ANAEROBIC CULTURES. 57 
or even two or three in series, will completely remove it, since the bubbles of 
gas are in contact with the fluid only at their surface and for a very brief time. 
Hydrogen must be passed through 5 wash-bottles of sodium hydroxide and pyrogallol 
if every trace of oxygen is to be removed. From nitrogen or carbon dioxide the 
last traces of oxygen may be removed by passing it over copper filings inclosed 
in a piece of gas-pipe which is heated red hot in a small furnace containing about 
20 Bunsen flames in series. ‘The gas-pipe may be 0.75 inch in diameter and about 
3 feet long, plugged at the ends with tight-fitting rubber stoppers, the middle 2 feet 
filled with the copper fragments. The gas should be allowed to flow only in rapid 
bubbles, not in a stream (Dr. Day). 
The test-tube cultures may be placed in Novy jars, securely waxed (fig. 53), 
or in large, thick-walled test-tubes made impervious 
with sealing wax (see Sternberg, Manual, fig. 53; 
Text-Book, fig. 53). Media designed for use in any 
» of these gases should be resteamed immediately 
before inoculation, and if one is experimenting with 
unknown or with very sensitive anaerobes the boiled 
media should be allowed to cool in an atmosphere 
of hydrogen. Francis Darwin’s wax-mixture has 
been found useful for luting. 
When large Novy jars are used (fig. 54), the 
thoroughly waxed gaskets must be clamped down 
securely and tested for leaks by preliminary exhaus- 
tions. If any are discovered, additional wax must 
be used and the clamps must be screwed tighter. ‘To 
determine whether there is any subsequent entrance 
of air it is always best to include along with the 
cultures one or more tubes containing some sub- 
stance which is reduced in the absence of free oxygen, but which readily oxidizes 
to some different color as soon as traces of air are mixed with the gas in the jar. 
Methylene blue in recently steamed bouillon or gelatin with 5 per cent grape-sugar 
is one of the best pigments for this purpose. In the absence of free oxygen it 
becomes a colorless substance ; with the entrance of traces of air it becomes blue. 
Usually, however, the fluid or solid holds on to a trace of color at its surface. A 
solution of bilirubin is also said to be very sensitive to free oxygen and a good test 
for its presence. 
Some care is necessary in order to avoid erroneous conclusions when pyro- 
gallol and caustic potash are used to absorb the oxygen. The vessel must not 
leak, enough of the mixture must be used to absorb all the oxygen, and the action 
must be rapid enough so that the oxygen will have been removed completely before 
visible growth of the organism can possibly have taken place. Neglect of these 
*Fic. 53—Novy jar. Small size (wide mouth) for test-tube cultures. Only those with mouths 
at least 2%4 inches wide are serviceable. Height to mouth of jar, 7%4 inches. 
