156 MANUAL OF MICROBIOLOGICAL METHODS 



The test for indole may be performed by the technic of EhrUch-Bohme, 

 by either the Gore or the Kovacs modification of the same or by the 

 Gnezda technic. The Kovacs method is especially simple and con- 

 venient. These procedures are as follows: 



Bohme (1905) called for the following solutions: 



Solution 1 



Para-dimethyl-amino-benzaldehyde 1 g 



Ethyl alcohol (95%) 95 ml 



Hydrochloric acid, concentrated 20 ml 



Solution 2 

 Saturated aqueous solution of potassium persulfate (K2S2O8) 



To about 10 ml of the culture fluid add 5 ml of solution 1, then 5 ml of solution 2, and 

 mix well by rotating between the hands; a red color appearing in 5 min indicates a 

 positive reaction. This test may also be performed (and sometimes more satisfac- 

 torily) by first mixing up the culture with ether and adding solution 1 (Ehrlich's 

 reagent) dropping down the side of the tube so that it spreads out as a layer between 

 the ether and the culture fluid. After this method of applying, solution 2 seems to be 

 unnecessary. 



The Gore (1921) test uses these same solutions, but the method of application is as 

 follows: Remove the plug of the culture tube (which must be of white absorbent 

 cotton), moisten it first with 4-6 drops of solution 2, then with the same amount of 

 solution 1. Replace the plug and push down until 1 or IJ-^ in. above the surface of 

 the culture. Place the tube upright in a boiling-water bath, and heat for 15 min 

 without letting the culture solution come in contact with the plug. The appearance 

 of a red color on the plug indicates the presence of indole. 



The Kovd,cs (1928) test is a simplification of that of Bohme, using only one solution; 

 it is now the method of choice in many laboratories: 



Para-dimethyl-amino-benzaldehyde 5 g 



Amyl or butyl alcohol 75 ml 



Hydrochloric acid, concentrated 25 ml 



This reagent may be used as in the Bohme test, but no solution 2 is 

 required. The red color appears in the alcohol layer. 



The Gnezda (1899) oxalic acid test is made as follows: dip a strip of filter paper in a 

 warm saturated solution of oxalic acid; on cooling, this is covered with crystals of the 

 acid. Dry the strip of paper thoroughly (sterilization by heat seems unnecessary), 

 and insert into the culture tube under aseptic conditions, bent at such an angle that it 

 presses against the side of the tube and remains near the mouth. Reinsert the plug, 

 and incubate the culture. If indole is formed, the oxalic acid crystals take on a pink 

 color. 



It is recommended that the Gore or the Kovacs test be used in a routine 

 way. In interpreting the results obtained it must be remembered that 

 when the reagents are added directly to the medium, they react with 

 alpha-methyl-indole as well as with indole itself, but as the former com- 

 pound is nonvolatile, it cannot react to the Gor^ or Gnezda tests. Hence 

 the Ehrlich test unmodified is less specific for indole than the Gore modi- 

 fication or the Gnezda test. 



