absolute alcohol with sterile
forceps; burn off the alcohol in the gas flame.

4. Lower the now sterile cover-slip carefully on to the inoculated
surface of the medium, carefully excluding air bubbles, and press it
down firmly with the points of the forceps. (A sterile disc of mica may
be substituted for the cover-slip.)

5. Incubate.

~Method III~ (Roux's Physical Method).--

1. Prepare tube cultures of fluid media (or solid media rendered fluid
by heat) in the usual way.

2. Aspirate some of the inoculated media into capillary pipettes.

3. Seal both ends of each pipette in the blowpipe flame.

4. Incubate.

~Method IV~ (Roux's Biological Method).--

1. Plant a deep stab, as in method I.

2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous
aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the
surface of the medium; or an equal depth of liquefied gelatine, which is
then inoculated with the aerobic organism.

3. Incubate.

The growth of the aerobe will use up all the oxygen that reaches it and
will not allow any to pass through to the medium below, which will
consequently remain in an anaerobic condition.

(B) ~Method V.~--

1. Prepare tube or flask cultivations in the usual way.

2. Replace the cotton-wool plug by an india-rubber stopper perforated
with one hole and fitted with a length of glass tubing which has a
constriction about 3 cm. above the stopper and is then bent at right
angles (Fig. 129). The stopper and glass tubing are sterilised by being
boiled in a beaker of water for five minutes.

[Illustration: FIG. 129.--Vacuum culture.]

3. Connect the tube leading from the culture vessel with a water or air
pump, interposing a Wulff's bottle fitted as a wash-bottle and
containing sulphuric acid.

4. Exhaust the air from the culture vessel.

5. Before disconnecting the apparatus, seal the glass tube from the
culture vessel at the constriction, using the blowpipe flame.

6. Incubate.

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

~Apparatus and Solutions Required.~--

Buchner's tube (a stout glass 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
s