164 MANUAL OF MICROBIOLOGICAL METHODS 



Tubes should be filled with 5 ml each, and each culture should be inocu- 

 lated into duplicate (or triplicate) tubes for each of the two tests. Incu- 

 bation should be at optimum temperature of the organism under investi- 

 gation, and tubes should be incubated 2-7 days, according to the rate of 

 growth of the organism in question. Although the same medium is used 

 for both the methyl red and the Voges-Proskauer tests, they must be per- 

 formed in separate tubes. The latter test depends upon the production 

 of acetyl-methyl-carbinol from the glucose. Fabrizio and Weaver 

 (1947) show the possibility of a rapid test for the production of this 

 compound ; Cowan (1953) agrees as to its practicability. A similar micro- 

 test for the methjd red reaction proves more difficult. 



A positive methyl red reaction is regarded as being present when the 

 culture is sufficiently acid to turn the methyl red (0.1 g dissolved in 

 300 ml of 95 per cent ethyl alcohol and diluted to 500 ml with distilled 

 water) a distinct red; a yellow color with the methyl red indicator is 

 regarded as a negative reaction, while intermediate shades should be 

 considered doubtful. 



For the Voges-Proskauer reaction, according to the ''Standard Meth- 

 ods" of the APHA (1946), to 1 ml of culture add 0.6 ml of 5 per cent 

 a-naphthol in absolute alcohol and 0.2 ml of 40 per cent KOH. It is 

 important to shake for about 5 sec after addition of each reagent. The 

 development of a crimson to ruby color in the mixture from 2 to 4 hr 

 after adding the reagents constitutes a positive test for acetyl-methyl- 

 carbinol. Results should be read not later than 4 hr after addition of the 

 reagents. 



Various other tests have been suggested for this reaction, both to obtain results 

 more quickly and because some organisms apparently give different results with dif- 

 ferent tests. In any case, weakly positive reactions may be obscured by the color of 

 the reagent. A procedure which has given excellent results with many thousand cul- 

 tures run by a member of the committee (CAS) is the creatine test of O'Meara, as 

 modified by Levine, Epstein, and Vaughn (1934). In this procedure the test reagent 

 added to the culture is 0.3 per cent creatine in 40 per cent KOH. This reagent deterio- 

 rates rapidly at temperatures over 50°C but may be kept 2 weeks at room temperature 

 (22-25°C) or for 4 to 6 weeks in a refrigerator. 



A recent modification of Coblentz (1943) is similar to the APHA method but uses 

 a massive inoculum in broth from an infusion-agar slant culture, followed by incuba- 

 tion of the broth for 6 hr. Also, the 40 per cent KOH has 0.3 per cent of creatine 

 added to it to intensify the reaction. After addition of the reagents (a-naphthol and 

 KOH-creatine) the culture is shaken vigorously for 1 min; a positive reaction is charac- 

 terized by an intense rose-pink color developing in a few seconds to 10 min. 



The microtest of Fabrizio and Weaver (1947) calls for inoculation with a loopful of 

 growth from a 6- to 12-hr infusion agar slant into 0.5 ml of infusion medium with 1 

 per cent trypticase and 0.5 per cent NaCl, placed in small tubes and preheated in a 

 30° water bath. It is then incubated at 30°C in a water bath for 90 min. It is then 

 tested for acetyl methyl carbinol by the same method as given above, except that 

 smaller quantities of the reagent (0.15 and 0,05 ml, respectively) are added. 



