34 BACTERIOLOGY 



ism in question introduced, to which is added 5 cc. of concen- 

 trated hydrochloric acid. About 15 cc. are distilled, and the 

 distillate divided into three portions, to be tested as follows : 

 1. To one portion is added a few drops of Millon's reagent, and 

 the mixture heated to boiling ; the development of a red color 

 indicates the presence of phenol. 2. To another portion add a 

 few drops of strong bromine water ; a turbidity develops in the 

 presence of phenol. 3. To the third portion add a few drops of 

 a very dilute solution of ferric chloride ; a violet color develops 

 in the presence of phenol. 



In recording the development of phenol in cultures, the age 

 of the culture and temperature of growth should be stated. 

 When indol and phenol occur together in the same culture, their 

 separation is advisable before applying the tests. For this, the 

 method as proposed by Hoppe-Seyler can be used. Distil 200 cc. 

 of the culture with 50 cc. of concentrated HC1 until 50-70 cc. 

 passes over. The distillate will contain both indol and phenol. 

 Render the distillate strongly alkaline with caustic potash and 

 distil ; the indol will be found in the distillate, the phenol in the 

 residue. When the residue is cold, saturate with carbon dioxide, 

 and distil ; the phenol will pass over into the distillate. 



The Reduction of Nitrates to Nitrites 



For the study of the reduction of nitrates to nitrites a special 

 medium is desirable, composed of Witte's peptone 10 g., nitrate of 

 soda .02 g., and water 1000 cc. It is important to have the 

 medium originally free from nitrites, and since distilled water 

 frequently contains considerable quantities of nitrous acid, it is 

 better to use well or spring water, which gives no reaction for 

 nitrites. Furthermore, nitrous acid is present in the atmosphere, 

 some of which will be absorbed by the culture during its growth. 

 Hence it is necessary to have blank, un-inoculated tubes kept 

 under the same conditions as those inoculated, which shall also 

 be tested for nitrites. For the test two solutions are necessary. 



