elted 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;
glass 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