240 LABORATORY EXERCISES IN BACTERIOLOGY. 



time should be avoided in practice, and the reaction accounted positive 

 only when occurring within the first hour; if strong, it will probably have 

 occurred within a few seconds. Should failure occur with one to fifty 

 dilution, a one in twenty-five dilution may be tried, and failures with this 

 may be accounted negative. If very strong, bacteriolysis of the typhoid 

 germs may also be noted, the destruction beginning with vacuolization 

 and erosion of the bacteria. 



The same result may often be obtained in a tube by the addition of 

 large amounts (in same proportions as above) of the serum, the agglutin- 

 ated masses forming a flocculent precipitate visible to the unaided eye, and 

 the previously turbid bouillon becoming clear between the tiny flakes. In 

 the course of twenty -four hours the effect of agglutination and stoppage of 

 motion will usually be found to have disappeared, the agglutinating agent 

 having apparently been exhausted from the serum employed. 



10. Indol and Phenol Production. Among the ptomaines indol and phenol 

 are substances of importance in that they are demonstrable with little difficulty, 

 and, being more or less characteristic of certain organisms, serve to aid in their identi- 

 fication. Aside from the last feature it is not known that these substances are of 

 importance. 



Indol occurs particularly in the old cultures of bacteria of the colon group and 

 of the various microspirae and spirilla. It is also found in old cultures of Mycobac- 

 terium diphtherice and bacterium mallei. It is shown by adding to a tube of bouillon 

 free from sugar (or the ordinary peptone solution), in which one of the indol-producers 

 has been grown for eight to ten days, an equal amount of weak nitric or sulphuric 

 acid (twenty per cent.). In case the germ has also produced nitrites in the medium, 

 or if nitrites be present in the nitric acid used, the reaction, consisting in the production 

 of a red color (probably due to the formation of nitroso-indol nitrate), occurs. But 

 in the absence of the nitrites it will be necessary to add a drop of a one per cent, solution 

 of sodium or other nitrite. Should the reaction take place without the addition of 

 nitrite, upon the addition of sulphuric acid alone, it may be tentatively inferred that 

 the organism is also a nitrite-producer. 



Exercise 68. Grow in sugar-free bouillon or peptone solution a culture of 

 Bacillus coli for eight or ten days. To this culture add an equal amount of 

 twenty per cent, pure sulphuric acid, and if needed slightly warm for a few 

 minutes. Now add a drop of a one per cent, solution of sodium nitrite 

 and continue to add and warm until the color is at its intensity. Tco 

 great an addition of nitrite will cause a yellowish tint to be assumed. 



Repeat with cultures of Micrococcus pyogenes, Bacillus subtilis, Bacillus 

 typhosus, and Mycobacterium diphtheria. 



Exercise 69. To a similar culture of the cholera organism add the acid 

 alone, the color being produced without the addition of the nitrite, showing 

 the production of the nitrite as well as of indol by this bacterium. The 



