ALKALI-FOEMING BACTEKIA FOUND IN MILK. 3 
incubation at 30° C. and even then there was not a sufficient amount 
formed to affect the titration appreciably. 
That citric acid was used up as the reaction became alkaline is 
shown by the results in Table 1. Since culture No. 12 was the only 
one which could produce even a trace of ammonia during incubation 
for 7 days at 30° C, it is assumed that the alkaline change of about 
16 cubic centimeters tenth-normal alkali was due to the production of 
carbonates resulting from the oxidation of the citric acid. It may be 
observed, however, that in order to account for the alkaline change it 
would be necessary to oxidize to carbonates about one-half of the citric 
acid actually found to have been used up. Since the quantity of 
citric acid fermented, if calculated as carbonate, would be about 
twice as much as is necessary to account for the reduction in acidity 
of the milk, it seems evident that about one-half of the citric acid is 
oxidized to carbonates while the remainder is probably converted 
into other organic acids and is not carried so far as the alkaline 
carbonates. 
Table 1. — Alkaline change in milk due to oxidation of the citric acid. 
Titration (N/10 NaOH 
per 100 c. c. of milk). 
Alkaline 
change 
(c.c. of N/10 
alkali per 
100 c. c. 
of milk. 
Citric acid in 100 c. c. 
of milk. 
Citric acid 
necessary 
to produce 
enough 
carbonate 
to account 
for alkaline 
change 
in milk. 
Citric acid 
Culture No. 
Control. 
After 
7 days' 
incubation. 
Control. 
After 
7 days' 
incubation. 
fermented 
but not 
oxidized to 
carbonates. 
12 
C.c. 
19.20 
19.20 
19.20 
C.c. 
2.70 
3.20 
2.60 
C.c. 
16.5 
16.0 
16.6 
Gram. 
0. 2013 
.2013 
.2013 
Gram. 
0.0 
0.0 
0.0 
Gram. 
0. 1056 
.1024 
.1063 
Gram. 
0. 0957 
60 
.09S9 
130 
.0950 
The citric acid was determined according to the modification of 
Beau's (3) method. Denige's reagent was used but changed slightly 
as follows: To 50 grams of red oxid of mercury in 500 cubic centi-. 
meters distilled water add 110 cubic centimeters of concentrated 
sulphuric acid little by little and shake well until the solution is 
complete ; dilute to 1 liter, heat to boiling, and filter when cold. The 
method of analysis was as follows: Place 50 cubic centimeters of milk 
in a 200 cubic centimeter graduated flask, add 75 cubic centimeters 
distilled water and 50 cubic centimeters of reagent, agitate gently, 
fill to mark, and mix well. Allow to settle and filter through a dry 
Swedish filter 12.5 centimeters No. until only a faint opalescence 
remains. Then oxidize 100 cubic centimeters of the filtrate which 
corresponds to 25 cubic centimeters of milk at 75° C. by adding 1 per 
cent solution of potassium permanganate drop by drop until the 
precipitate acquires a light-brown color and settles rapidly. About 
