ROUTINE TESTS FOR THE DESCRIPTIVE CHART v«-21 



For the Vogcs-Proskaucr reaction, according to the "Standard 

 Methods" of the A. P. H. A. (1946), to 1 ml. of culture add 0.6 ml. 

 of 5% a-naphthol in absolute alcohol and 0.^2 ml. of 40% KOII. The 

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

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

 methyl-carbinol. Itesults should be read not later than 4 hours 

 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 (C.A.S.) 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% creatine in 40% KOH. This reagent deteriorates rapidly 

 at temperatures over 50°C. but maybe 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 A. P. H. A. method, but 

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

 incubation of the broth for 6 hours. Also, the 40% KOH has 0.3% creatine added to 

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

 creatine) the culture is shaken vigorously for one minute; a positive reaction is charac- 

 terized by an intense rose-pink color developing in a few seconds to ten minutes. 



A more detailed and accurate procedure for determining acetyl- 

 methyl-carbinol is given in Leaflet VI. 



Acid Production in Milk 



Acid production in milk may be determined very simply; but 

 the opacity of the milk must be taken into account if accurate de- 

 terminations are desired. The milk must be considerably diluted 

 before adding indicator for comparison with a buffer standard. 



Indicator milk is often useful. Litmus has been used most fre- 

 quently, as it indicates reduction as well as pH changes (although 

 roughly). Neutral litmus milk (about pH 6.8) has a lavender color, 

 which becomes red with acid production or blue with production of 

 alkalinity. Reduction is indicated by a partial or complete fading 

 of the color. The use of litmus milk has been seriously criticized 

 because of the inaccurate nature of litmus as a pll indicator; never- 

 theless the differences it brings out have enough practical value so 

 that it has not yet been superceded by any other indicator in milk. 



The use of brom cresol puri:)le, as was reconmien<led by 

 Clark and Lubs (1917) does not show changes in 0-R potential. 



