4 BULLETIN" 782, U. S. DEPARTMENT OF AGRICULTURE. 



4.5 cubic centimeters of permanganate solution is sufficient. After 

 boiling from 1 to 2 minutes the precipitate becomes almost white. 

 If the precipitate remains colored, add from 2 to 3 drops of hydrogen 

 peroxid solution and shake well 1 or 2 minutes. When cold, filter by 

 weak suction in a Gooch crucible, wash with distilled water, once 

 with alcohol, dry at 100° C, and weigh. The weight of the precipitate 

 multiplied by 0.271 equals anhydrous citric acid. The method is 

 accurate in milk, as shown by the fact that triplicate determinations 

 upon a sample of milk gave the following results: No. 1, 0.2246 gram. 

 No. 2, 0.2270 gram, and No. 3, 0.2285 gram of citric acid to 100 

 cubic centimeters of milk. The amount of citric acid found in the 

 three analyses differs only by 4 milligrams. 



That ammonia played little or no part in causing the alkaline 

 reaction, especially during the first 7 days of the incubation, is shown 

 in Table 2. Experiments were carried on to determine the amount 

 of am m onia produced after periods of 7, 14, and 30 days of incubation 

 at 30° C. Three sets of tubes of skim milk were inoculated with 

 cultures of' the alkali-forming bacteria and the ammonia was deter- 

 mined together with the reaction of the milk in each of the sets after 

 the various periods of incubation. Each tube of milk contained 10 

 cubic centimeters and was marked so as to show the total volume. 

 The amount of water lost by evaporation was made up at the time 

 of the determination of ammonia and acidity. Five cubic centimeters 

 or one-half of the 10 cubic centimeters was then removed for the 

 ammonia test and the remaining 5 cubic centimeters portion was 

 then titrated in order to determine the reaction. The ammonia was 

 determined by the Folin method (6) with 0.2 cubic centimeter of oxyl 

 alcohol added to prevent foaming. When the quantity of ammonia 

 and the change in reaction of the milk was known, it was possible 

 to calculate the amount of change in reaction due to the ammonia 

 formed, and the difference between that and the total change was 

 assumed to be due to formation of sodium carbonate or bicarbonate. 



As may be seen from the results in Table 2, the reduction of the 

 acidity in 58 of the 68 cultures during the first 7 days' incubation 

 was due entirely to the formation of alkaline carbonates. In 10 

 cultures there was slight amount of ammonia produced, which as 

 the results show accounted for only a very small part of the alkali 

 produced. Generally speaking, the small amount of ammonia pro- 

 duced by the alkali-forming bacteria shows that the reduction of 

 acidity in milk after 7 days at 30° C. was due almost entirely to 

 carbonates or bicarbonates. 



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