osite-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 deg. C. and at 20 deg. 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).

_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 pr