54 DIFCOMANUAL 



then with the same amount of Solution I. The plug is replaced in the tube and 

 is pushed down until it is about one inch above the surface of the medium. The 

 tube is heated in a boiling water bath for 15 minutes without allowing the 

 medium to touch the plug. The appearance of a red color on the plug indicates 

 the presence of indole. 



The Gnezda test is carried out as follows : A strip of filter paper is dipped into 

 a warm saturated solution of oxalic acid and is then dried thoroughly. The strip 

 is bent at an angle and is inserted into the culture tube in such a manner that it 

 presses against the sides and remains immediately below the cotton plug. Although 

 it is not necessary to sterilize the paper, aseptic precautions should be observed 

 when it is placed in the tube. The cotton plug is reinserted into the tube which 

 is then incubated at 37 °C. Development of a pink color on the paper indicates 

 the formation of indole. 



1 Standard Methods for the Examination ^ Centr. Bakt. I Abt. Orig., 40:129:1905. 

 of Water and Sewage, gth Edition: 230: 1946. ^ Indian J. Med. Research, 8:505:1921. 



2 Standard Methods for the Examination ' Compt. rend. acad. sci., 128:1584:1899. 



of Dairy Products, gth Edition: 92: 1948. ^ Manual of Methods of Pure Culture Study 



sZeit. Immunitaetsf. Exper. Therap., of Bacteria, 7th Edition: Leaflet V, 



55:311:1928. page 9, 1939. 



4 Diagnostic Procedures and Reagents, 



2nd Edition: 53: 1945. 



BACTO 



M.R.-V.P. MEDIUM (B16) 



DEHYDRATED 



Buffered Peptone 7 g. 



Bacto-Dextrose 5 g. 



Dipotassium Phosphate 5 g. 



Bacto-M.R.-V.P. Medium is recommended for the performance of the Methyl 

 Red and Voges-Proskauer Tests in differentiation of the coli-aerogenes group. 

 The use of this medium is recommended in Appendix I of "Standard Methods 

 for the Examination of Water and Sewage"^ for this purpose. 



Clark and Lubs^ first pointed out that in a suitable medium coli organisms 

 produced a high acidity which was constant, while the aerogenes group produced 

 a much less acid reaction, and on continued incubation became more alkaline. 

 This difference in the acidity produced in the cultivation of coli or aerogenes 

 could be recognized by the addition of the indicator methyl red. This Clark and 

 Lubs test has become known as the Methyl Red Reaction. 



Closely associated with the Methyl Red Reaction is the test described by Voges 

 and Proskauer,^ who noted that a color reaction took place if certain cultures in 

 a suitable medium were treated with potassium hydroxide and allowed to stand 

 for some time. This color reaction develops particularly in that part of the 

 medium exposed to the air, and is very similar to that of a dilute alcoholic solu- 

 tion of eosin. The development of the color reaction upon treatment of cultures 

 with potassium hydroxide was found to be due to the presence of acetyl-methyl- 

 carbinol, (3-hydroxy-2-butanone). Levine* recommended that the term "Voges- 

 Proskauer Reaction" be restricted to designate the formation of acetyl-methyl- 

 carbinol from dextrose. 



Bacto-M.R.-V.P. Medium was developed as a simple and reliable medium for 

 use in the performance of the Methyl Red and Voges-Proskauer Tests. Ruch- 

 hoft, Kallas, Chinn, and Coulter^ reported that Bacto-M.R.-V.P. Medium is 

 uniform and superior to laboratory-made media for these tests. 



For performance of the Methyl Red and Voges-Proskauer Tests, each tube 

 should be inoculated with a pure culture. 



