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 jai's, 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 nuist be clamped down 

 securel}' 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 ver)' 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 renioved completely before 

 visible growth of the organism can possibly have taken place. Neglect of these 



Fig. 53.^ 



*Fic. S3. — Novy jar. Small size (wide mouth) for test-tube cultures. Only those with mouths 

 at least 2}4 inches wide are serviceaible. Height to mouth of jar, 7J4 inches. 



