METHODS FOR THE MICROSCOPIC STUDY OF BACTERIA 219 



tophan, tyrosine, histidin give colored compounds with various 

 reagents because they contain the chromophoric group, C = C. The 

 formation of indol from tryptophan (see page 74 for chemistry) has 

 long been used as a diagnostic test for Bacillus coli and other bacteria. 

 The test depends upon the removal of alanin from the tryptophan 

 molecule by the activity of the organism, and the addition of an auxo- 

 chromic group, NO 2 in the beta position of the pyrrol ring, previously 

 occupied by alanin. In an acid medium the compound, betanitroso- 

 indol, is brownish red. 



Procedure, Indol Test. To a three-day plain broth culture of Bacil- 

 lus coli (or other organism) add 1 c.c. of concentrated hydrochloric 

 acid. 1 Mix thoroughly and overlay the acid broth with 1 to 2 c.c. of a 

 0.1 per cent, solution of sodium nitrite. 2 At the junction of the two 

 solutions a brownish-red ring of nitroso indol develops. 



(b) Carbohydrate Broths. The addition of sugars, as dextrose, lac- 

 tose, saccharose, or of alcohols, as glycerol, to plain broth media, 

 greatly enriches the medium in non-nitrogenous substances which 

 may be readily utilizable sources of energy for bacteria. It is hardly 

 necessary to emphasize the importance of purity in all sugars and 

 other carbohydrates intended for bacterial purposes, nor the fallacy of 

 attempting to determine the action of bacteria upon specific carbohy- 

 drates in media not freed from muscle sugar (dextrose). The use of 

 serum as a basis for fermentation media frequently introduces a source 

 of error, because blood serum normally contains about 0.08 per cent, 

 of dextrose, an amount quite sufficient to give rise to considerable 

 amounts of acid. 3 



The observations made in carbohydrate media are usually restricted to : 



(a) Change in reaction. 



(6) Gas formation, and in fermentation tubes, to growth in the 

 closed arm as well. 



1 Any strong mineral acid will answer the purpose. 



2 Best accomplished by running the nitrite solution carefully down the side of the 

 tube held in a slanting position; a stratification of the two liquids should be obtained. 



3 The significance of fermentation media for the classification and identification of 

 bacteria depends upon their content both of protein and carbohydrate. Bacteria derive 

 their energy requirements from carbohydrate, if it is utilizable, but of course they must 

 obtain their "Bausteine" from the nitrogenous constituents. If the carbohydrate 

 cannot be utilized, both structural and energy requirements are derived from the protein 

 constituents. Bacteria vary greatly in their ability to ferment carbohydrates; some 

 types, as Bacillus alcaligenes, do not appear to ferment even dextrose. Bacillus lactis 

 aerogenes, on the contrary, can ferment hexoses, bioses, and even starches. The fer- 

 mentability of a carbohydrate depends apparently upon its stereo-isomeric configuration, 

 and relatively slight differences in the configuration of similar carbohydrates may 

 determine their value for specific organisms as sources of energy. This point is discussed 

 somewhat later in this section. 



