244 LABORATORY EXERCISES IN BACTERIOLOGY. 



1000 kept in clean glass-stoppered bottle; sulphanilic acid 0.5 gram, dilute [1:16] 

 acetic acid 150 cubic centimeters also kept separate in glass-stoppered bottle. Mix 

 equal quantities of the two solutions for use. Add to first inoculated tube and control 

 tube two cubic centimeters each of the mixture). The tubes are closed with rubber 

 stoppers and allowed to stand for about half an hour, warming hastening the reaction. 

 If nitrites be present from the reduction of the nitrate, a pink or red color develops, 

 the control tube remaining colorless or perhaps slightly pink if it has absorbed a trace 

 of nitrite from the atmosphere. If no nitrites are thus found, no reduction may have 

 taken place, the nitrate remaining unchanged in the medium ; or the reduction may 

 have advanced beyond the formation of nitrite to the formation of nitrogen or ammonia 

 To determine these points the second inoculated tube is taken up. One-half its con- 

 tents is poured into a clean test-tube and a strip of paper wet with Nessler's reagent 

 suspended over the medium, when if fumes of ammoniacal character arise, the paper 

 will slowly assume a yellowish or brownish-red color. The remaining half of the 

 second inoculated medium is evaporated to dryness either in the tube or in a porcelain 

 dish, and to the residue is added a drop of phenol-sulphonic acid (cone, sulphuric 

 acid, c. p., 74 cubic centimeters; water, 6 cubic centimeters; and pure carbolic acid, 

 12 grams), and water added to dilute the mixture to one or two cubic centimeters. 

 This is then alkalinized by addition of sodium hydroxide solution. A yellow color 

 indicates the persistence of the nitrate in the medium. 



