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 20C.), 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).

_Reagents Required_:

Sulphuric acid (25 per cent.).

Metaphenylene diamine, 5 per cent. aqueous solution.

METHOD.--

1. Prepare tube cultivations and incubate together with control tubes (i. e., uninoculated tubes of the same medium, placed under identical conditions as to environment).

This precaution is necessary as the medium is liable to take up nitrites from the atmosphere, and an opinion as to the absence of nitrites in the cultivation is often based upon an equal colouration of the medium in the control tube.

Test both the culture tube and the control tube for the presence of nitrites.

2. Add a few drops of sulphuric acid to the medium in each of the tubes.

3. Then run in 2 or 3 c.c. metaphenylene diamine into each tube.

Brownish-red colour = nitrites.

The depth of colour is proportionate to the amount present.

~8. Gas Production.~--

(A) _Carbon Dioxide and Hydrogen._--

_Apparatus Required_:

Fermentation tubes (_vide_ page 161) containing sugar bouillon (glucose, lactose, etc.). The medium should be prepared from inosite-free bouillon (_vide_ page 183).

_Reagent Required_:

n/2 caustic soda.

METHOD.--

1. Inoculate the surface of the medium in the bulb of a fermentation tube and incubate.

2. Mark the level of the fluid in the closed branch of the fermentation tube, at intervals of twenty-four hours, and when the evolution of gas has ceased, measure the length of the column of gas with the millimetre scale.

Express this column of gas as a percentage of the entire length of the closed branch.

3. To a.n.a.lyse the gas and to determine roughly the relative proportions of CO_{2} and H_{2}, proceed as follows:

Fill the bulb of the fermentation tube with caustic soda solution.