CULTURAL CHARACTERS OF BACTERIA 35 



I. Naphthylamine i-O g. 



Distilled water 100 cc. 



II. Sulphanilic acid 0.5 g. 



Dilute acetic acid 1 50 cc. 



These solutions are kept separate in glass-stoppered bottles. 

 Three cubic centimetres of each of solution I and II are placed 

 in a test-tube, and mixed. Two cubic centimetres of this mixture 

 are added to the cultures, and the same quantity to the blank 

 tube. The tubes should be of the same capacity, and the fluid 

 should be of the same height in both. The tubes are then 

 allowed to stand for half an hour, keeping them closed with 

 rubber stoppers. If a slight pink color develops in the blank 

 tube, it may be due to nitrous acid originally present or absorbed. 

 If appreciable amounts of nitrates have been reduced to nitrites, 

 the pink color in the culture tubes will be deeper, and pro- 

 portionate to the quantity of nitrites present. The absence of 

 nitrites may not indicate non-reduction of nitrates to nitrites, 

 since the nitrites previously formed may have already been 

 reduced to free nitrogen, or to ammonia. In that case the nitrates 

 will have disappeared. To test the presence or absence of 

 nitrates, evaporate 10 cc. of the culture to dryness, and add to 

 the residue i cc. of phenolsulphonic acid, composed of concen- 

 trated sulphuric acid 74 cc, water 6 cc, and phenol 1 2 g. Dilute 

 with water, and transfer to a Nessler jar. Add enough concen- 

 trated caustic soda solution to make alkaline, and make up to 

 50 cc. If nitrates are present, the contents of the jar become 

 a decided yellow. Since bouillon alone, when treated in this 

 way, will give a slight yellow color, it is important, in case only 

 a faint reaction for nitrates is obtained, to have a blank test 

 made with a simple bouillon, and compare the color tints. A 

 •marked excess of color with the culture shows the presence of 

 nitrates. 



