LABORATORY AND GREENHOUSE STUDIES. 85 



TEST 1 OF D. RIVAS. 



One-fourth c. c. of a 48-liour culture in neutral dextrose bouillon was 

 rapidly boiled in about 5 c. c. of a 10 per cent solution of NaOH, Tests 

 made with both the coconut organism and Bacillus coli gave the 

 typical clear lemon-yellow color reaction of this test. 



The color in this reaction is discharged by acid and restored by 

 alkali. This reaction depends upon the biological action of the bac- 

 teria upon the sugar. 



This experunent was also tried using beef bouillon +14 instead of 

 neutral, with the same results. Cultures in 1 per cent peptone with 2 

 per cent dextrose, titrating + 3 likewise gave the same lemon-yellow 

 reaction. 



TEST 3 OP D. KIVAS. 



According to Dr. Rivas,^ Bacillus coli does not exhaust all the sugar 

 from a medium, at least if there is any large amount. On this ground 

 he would separate this organism from closely allied ones which he 

 would place in a so-called saccharolytic group, i. e., those capable of 

 exliausting all the sugar. So incomplete is the exhaustion of sugar by 

 Bacillus coli that it is inadvisable to use it for the purpose of freeing 

 beef bouillon from the small amount of muscle sugar it may contain. 

 Bacillus cloacae is said to be much preferable. At least a partial ex- 

 planation of this condition is that Bacillus coli produces so much acid 

 in the presence of sugar that it prevents the extensive growth that 

 would otherwise take place. 



For the purpose of identifying the coconut organism with Bacillus 

 coli tests were made of cultures in sugar solutions to ascertain the 

 relative amount of sugar used in the growth of the organisms. 



Two methods were used for determining the amount of sugar remain- 

 ing in the cultures after a certain amount of growth. Fehling's solu- 

 tion was diluted with an equal amount of water and divided among a 

 number of small test tubes, 1 cubic centimeter being placed in each. 

 To these the cultures were added in increasing amounts, begimiing 

 with one, two, three, etc., drops up to 1 cubic centmieter, and the 

 mixture was then boiled. In the other experiments a less accurate 

 method was used. Fehling's solution was added directly to each 10 

 c. c. of the culture tubes. Amounts from 2 to 3 c. c. were added at a 

 time and then boiled to bring about the reduction. Fehling's was 

 added only until the light orange-red color of the heated solution began 

 to change to a greenish tinge. 



(1) Cultures of February 26 in medium 4192, tested after 55 days 

 in Dunliam's solution with 2 per cent dextrose. The average of six 



I Rivas, D. Contribution to the Differentiation of Bacillus Coli Communis from Allied Species in Drink- 

 ing Water. Journal of Medical Research, vol. 18, 1908, pp. 81-91. 



228 



