of 2 samples.] 
Period of incubation. 
lday. 
3 days 
4 days 
5 days 
7 days 
9 days 
N added. 
Mg. 
94.3 
94.3 
94.3 
94.3 
94.3 
94.3 
N found 
asNH 3 . 
Mg. 
0.0 
.6 
2.5 
9.7 
56.1 
58.7 
N precipi- 
tated. 
Mg. 
90.4 
91.1 
75.9 
29.6 
13.8 
17.6 
Per cent of 
N ammoni- 
fied. 
0.00 
.64 
2.65 
10.28 
59. 53 
62.25 
Per cent of 
N hydro- 
lyzed. 
4.13 
3.39 
19.51 
68.61 
85.36 
81.32 
Active ammonification set in after the fourth day and reached a 
practical maximum on the seventh day, when 59.53 per cent of the 
nitrogen had been converted into ammonia. Active hydrolysis set 
in after the third day and was completed by the seventh day. By 
this time the solutions had become quite opalescent, due to the 
abundance of cells of bacteria and fungi, and no precipitate was 
Jour. Biol. Chem., 11 (1912), pp. 267-305. 
