Standard methods for the examination of water and sewage

by American Public Health Association. Laboratory Section; American Chemical Society; Association of Official Agricultural Chemists

(3) After 48 hours record gas formation in adonite and saccharose broths. Determine indol formation in tryptophane broth by adding drop by drop, to avoid mixing with the medium, about 1 cc. of a 2 per cent. alcoholic solution of p-dimethyl amido-benzaldehyd, then a few drops of concentrated hydrochloric acid. The presence of indol is indicated by a red color which is soluble in chloroform. There may be some unconverted tryptophane still present which will give a distinctly blue color which is insoluble in chloroform. A mixture of the two will be either blue or violet. If from such a mixture of colors the red of indol be extracted with chloroform proof of the presence of indol will be complete.

(4) After 5 days apply methyl red test and Voges-Proskauer test to dextrose broth.

Methyl Red Test.[^[J]]

Indicator solution.—Dissolve 0.1 gram methyl red in 300 cc. alcohol and dilute to 500 cc. with distilled water.

[J]. (b) Synthetic Medium for the Methyl Red Test. To Make One Liter. Dissolve 7 grams Na₂HPO₄ (anhydrous) or 8.8 grams Na₂HPO₄.2H₂O, 2 grams KHphthalate, 1 gram aspartic acid, and 4 grams dextrose in about 800 cc. of warm distilled water. When solution is complete, cool and make up to 1 liter at room temperature. Heat in an autoclave for 15 minutes after the pressure has reached 15 pounds, provided the total time of exposure to heat is not more than one-half hour. The hydrogen-ion concentration of the medium is fixed by the composition. It should be very close to P_H 7.0, slightly red with phenol red. All materials should be recrystallized or if used from stock furnished by manufacturers, should be carefully examined. The di-sodium hydrogen phosphate may be used either as the anhydrous salt obtained by dessication in vacuo at 100° C. or else as the salt containing two molecules of water of crystallization. This is obtained by exposing the recrystallized Na₂HPO₄.12H₂O for two weeks. Use 0.88 per cent. of Na₂HPO₄.2H₂O.

Procedure in test.—1. To 5 cc. of each culture add 5 drops of methyl red solution.

2. Record distinct red color as methyl red +, distinct yellow color as methyl red -, and intermediate colors as ?.

Voges-Proskauer Test.[^[216]]

To the remaining 5 cc. of medium add 5 cc. of a 10 per cent. solution of potassium hydroxide. Allow to stand over night. A positive test is indicated by an eosin pink color.

(5) Gelatin tubes should not be pronounced negative until they have been incubated at least 15 days.