To 1,000 cc. of distilled water add 0.3 gram tryptophane, 5 grams dipotassium hydrogen phosphate (K_{2}HPO_{4}), and 1 gram peptone. Heat until ingredients are thoroughly dissolved, tube (6 to 8 cc.), and sterilize in autoclave for 15 minutes after the pressure reaches 15 pounds. Some American peptones are standardized to contain a uniform amount of tryptophane. If such peptone is used the tryptophane in the above formula may be omitted and the peptone increased to 5 grams. (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. Footnote J: (b) _Synthetic Medium for the Methyl Red Test._ To Make One Liter. Dissolve 7 grams Na_{2}HPO_{4} (anhydrous) or 8.8 grams Na_{2}HPO_{4}.2H_{2}O, 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 Na_{2}HPO_{4}.12H_{2}O for two weeks. Use 0.88 per cent. of Na_{2}HPO_{4}.2H_{2}O. 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.