NOTE.--In place of performing the test in two stages as given above, 2 c.c. concentrated _commercial_ sulphuric, hydrochloric, or nitric acid (all of which hold a trace of nitrite in solution), may be run into the cultivation. The development of a red colour within twenty minutes will indicate the presence of indol. ~5a. Phenol Production.~-- _Medium Required_: Nutrient bouillon. _Reagents Required_: Hydrochloric acid, concentrated. Millon's reagent. Ferric chloride, 1 per cent. aqueous solution. METHOD.-- 1. Prepare cultivation in a Bohemian flask containing at least 50 c.c. of medium, and incubate. Test for phenol in the following manner: 2. Add 5 c.c., 25 per cent. sulphuric acid to the cultivation and connect up the flask with a condenser. 3. Distil over 15 to 20 c.c. Divide the distillate into three portions a, b and c. 4. Add to (a) 0.5 c.c. Millon's reagent and boil. Red colour = phenol. 5. Add to (b) about 0.5 c.c. ferric chloride solution. Violet colour = phenol. (If the distillate be acid the reaction will be negative.) 6. Add to (c) bromine water. Crystalline white ppt. of tribromo-phenol = phenol. NOTE.--If both indol and phenol appear to be present in cultivations of the same organism, it is well to separate them before testing. This may be done in the following manner: 1. Prepare inosite-free bouillon cultivation, say 200 or 300 c.c., in a flask as before. 2. Render definitely acid by the addition of acetic acid and connect up the flask with a condenser. 3. Distil over 50 to 70 c.c. Distillate will contain both indol and phenol. 4. Render the distillate strongly alkaline with caustic potash and redistil. Distillate will contain indol; residue will contain phenol. 5. Test the distillate for indol (_vide ante_). 6. Saturate the residue, when cold, with carbon dioxide and redistil. 7. Test this distillate for phenol (_vide ante_). ~6. Pigment Production.~-- 1. Prepare tube cultivations upon the various media and incubate under varying conditions as to temperature (at 37° C. and at 20°C.), atmosphere (aerobic and anaerobic), and light (exposure to and protection from). Note the conditions most favorable to pigment formation. 2. Note the solubility of the pigment in various solvents, such as water (hot and cold), alcohol, ether, chloroform, benzol, carbon bisulphide. 3. Note the effect of acids and alkalies respectively upon the pigmented cultivation, or upon solutions of the pigment. 4. Note spectroscopic reactions. ~7. Reducing Agent Formation.~-- (a) _Colour Destruction._-- 1. Prepare tube cultivations in nutrient bouillon tinted with litmus, rosolic acid, neutral red, and incubate. 2. Examine the cultures each day and note whether any colour change occurs. (b) _Nitrates to Nitrites._-- _Medium Required_: Nitrate bouillon (_vide_ page 185). Or nitrate peptone solution (_vide_ page 186).