200 BIOLOGY AND TECHNIQUE 



violet which grows deeper when the medium, is heated or allowed 

 to stand. 



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

 the coarse of protein 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 Milloii's reagent (solu- 

 tion 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 151). 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. 3 



In bacteriological work, 4 c.c. of the culture fluid are poured into 

 a clean test tube, and to it are gradually added 2 c.c. of the mixed 

 test solutions. A pink color indicates the presence of nitrites, the 

 intensity of the color being proportionate to the amount of nitrite 

 present. 



The reducing powers of bacteria may also be shown by their 

 ability to decolorize litmus, methylene-blue, and some other anilin 

 dyes, which on abstraction of oxygen form colorless leukobases. 



8 We are indebted to Dr. J. P. Mitchell, of Stanford University, for the follow- 

 ing technique for nitrite tests: 



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

 (Acetic acid of 1.04 prepared by diluting 400 c.c. of cone, of sp. gr. 1.75 with 

 700. c.c. of water.) 



II. A-Naphthylamin. Dissolve 0.1 gr. in 20 c.c. of water, boil, filter (if neces- 

 sary), and to clear filtrate add 180 c.c. of acetic acid, Sp. Gr. 1.04. 



The solutions are kept separate and mixed in equal parts just before use. 



In carrying out the test, put 2 c.c. of each reagent in a test tube and add 

 substance to be tested. (In ordinary water analysis use 100 c.c.) Cover tube 

 with watch glass and sot in warm water for 20 minutes. Observe presence or 

 absence of pink color promptly. Always run a blank on the distilled water used 

 for rinsing to avoid errors due to nitrites in the water, or in the air of the 

 laboratory. 



