ALKALI-rORMING BACTERIA FOUITD IF 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 

 1 6 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-haK 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 ia 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 



.0989 



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 



