ANAEROBIC METHODS 17 
but this should be used with care since the hottom of the pipette may 
chip off. 
The counting chamber should be washed with dilute alkali or soap. 
It should be thoroughly rmsed in distilled water and wiped dry and 
should not be sterilized by heating because the cement which holds 
the disk will be melted. 
ANAEROBIC METHODS 
For the cultivation of those microorganisms which grow under 
anaerobic conditions, some special apparatus and methods are neces- 
sary to remove free oxygen. Many procedures have been proposed, 
a few of which will be given here. 
Boiling. Air is very soluble in water at room temperature. Wink- 
ler has found that 1 liter of water at 20° C. will dissolve about 9 mg. 
of oxygen. By boiling water for some time this dissolved oxygen may 
be driven off. Special methods are then necessary to prevent re- 
absorption of air. In liquid media where heating will cause no change, 
this may be done by a layer of sterile vaseline or ligroin. 
Displacement by an Inert Gas. For this purpose several gases, 
such as nitrogen, hydrogen, carbon dioxide, coal gas, etc., have been 
used. Hydrogen has had the widest application on account of its 
ease of preparation and lack of harmful effects on bacterial develop- 
ment. When these gases are used, they are bubbled through the 
medium and should be carefully washed by the proper solutions to 
render them pure. Gas wash bottles of the Bunsen or Friedrich types 
will allow satisfactory results. 
Absorption of Oxygen. For this purpose alkaline pyrogallol which 
has great affinity for oxygen is usually used. It becomes reduced 
to compounds which are not definttely known. In applying this 
chemical to the growth of anaerobes many devices have been used. 
Any one is quite satisfactory which prevents the ingress of more oxygen 
after the initial oxygen has been absorbed. 
Buchner’s Method. In this method the culture tube is placed 
in a larger one which contains the alkaline pyrogallol. It has the 
disadvantage that if some special support is not used to keep the cul- 
ture tube above the surface of the pyrogallol, it is difficult to follow 
erowth during incubation. These tubes may be prepared as follows: 
Introduce a few grams of pyrogallol into the bottom of a large 
test tube and place therein a support for the culture tube. This may 
be made from wood, cotton, glass, ete. After the culture tube has been 
