98 PRINCIPLES OF BACTERIOLOGY 



duced nitrite abundantly; once only was ammonia observed. In a 

 number of bacteria studied by Rubner only one failed to produce nitrite. 

 The test for nitrites is made as follows : Two bouillon tubes containing 

 nitrates are inoculated, and, along with two uninoculated tubes, are 

 allowed to remain in the incubator for several days; then to the cultures 

 and control test is added a small quantity of colorless iodide of starch 

 solution (thin starch paste containing 0.5 per cent, potassium iodide) 

 and a few drops of pure sulphuric acid. The control tubes remain 

 colorless or become gradually slightly blue, while if nitrites are present 

 a dark-blue or brown-red coloration is produced. 



The demonstration of ammonia is made by the addition of Nessler's 

 reagent to culture media free from sugar. In bouillon, if ammonia be 

 present, Nessler's reagent is almost immediately reduced to black 

 mercurous oxide. A strip of paper saturated with the reagent can also 

 be suspended over the bouillon tube, or this can be distilled at a low 

 temperature with the addition of magnesium oxide and the distillate 

 treated with Nessler's reagent. A yellow to red coloration indicates the 

 presence of ammonia. Controls are necessary. 



Aromatic Products of Decomposition. Many bacteria produce aromatic 

 substances as the result of their growth. The best known of these are 

 indol, skatol, phenol, and tyrosin. Systematic investigations have only 

 been made with regard to the occurrence of indol and phenol. 



TEST FOR INDOL. To a bouillon culture, which should, if possible, 

 be not under eight days old and free from sugar, is added half its volume 

 of 10 per cent, sulphuric acid. If in heating to about 80 C. a pink or 

 bluish-pink coloration is immediately produced it indicates the presence 

 of both indol and nitrites, the above-described nitroso-indol reaction 

 requiring the presence of both of these substances for its successful 

 operation. This is the so-called " cholera-red reaction," but it may be 

 applied to many other spirilla besides cholera. As a rule, however, the 

 addition of sulphuric acid alone is not sufficient, and a little nitrite must 

 be added; this may be done later, the culture being first warmed with- 

 out nitrite, when, if there is no reaction or a doubtful one, 1 to 2 c.c. 

 of 0.005 per cent, solution of sodium nitrite is added until the maxi- 

 mum reaction is obtained. The addition of strong solutions of nitrite 

 colors the acid liquid brownish-yellow and ruins the test. Out of sixty 

 species examined by Lehmann, twenty-three gave the indol reaction. 



Decomposition of Fats. Pure melted butter is not a suitable culture 

 medium for bacteria. The rancidity of butter is brought about (1) as 

 the result of a purely chemical decomposition of the butter by the 

 oxygen of the air under the influence of sunlight, and (2) through the 

 formation of lactic acid from the milk-sugar left in the butter. Fats 

 are, however, attacked by bacteria when mixed with gelatin and used 

 as culture media, with the consequent production of acid. 



Putrefaction. By putrefaction is understood in common parlance 

 every kind of decomposition due to bacteria which results in the pro- 

 duction of malodorous substances. Scientifically considered, putrefac- 

 tion depends upon the decomposition of complex organic compounds, 



