4. Close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water.

[Ill.u.s.tration: 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.]

[Ill.u.s.tration: FIG. 133.--Improved gas apparatus; the metal is contained in a perforated gla.s.s 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 gla.s.s 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 pa.s.s 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 gla.s.s dish 20 cm. diameter and 8 cm. deep. Flat leaden cross slightly shorter than the internal diameter of the gla.s.s dish.

Bell gla.s.s about 15 cm. diameter and 20 to 25 cm. high.

Metal frame for plate cultivations.

_Or_, gla.s.s battery jar for tube cultivations.

Cylinder of compressed hydrogen.

Rubber tubing.

Two pieces of ~U~-shaped gla.s.s tubing (each arm 8 cm. in length).

Half a litre of glycerine (or metallic mercury).

METHOD.--

1. Place the leaden cross inside the gla.s.s dish, resting on the bottom.

2. Prepare the cultivations in the usual way.

3. Place the tube cultivations in a gla.s.s 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 gla.s.s 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 gla.s.s dish. Fix a short length of rubber tubing clamped with a metal clip to each of the outside arms (Fig. 134).

6. Fill the gla.s.s dish with glycerine or metallic mercury to a depth of about 5 cm.

[Ill.u.s.tration: FIG. 134.--Botkin's apparatus.]

7. Connect up one U-shaped tube with the hydrogen cylinder (or gas apparatus) by means of rubber tubing. Replace the atmospheric air by hydrogen, as in method IX.

8. Clamp the tubes and disconnect the gas apparatus.

9. Incubate.

~Method XI~ (Novy's Method).--

~Apparatus Required.~--

Jar for plate cultivations (Fig. 135).

_Or_, jar for tube cultivations (Fig. 136).

Lubricant for stopper of jar.

Rubber tubing.

Cylinder of compressed hydrogen.

METHOD.--

1. Prepare cultivations in the usual way.

2. Place these inside the jar.

3. Lubricate the stopper and insert it in the mouth of the jar, with the handle in a line with the two side tubes.

4. Connect up the delivery tube a with the hydrogen gas supply by means of rubber tubing.

[Ill.u.s.tration: FIG. 135.--Novy's jar for plate cultivations.]

[Ill.u.s.tration: FIG. 136.--Novy's jar for tube cultivations.]

5. Attach a piece of rubber tubing to the exit tube b and collect samples of the issuing gas (over water) and test from time to time.

6. When the air is completely displaced by hydrogen, turn the handle of the stopper at right angles to the line of entry and exit tubes; this seals the orifice of both tubes.

7. Disconnect the gas apparatus and incubate.

(E) ~Method XII~ (Bulloch's Method).--

~Apparatus Required.~--

Bulloch's jar.

Pot of resin ointment.

Small gla.s.s dish 14 cm. diameter by 5 cm. deep.

Vessel for tube cultures or metal rack for plate cultures.

Pyrogallic acid tablets.

Cylinder of compressed hydrogen.

Geryk or other air pump.

Rubber pressure tubing.