(C) ~Method VI~ (Buchner's Method).

~Apparatus and Solutions Required.~--

Buchner's tube (a stout gla.s.s test-tube 23 cm. long and 4 cm. in diameter, fitted with india-rubber stopper, Fig.

130).

Pyrogallic acid in compressed tablets each containing 1 gram.

Dekanormal solution of caustic soda.

METHOD.--

1. Prepare the tube cultivation in the usual way.

2. Moisten the india-rubber stopper of the Buchner's tube with water and see that it fits the mouth of the tube accurately.

3. Remove the stopper from the caustic soda bottle.

4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube (roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity of the receiving vessel).

5. Add about 1 c.c. of the soda solution.

6. Place the inoculated tube inside the Buchner's tube. The pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the Buchner's tube even should it be slipped in hurriedly.

7. Fit the india-rubber stopper tightly into the mouth of the Buchner's tube.

[Ill.u.s.tration: FIG. 130.--Buchner's tube.]

The pyrogallic acid tablet dissolves slowly in the soda solution and its oxidation proceeds very slowly at first so that ample time is available when this method is adopted.

8. Restopper the caustic soda bottle.

9. Place Buchner's tube in a wire support, and incubate.

~Method VII~ (Wright's Method).--

1. Prepare tube cultivation in the usual way.

2. Cut off that portion of the cotton-wool plug projecting above the mouth of the tube with scissors, then push the plug into the tube for a distance of 2 or 3 cm.

3. By means of a pipette drop about 1 c.c. of pyrogallic acid 10 per cent. aqueous solution on to the plug. It will immediately be absorbed by the cotton-wool.

4. With another pipette run in an equal quant.i.ty of the caustic soda solution.

5. Quickly close the mouth of the tube with a tightly fitting india-rubber stopper.

6. Incubate.

[Ill.u.s.tration: FIG. 131.--McLeod's anaerobic plate base with half petri dish inverted _in situ_]

~Method VIII~ (McLeod's Method).--

~Apparatus and Solutions Required.~--

McLeod's plate base (a hollow glazed earthenware disc 9 cm.

in diameter and 2 cm. deep: the upper surface is pierced by a central hole, 2 cm. in diameter, giving access to the interior, the lower part of which is divided into two by a low part.i.tion. A shallow groove encircles the upper surface near to the edge).

Plasticine.

Pyrogallic acid (1 gramme) compressed tablets.

Sodic hydroxide (0.4 gramme) compressed tablets.

Wash bottle of distilled water.

Surface plates of one or other agar medium (in petri dishes of 8 cm. diameter).

Surface plate spreader.

METHOD.--

1. Roll out a long cylinder of plasticine and fit it into the groove on the upper surface of the earthenware base.

2. Place a tablet of pyrogallic acid in one division of the interior of the plate base, and two tablets of sodic hydroxide in the other.

3. Prepare surface plate culture of the organism to be cultivated.

4. Run a few cubic centimetres of distilled water into that division of the plate base containing the sodic hydroxide.

5. Invert the bottom half of the surface plate over the plate base and press its edges firmly down into the plasticine filling the groove.

6. Label and incubate.

(D) ~Method IX.~--

~Apparatus Required.~--

Small Ruffer's or Woodhead's flask (Fig. 33).

Sterile india-rubber stopper.

India-rubber tubing.

Gla.s.s tubing.

Metal screw clips.

Cylinder of compressed hydrogen; or hydrogen gas apparatus

METHOD.--

1. Sterilise a gla.s.s vessel, shaped as in a Ruffer's or Woodhead's flask, in the hot-air oven. (The tubulure and the side tubes are plugged with cotton-wool.) After sterilisation, fix a short piece of rubber tubing occluded by a metal clip to each side tube.

2. Inoculate a large quant.i.ty (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 gla.s.s vessel.