DETERMINING BIOLOGICAL ACTIVITIES OF RACTERIA 167 



Indol Production by Bacteria. Many bacteria possess the power of 

 producing indol. Though formerly regarded as a regular accompani- 

 ment of proteid decomposition, later researches have shown that indol 

 production- is not always coexistent with putrefaction processes and 

 occurs only when pepton is present in the pabulum. 



Indol formation by bacteria is determined by the so-called nitroso- 

 indol reaction. Organisms are grown in sugar-free pepton broth or in the 

 pepton-salt bouillon of Dunham. (See page 126.) Media containing 

 fermentable substances are not favorable for indol production since acids 

 interfere with its formation. The cultures are usually incubated for three 

 or four days at 37 C. At the end of this time, ten drops of con- 

 centrated sulphuric acid are run into each tube. If a pink color 

 appears, indol is present, and we gather the additional information 

 that the microorganism in question has been able to form nitrites 

 by reduction (e.g., cholera spirillum). If the pink color does not 

 appear after the addition of the sulphuric acid alone, nitrites must 

 be supplied. This is done by adding to the fluid about 1 c.c. of a 0.01 

 per cent aqueous solution of sodium nitrite. The sodium nitrite solu- 

 tion does not keep for any length of time and should be freshly made up 

 at short intervals. 



Phenol Production by Bacteria. Phenol is often a by-product in the 

 course of proteid cleavage by bacteria. To determine its presence in 

 cultures, bacteria are cultivated in flasks containing about 50-100 c.c. 

 of nutrient broth. After three to four days' growth at 37 C., 5 c.c. of 

 concentrated HC1 are added to the culture, the flask is connected with a 

 condenser, and about 10-20 c.c. are distilled over. 



To the distillate may be added 0.5 c.c. of Millon's reagent (solution of 

 mercurous nitrate in nitric acid), when a red color will indicate phenol; 

 or 0.5 c.c. of a ferric chloride solution, which will give a violet color if 

 phenol is present. 



Reducing Powers of Bacteria. The power of reduction, possessed by 

 many bacteria, is shown by their ability to form nitrites from nitrates. 

 This is easily demonstrated by growing bacteria upon nitrate broth 

 (see page 126). Bacteria are transferred to test tubes containing this 

 solution and allowed to grow in the incubator for four or five days. 

 The presence of nitrites is then chemically determined. 1 



1 We are indebted to Dr. J. P. Mitchell, of Stanford University, for the following 

 technique for nitrite tests: 



I. Sulphaiiilic Acid, Dissolve 0.5 g. in 150 c.c. of acetic acid of Sp. Gr. 1.04, 



