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 dissolve in the hot gelatine. 3. Filter through papier Chardin. 4. Tube, and sterilise as for nutrient gelatine. ~Sugar Agar.~-- 1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out 1000 c.c. 2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the clear agar. 3. Tube, and sterilise as for nutrient agar. NOTE.--Other "sugar" media are prepared by substituting a corresponding percentage of lactose, maltose (or any other of the substances referred to under "Sugar Media," page 177) for the glucose. ~Iron Bouillon.~-- 1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 141, sections 1 to 6). 2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve it in the bouillon. 3. Tube, and sterilise as for bouillon. NOTE.--The lactate of iron may be substituted for the tartrate. ~Lead Bouillon.~-- 1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1 to 6).