368 METHODS USED IN SYSTEMATIC BACTERIOLOGY 



Solution A: 



joarft-dimethylaminobenzaldehyde 4 gm. 



96 per cent. Alcohol . . 380 „ 



Concentrated HCl . . . 80 „ 



Solution B : 



Saturated watery solution of potassium persulphate. 



An alternative method, depending on the volatility of indole at 37° C, is recom- 

 mended by Holman and Gonzales (1923). It consists in placing a strip of filter 

 paper, soaked in a saturated watery solution of oxalic acid and subsequently dried . 

 between the cotton-wool plug and the tube. The paper should be carefully folded 

 so as to present the maximum surface to the volatilizing indole, which turns it a pink 

 colour. 



Some organisms form indole, but break it down more rapidly than they produce 

 it, and hence may give a false negative reaction (Reed 1942). 



Methyl-Red Test {M.R.). — ^Tested by adding 5 drops of an 0-04 per cent, solution 

 of methyl red to a culture in glucose phosphate medium (peptone 0-5 gm., 

 K2HPO4 0-5 gm., glucose 0-5 gm., water 100 ml., pH 7-5). Culture grown 

 for 5 days at 30° C or 3 days at 37° C. 



Red colour = positive. 



Yellow colour = negative. 



Voges-Proskauer Test {V.P.). — Tested by adding 1 ml. of a 10 per cent, solution 

 of KOH to a glucose phosphate culture grown for 5 days at 30° C. or 2 days 

 at 37° C. The colour develops slowly, and the test should be read after 

 18 to 24 hours. 



Pink fluorescence = positive. 

 No coloration = negative. 



A higher proportion of positive reactions is obtained by the use of O'Meara's 

 (1931) modification. A knife point of creatine is added to the culture, followed by 

 5 ml. of 40 per cent, sodium hydroxide. The tube is shaken thoroughly for 2 to 5 

 minutes. A positive reaction is characterized by the appearance of a pink colour 

 within about 2 minutes, unaccompanied by fluorescence ; the development of 

 the colour may, however, be delayed for an hour or longer. 



An even more sensitive test for acetylmethylcarbinol is that described by 

 Barritt (1936). It consists in adding 0-6 ml. of a 5 per cent, alcoholic solution 

 of a-naphthol and 0-2 ml. of 40 per cent. KOH solution to 1 ml. of culture. In 

 a positive reaction a pink colour appears in 2-5 minutes, deepening to magenta 

 or crimson in half an hour. In a negative reaction the mixture remains colour- 

 less for an hour or so, when it may become copper-coloured owing to the action 

 of KOH on the a-naphthol. Traces of pink coloration are best neglected. 



Nitrate Reduction. — Tested on a broth culture containing 0-1 per cent. KNO3, 

 grown for 5 days at 37° C, by the Griess-Ilosva method. 



Solution A : 



a-naphthylamine ... 1 gm. 



Water 22 ml. 



Dissolve, filter, and then add 180 ml. of dilute acetic acid (sp. gr. 1-04). 



