4. Spread a drop of fluid cultivation (or an emulsion of growth from a solid medium) over the upper surface of the agar block as if making a cover-slip film.

5. Place the slide and block covered by the bell gla.s.s in the incubator at 37 C. for ten minutes to dry slightly.

6. Take a clean dry sterile cover-slip in a pair of forceps, and with the help of a second pair of forceps lower it carefully on the inoculated surface of the agar (avoiding air bubbles), so as to leave a clear margin of cover-slip overlapping the agar block.

7. Invert the preparation and with the blade of the scalpel remove the slide from the agar block.

8. With a platinum loop run a drop or two of melted agar around the edges of the block. This solidifies at once and seals the block to the cover-slip.

9. Prepare a sterile hanging-drop slide, and smear hard vaseline or melted white wax on the rim of the metal cell.

10. Invert the cover-slip with the block attached on to the hanging-drop slide, and seal the cover-slip firmly in place.

11. Observe as for hanging-drop cultivations.

ANAEROBIC CULTIVATIONS.

Numerous methods have been devised for the cultivation of anaerobic bacteria, the majority requiring the employment of special apparatus.

The principle upon which any method is based and upon which it depends for its success falls under one or another of the following headings:

(a) ~Exclusion of air~ from the cultivation.

(b) ~Exhaustion of air~ from the vessel containing the cultivation by means of an air pump--i. e., cultivation _in vacuo_.

(c) ~Absorption of oxygen~ from the air in contact with the cultivation by means of pyrogallic acid rendered alkaline with caustic soda--i. e., cultivation in an atmosphere of nitrogen.

(d) ~Displacement of air~ by an indifferent gas, such as hydrogen or coal gas--i. e., cultivation in an atmosphere of hydrogen.

(e) A combination of two or more of the above methods.

A selection of the simplest and most generally useful methods is given here.

Whenever possible, the nutrient media that are employed in any of the processes should contain some easily oxidisable substance, such as sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per cent.), which will absorb all the available oxygen held in solution by the medium. The further addition of glucose, 2 per cent., favors the growth of anaerobic bacteria (_vide_, pages 189-190).

Further, it is advisable to seal all joints between india-rubber stoppers and tubulures or the mouths of the tubes with melted paraffin; gla.s.s stoppers and taps should be lubricated with resin ointment or a mixture of beeswax 1 part, olive oil 4 parts.

(A) ~Method I~ (Hesse's Method).--

1. Make a stab culture in gelatine or agar, choosing for the purpose a straight tube containing a deep column of medium, and thrusting the inoculating needle to the bottom of the tube.

2. Pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1 or 2 cm. deep, upon the surface of the medium.

3. Incubate.

~Method II.~--This method is only available when dealing with pure cultivations.

1. Liquefy a tube of gelatine (or agar) by heat, pour it into a Petri dish, and allow it to solidify.

2. Inoculate the surface of the medium in one spot only.

3. Remove a cover-slip from the pot of 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 subst.i.tuted 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 pa.s.s 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 gla.s.s tubing which has a constriction about 3 cm. above the stopper and is then bent at right angles (Fig. 129). The stopper and gla.s.s tubing are sterilised by being boiled in a beaker of water for five minutes.

[Ill.u.s.tration: 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 gla.s.s tube from the culture vessel at the constriction, using the blowpipe flame.

6. Incubate.