114 LABORATORY GUIDE IN BACTERIOLOGY 



7. Fill fermentation tubes, taking care to label 

 each one properly, and sterilize in the arnold on 3 con- 

 secutive days for 20 minutes or in the autoclave for 

 5 minutes. 



All gas must be carefully tilted out of the closed 

 arm of the tube while the fluid is warm. When sterili- 

 zation is completed, inoculate one set of the fermenta- 

 tion tubes with B. coli, another set with B. coli anaero- 

 genes, and a third set with B. aerogenes. Inoctilate 

 with the straight or looped needle. 



Directions for measuring gas formation in fermenta- 

 tion tubes and for analyzing the gas are given on p. 81. 

 The reaction in the closed arm is not always the same 

 as in the bulb. This may be ascertained by adding a 

 small amount of Utmus solution by means of a suitably 

 bent glass tube. 



3. Special study. — Test for indol and nitrites. 



a) Test for nitrites: Add to a culture in Dunham's 

 solution, or, better, in sugar-free broth, successively i 

 drop of each of the following solutions: 



(i) Sulphanilic acid 0.5 gram 



Acetic acid (25 per cent) 150 c.c. 



(2) o Naphthylamine chlorid o.i gram 



Distilled water 20 c.c. 



Acetic acid (25 per cent) 150 c.c. 



A yellowish-red or rose color shows the presence of 

 nitrites. 



b) Test for nitrites and indol combined. 



(i) Add to a culture in Dunham's solution, or sugar- 

 free broth, I or 2 drops pure sulphuric acid. 



(2) Heat gently. Rose color shows the presence of 

 nitrites and indol. If no reaction takes place, add — 



