20 



portion of the I)ott]e may l)e free from nitrites, or vice versa. 

 This, togetlier witli further evidence to ho presented, jilainly indi- 

 cates why the indol reaction may he obtained with one lot of 

 media by the addition of chemically pure sulphuric acid alone 

 Avhile with another the addition of nitrite is necessary. 



All these "peptones" give a strong Biuret reaction, Init only 

 one was tested for the presence of peptones. Ten grams from 

 bottle No. 5 were dissolved in pui-e ^\•atcr and i)laced in a parch- 

 ment dialyzer with all di\e precautions against introducing nitrites. 

 After six hours at 28° the diffusate (about 150 cubic centimeters) 

 was tested. It did not give the Biin-et reaction, and 10 cubic 

 centimeters gave a reaction for nitrites equal to 0.0000005 gram 

 nitrogen. 



Further, it was necessary to wash filter paper free from nitrites — 

 e. g., a filter paper (Schleicher and Schiills) was washed with 

 200 cubic centimeters nitrite-free water; 10 cubic centimeters of 

 the filtrate gave a reaction for nitrites corresponding to about 

 0.0000005 gram nitrogen. 



The sulphuric acid used throughout these experiments was 

 tested to prove the absence of nitrites. 



(II) EXPERIMENTS ON THE DEMONSTRATION OF THE 

 INDOL AND CHOLERA-RED REACTIONS IN DUNHAM'S 

 PEPTONE SOLUTION. 



(l) IN UXDIALYZED PEPTONE SOLUTION. 



Dunham's peptone solution, containing 1 per cent Witte's 

 "peptone" (bottle No. 5) and 0.5 per cent sodium chloride, in redis- 

 tilled water, was prepared with special precautions to avoid intro- 

 ducing nitrates and nitrites.^ 



Half of the medium was distributed in test tulies (10 cubic 

 centimeters per tube) and marked "peptone solution only." To 

 the other half was added 0.01 per cent of a freshly prepared solution 

 of sodium nitrate, and this Avas then distributed in a similar 

 manner. Both sets were autoclaved for half an hour at 120°. 

 Final reaction, 0.5 per cent acid to ])henolphthalein. A tube of 



' A large glass beaker was found to be especially convenient for boiling 

 medium, as one can more easily mark the initial height of the latter and 

 watch the completeness of solution than when the cooking is performed in 

 an agate-ware boiler. The albumoses do not all go into solution in the 

 presence of 0.5 per cent sodium chloride. 



