NITRITE FORMATION 141 



bacterium to be investigated and kept in the incubator, together with 

 non-inoculated control tubes. If hydrogen sulphide is formed, a 

 brownish-black to black precipitate appears. 



Ammonia. The detection of ammonia is more complicated. It 

 requires distillation after the culture has been grown in several hun- 

 dred cubic centimeters of the culture medium, and the media, of 

 course, must be prepared with ammonia-free reagents, including 

 ammonia-free water. The distillate is tested with Nessler's reagent, 

 which gives a yellow color in the presence of free ammonia. The 

 intensity of the color depends upon the amount of ammonia formed, 

 and the test can be arranged as a colorimetric quantitative method. 



Indol. Indol is not infrequently produced in the growth of certain 

 bacteria. These must be inoculated into Dunham's peptone water 

 (see above) kept in tubes which have been incubated for several days. 

 Before making the test the tubes are cooled in running water and then 

 the following reagents are added: (1) A few drops of a 1 to 10,000 

 sodium nitrite watery solution; (2) chemically pure sulphuric acid, 

 drop by drop, until about 1 c.c. has been added to the 10 c.c. con- 

 tained in the culture tube. Red discoloration indicates the presence 

 of indol. If a nitrite has been formed in the growth the red color 

 will appear on the addition of the pure strong sulphuric acid alone. 



Phenol, or Carbolic Acid. Some bacteria also produce phenol, or 

 carbolic acid. To ascertain its formation about 100 c.c. of a nutrient 

 bouillon are inoculated. After growth has continued for several days, 

 approximately one-fifth to one-sixth of the fluid is distilled over. The 

 distillate contains the carbolic acid. It is tested by adding first a 

 few drops of lactic acid and then gradually a dilute solution of the 

 sesquichloride of iron. If phenol is present an amethyst-violet color 

 is produced. 



Nitrite Formation. Some bacteria possess the power to reduce 

 nitrates to nitrites. This characteristic can be ascertained by inocu- 

 lating the nitrate solution described in the preceding chapter with 

 such bacteria. The cultures are best kept for one week at a temper- 

 ature of 28 C. Two solutions are necessary for the nitrite test: 



1. Sulphanilic acid 0.5 gram, dissolved in 150 c.c. acetic acid of 

 specific gravity 1.04. 



2. Amidonaphthalin acetate 0.1 gram, boiled in 20 c.c. distilled 

 water, then filtered through cotton and diluted with 180 c.c. dilute 

 acetic acid. 



Before use, mix equal quantities of 1 and 2, and add 2 c.c. of the 

 mixture to 3 c.c. of the culture. A red color indicates the formation 

 of nitrites, and its intensity corresponds to the smaller or greater 

 amount of nitrites present. In this, as in other similar tests, control 

 tests must be made with non-inoculated tubes, which are kept in 

 the incubator for the same length of time. 



