21 



each of the media tested for nitrites gave a reliction e(|ual to ahout 

 0.0000005 gram nitrogen. 



(a) One tul)e of each medivmi was inoculated with cholera 579.' 

 After eighteen liours' growth at 36° to 37°, 0.5 cubic centimeter 

 of pure suli)liuric acid was added to each culture. Tlic culture 

 marked "peptone solution only" yielded an immediate yuridc- 

 colored indol reaction. The culture marked "peptone solution 

 only -^0.01 per cent NaNO," did not give an innnediate reaction, 

 but in about half an hour developed an intense red color (cholera- 

 red reaction). 



(h) That'this "peptone solution only" is eminently suitable for 

 the production of indol by B. coli 577'-' is shown by the following 

 experiment: 



Three tubes were inoculated each day for eight successive days 

 and the cultures kept at 36° to 37°. Bearing in mind the obser- 

 vation of A. W. Peckham,^ that with different cultures of Bacillus 

 coli the maximum amount of indol could be decided only by eor- 

 res})onding variations in the proportions of nitrites and acid used, 

 I experimented to see whethei- a similar variation in the amount 

 of nitrites and acid was necessary to elicit the maximum indol 

 reaction in cultures of the same organism — for example, of two and 

 eight days' growth. 



To each series of three tubes, then, one-half, 1, and 2 cul)ic 

 centimeters of a 0.01 per cent sodium nitrite solution and corres- 

 ponding amounts of pure sulphuric acid were added. Throughout 

 the series the most intense reaction was elicited by using 0.5 culiic 

 centimeter of the test solutions, with the excej)tion of the seven- 

 day-old series, where 1 cubic centimeter yielded the best reaction. 



Two cubic centimeters invariably yielded enough nitrous acid 

 to ol>scure the reaction. It may be of interest to plot the gradual 



' Unfortunately I have l)een unable to test all the cholera cultures 

 (le5)cril)e(l in Bnllclni l!i of this Bureau, owing to the accidental death of 

 nearly all my stock cultures. This strain of culture, 579, was reclaimed l)y 

 jtlating Dr. W. E. Musgrave's- amieba culture 11524 grown in pure sym- 

 biosis with this spirillum. Later, when I found there were other cultures of 

 the cholera spirillum in the laboratory, 1 did not have ojijiortunity to perform 

 these tests with them. 



'•'A culture kept in stock for two years, the fermentation tests of which are 

 given on page 24, Bnlletln 19, Biological Laboratory, Bureau of Gorernment 

 LahoratorifH, Manila, P. I. 



\fonr. Krper. Me<l., 1897, 2, 560. 



