242 LABORATORY EXERCISES IN BACTERIOLOGY. 



reaction has been long known in connection with this organism as the 

 i( cholera red reaction." 



Phenol production occurs with much the same groups of organisms as that of 

 indol. For its demonstration it is essential to use a larger quantity of growth in sugar- 

 free bouillon than in case of effort to demonstrate indol presence. To this is added 

 one-fifth its volume of pure hydrochloric acid and the mixture distilled. The distillate 

 contains the phenol, which, after neutralization of the distillate with calcium carbonate, 

 will strike a violet color upon the addition of neutral solution of chloride of iron (one 

 per cent.). 



Exercise 70. Make a culture of Bacillus coli in sugar-free bouillon or 

 peptone solution, using about fifty cubic centimeters of the medium. After 

 eight or ten days add ten cubic centimeters of hydrochloric acid, and in a 

 condenser (or retort) distil five or ten cubic centimeters from the mixture. 

 Carefully neutralize with powdered calcium carbonate (or calcined magne- 

 sia), and add drop by drop a one per cent, solution of ferric chloride to ob- 

 tain the violet color produced by phenol. A white flocculent precipitate 

 may be produced in a separate part of the distillate upon the addition of 

 bromine. 



ii. Nitrifying and Denitrifying Bacteria. Quite a large number of bacteria 

 apparently possess the power of forming nitrite in cultures, although it is not clear 

 how this is accomplished; and possibly, when it is found in traces only, it may have 

 been absorbed from the air. In the study of indol formation the lack of need of adding 

 nitrite to bring out the characteristic reaction in many examples is probable evidence of 

 its presence. As far as is known, certain pseudompnads found in the soil (Ps. Europa, 

 Ps. Javanensis) are alone able to convert, the first the nitrogen of ammonium com- 

 pounds into nitrite, the second nitrite into nitrate. A number of bacteria found 

 especially about the roots of certain plants (e. g., leguminosae) apparently absorb the 

 nitrogen of the air, preserving it in the soil as free nitrogen or transforming it into nitrites 

 or into ammonium. The general term nitrifying bacteria is applied to the forms en- 

 gaged in the above processes; these are of much importance to the agriculturist in 

 their role in enrichment of the soil. 



On the other hand, many bacteria possess the property of reducing nitrates to 

 nitrites by the abstraction of oxygen, or in the same way of reducing the nitrites to free 

 nitrogen, sometimes leading to the formation of ammonium. These are termed the 

 denitrifying bacteria. They are widely distributed in dung, soil, and putrefying organic 

 matter, and the reduction of nitrates to nitrites seems common to the majority of 

 bacteria. The reduction of nitrates to nitrites is usually determined by growing a 

 bacterium in question for seven days either in the room or incubator temperature, 

 as is best fitted, in a medium made up of water with but a small amount of peptone 

 added and with a little saltpeter (tap-water 1000, 1 gram dried peptone, 0.2 gram 

 sodium nitrate). To two tubes containing about three cubic centimeters, each thus 

 prepared, inoculated and grown, and to a control tube of the same size and same content 

 of the saltpeter broth and kept in the same conditions as the inoculated tubes, is added 

 a mixture of naphthylamine and sulphanilic acid (naphthylamine 1, distilled water 



