15 
the bottles was again drawn through a solution of sulphuric acid. 
Casein prepared by Hammarsten was employed. This was further 
purified by first dissolving in tenth-normal sodium hydroxid solution, 
then precipitating with 1 per cent acetic acid, filtering, and thoroughly 
washing' with alcohol and ether. After drying in vacuum over 
sulphuric acid, the product was found to contain only 13.50 per cent 
nitrogen, showing that considerable impurities still remained. (Pure 
casein contains 15.62 per cent N.) 
Five grams of the casein was mixed with 500 grams of silica sand 
and optimum moisture brought about by adding 50 cubic centimeters 
of a soil infusion prepared as in Series I. After incubating at 28° C. 
for one week the contents of the bottles were acidified with 1 per cent 
acetic acid, thoroughly shaken, filtered, and washed. The residue 
was then extracted with tenth-normal sodium hydroxid and the 
alkaline solutions acidified with 1 per cent acetic acid. No precipitate 
was formed and the solutions were combined with those above. Am- 
monia was determined in the solutions, sand residues, and the 
sulphuric acid by distilling with an excess of magnesia. 
Total amount of ammonia formed from casein in silica sand. 
Determination No. 
NasNH 3 
soluble 
in acetic 
acid. 
NasNH 3 
in the 
residue. 
NasNH 3 
volatil- 
ized. 
Total 
yield 
01NH3. 
Per cent of 
total N con- 
verted into 
ammonia. 
1 
Mg. 
271.6 
273.0 
324.8 
Mg. 
96.6 
96.6 
55. 7 
Mg. 
62.6 
76.4 
43.4 
Mg. 
430.8 
446.0 
423.9 
63.8 
2 
66.1 
3 
62.8 
A slightly higher yield of ammonia was obtained than previously, 
the average being 64.2 per cent of the total nitrogen. Since no casein 
could be precipitated from sodium hydrate solutions after bacterial 
action, the conclusion that the entire amount of casein added had 
undergone hydrolysis is justified. The nature of the undetermined 
balance remains to be determined. 
SERIES IX AMMONIFICATION AND HYDROLYSIS OF CASEIN. 
The purpose of this series was to study the relations between the 
rates of ammonification and hydrolysis of casein. A stock solution 
of casein purified as in the previous series was prepared by dissolving 
15 grams in 150 cubic centimeters of tenth-normal sodium hydroxid, 
then diluting to 2,100 cubic centimeters. One hundred cubic centi- 
meter portions were placed in 300 cubic centimeter Erlenmeyer flasks 
and 10 cubic centimeters of soil infusion added. After shaking, the 
flasks were loosely stoppered with cotton plugs and incubated at 
28° C. The rate of hydrolysis was measured by precipitating the 
