CHEMICAL CRITERIA OF ANAEROBIOSIS 27 



air soon after boiling. But large flasks of solution or very 

 slender deep tubes of such decolorized solutions remain decolor- 

 ized for some hours — sufficiently long, indeed, for anaerobic 

 growth to be initiated in suitable media heavily inoculated under 

 similar circumstances. In the Smith fermentation tube efficacy 

 for anaerobic culture, or maintenance of decolorization of meth- 

 ylene blue, depends largely upon the bore at the bend and satis- 

 factory results for either cannot be secured without the use of 

 a special seal, such as sand, tissues, etc. In some unprotected 

 tubes the color returned to the solution in the closed arm in 

 fifteen minutes, in others in sixty minutes and in still others 

 after several hours; with sand seals the dye color returned only 

 in the open arm and then gradually faded even here through 

 adsorption. 



The method of deep colony culture between the nested halves 

 of a Petri dish when tested with methylene blue showed a progres- 

 sive recoloration of the decolorized dye from the periphery inward, 

 except when protected by a paraffine or vasehne seal. 



There is no phase of the methylene blue problem to which 

 we have given more careful attention than its use in connection 

 with insoluble liquid seals, i.e., hydrocarbon oil, wax and grease. 

 The widespread use of these substances as a means of excluding 

 oxygen together with the theoretical and practical objections to 

 their use, especially in the case of oil, have justified a searching 

 examination of this matter. The results with methylene blue 

 only serve to support the conclusion that liquid hydrocarbons 

 are to a degree superfluous and inefficacious means of maintain- 

 ing anaerobiosis; on the other hand the waxes and semisolid 

 grease seals are more satisfactory for certain purposes, from the 

 standpoint of air exclusion, though inferior to mechanical seals 

 in the matter of convenience and cleanliness. 



In the first place an alkaline glucose solution of methylene 

 blue does not decolorize at 37°C. under 2.5 cm. depth of mineral 

 oil, although it may be mentioned here that it does so readily 

 in a few hours under the marble seal in a constricted tube, or 

 under a cover slip in a plain tube, or under a thin layer of paraffine. 



I have shown repeatedly with different samples of mineral 



