DEHYDRATED CULTURE MEDIA 55 



The Methyl Red Test is performed after 5 days incubation at 30° G. as recom- 

 mended by Vaughn, Mitchell and Levine^ and "Standard Methods."^ For the 

 Voges-Proskauer Test the culture should be incubated for 24 to 48 hours at 



Methyl Red Test 



To 5 ml. of culture add 5 drops of methyl red solution. A positive reaction is 

 indicated by a distinct red color, showing the presence of acid. A negative reac- 

 tion is indicated by a yellow color. The indicator solution is prepared by dissolv- 

 ing 0.1 gram Bacto-Methyl Red in 300 ml. of 95 per cent alcohol and diluting 

 to 500 ml. with distilled water. 



Voges-Proskauer Test 



To 5 ml. of culture add 5 ml. of a 10 per cent solution of potassium hydrox- 

 ide, mix well, allow to stand exposed to the air, and observe at intervals of 2, 

 12, and 24 hours. A positive test is indicated by the development of an eosin 

 pink color. This is the test as originally described. ^ 



Various other tests have been suggested as being excellently adapted to the 

 demonstration of the development of acetyl-methyl-carbinol, chief among which 

 are those of Werkman,'^ O'Meara,^ Levine, Epstein and Vaughn^ and Vaughn, 

 Mitchell and Levine.^ 



Werkman's tesf^ consists of the addition of 2 drops of a 2 per cent solution 

 of ferric chloride to 5 ml. of culture, followed by 5 ml. of 10 per cent sodium 

 hydroxide, and shaking the tube well to mix. A stable copper color appearing in 

 a few minutes is indicative of a positive test. 



O'Meara^ recommends the addition of approximately 25 mg. of solid creatine 

 to 5 ml. of culture and then adding 5 ml. concentrated (at least 40 per cent) 

 sodium hydroxide. The development of a red color in a few minutes, after 

 thorough agitation of the tube, is a positive test. Levine, Epstein and Vaughn^ 

 modified the O'Meara technique by dissolving the creatine in a concentrated 

 solution of potassium hydroxide. 



"Standard Methods"^ specifies the use of the Barritt^^ method as recom- 

 mended by Vaughn, Mitchell and Levine^ for the performance of this test. The 

 test is made by adding 0.6 ml. of 5 per cent alpha-naphthol in absolute ethyl 

 alcohol and 0.2 ml. of 40 per cent potassium hydroxide to 1 ml. of culture. 



To rehydrate the medium, dissolve 17 grams of Bacto-M.R.-V.P. Medium in 

 1000 ml. of distilled water. Distribute in 10 ml. quantities in test tubes. Sterilize 

 in the autoclave for 15 minutes at 15 pounds pressure (121°C.). The final reac- 

 tion of the medium will be pH 6.9. 



One pound of Bacto-M.R.-V.P. Medium will make 26.7 liters of medium. 



1 Standard Methods for the Examination ^ J. Am. Water Works Assoc, 3i:993:i939' 

 of Water and Sewage, gth Edition 1230: 1946. ' j, Bact., 20:121:1930. 



2 J. Infectious Diseases, 17:160:1915. ^ J. Path. Bact., 34:401:1931. 

 »Zeit. Hyg., 28:20:1898. » Am. J. Pub, Health, 24:505:1934. 

 *J. Bact., 1:153:1916. 10 J. Path. Bact., 42:441:1936. 



6 J. Bact., 22:125:1931. 



BACTO 



KOSER CITRATE MEDIUM (B15) 



DEHYDRATED 



Sodium Ammonium Phosphate 1-5 g. 



Monopotassium Phosphate 1 g. 



Magnesium Sulfate 0.2 g. 



Sodium Citrate 3 g. 



