2. Inoculate a large quantity (e. g., 200 c.c.) of the medium. Where solid media are employed they must first be liquefied by heat. 3. Remove the cotton-wool plug from the tubulure and pour the inoculated medium into the glass vessel. 4. Close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water. [Illustration: FIG. 132.--Kipp's hydrogen apparatus, (a) connected up to two washing bottles containing (b) lead acetate 10 per cent. solution, to remove H_{2}S and (c) silver nitrate solution to remove AsH_{3}. A third washing bottle containing pyrogallic acid 10 per cent. solution, rendered alkaline, to remove any trace of oxygen, is sometimes introduced.] [Illustration: FIG. 133.--Improved gas apparatus; the metal is contained in a perforated glass tube which is submerged in acid when the triangular bottle is upright (a), but is above the level of the liquid when the bottle is turned on its side (b).] 5. Connect up the india-rubber tubing on one of the side tubes with a cylinder of compressed hydrogen (or the delivery tube of a Kipp's Fig. 132 or other hydrogen apparatus, Fig. 133), interposing a short piece of glass tubing; and in like manner connect a long piece of rubber tubing which should be led into a basin of water, to the opposite side tube. 6. Open both metal clips and pass hydrogen through the vessel until the atmospheric air is replaced by hydrogen. This is determined by collecting some of the gas which bubbles through the water in the basin in a test-tube and testing it by means of a lighted taper. 7. Close the metal clip on the tube through which the gas is entering; close the clip on the exit tube. 8. Disconnect the gas apparatus. 9. Incubate. ~Method X~ (Botkin's Method).-- ~Apparatus Required.~-- Large glass dish 20 cm. diameter and 8 cm. deep. Flat leaden cross slightly shorter than the internal diameter of the glass dish. Bell glass about 15 cm. diameter and 20 to 25 cm. high. Metal frame for plate cultivations. _Or_, glass battery jar for tube cultivations. Cylinder of compressed hydrogen. Rubber tubing. Two pieces of ~U~-shaped glass tubing (each arm 8 cm. in length). Half a litre of glycerine (or metallic mercury). METHOD.-- 1. Place the leaden cross inside the glass dish, resting on the bottom. 2. Prepare the cultivations in the usual way. 3. Place the tube cultivations in a glass battery jar (or the plate cultivations on a metal frame), resting on the centre of the leaden cross. 4. Cover the cultivations with the bell jar. 5. Adjust the U-shaped pieces of glass tubing in a vertical position on opposite sides of the bell jar, one arm of each inside the jar, the other outside. These tubes are best held in position by embedding the U-shaped bends in two lumps of plasterine stuck on the bottom of the glass dish. Fix a short length of rubber tubing clamped with a metal clip to each of the outside arms (Fig. 134).