s, and incubate at 37 deg. C. for forty-eight hours.

4. Heat in the steamer at 100 deg. C. for twenty minutes to destroy the
bacilli and some of their products.

5. Estimate the reaction of the medium and if necessary restore to +10.

6. Inoculate the bouillon from a pure cultivation of the B. coli
communis and incubate at 37 deg. C. for forty-eight hours.

7. Heat in the steamer at 100 deg. C. for twenty minutes.

Now fill two fermentation tubes with the bouillon, tint with litmus
solution, and sterilise; inoculate with B. lactis aerogenes. If no acid
or gas is formed, the bouillon is in a sugar-free condition; but if acid
or gas is present, again make the bouillon in the flask +10, reinoculate
with one or other of the above-mentioned bacteria, and incubate; then
test again. Repeat this till neither acid nor gas appears in the medium
when used for the cultivation of either of the bacilli referred to
above.

8. After the final heating, stand the flask in a cool place and allow
the growth to sediment. Filter the supernatant broth through Swedish
filter paper. If the filtrate is cloudy, filter through a porcelain
filter candle.

9. Tube, and sterilise as for bouillon.

Bouillon prepared in the above-described manner will prove to be
absolutely sugar-free; and from it may be prepared nutrient sugar-free
gelatine or agar, by dissolving in it the required percentage of
gelatine or agar respectively and completing the medium according to
directions given on pages 166 and 167. The most important application of
inosite-free bouillon is its use in the preparation of sugar bouillons,
whether glucose, maltose, lactose, or saccharose, of exact percentage
composition.

~Sugar (Dextrose) Bouillon.~--

1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6) or sugar-free bouillon (_vide supra_).

2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and
dissolve in the fluid.

3. Tube, and sterilise as for bouillon.

Ordinary commercial glucose serves the purpose equally well, but is not
recommended, as during the process of sterilisation it causes the medium
to gradually deepen in colour.

NOTE.--In certain cases a corresponding percentage of
lactose, maltose, or saccharose is substituted for glucose.

~Sugar Gelatine.~--

1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
out 1000 c.c.

2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissol