CHEMICAL CRITERIA OF ANAEROBIOSIS 31 



up to 100°C. and layered upon the hot solution the color returned 

 to the latter in about half an hour as in the case of oil heated 

 with the solution in a boiling water bath. 



If one increases the depth of solution in a tube without oil, 

 several times over that of a similar tube with oil, both being 

 equally decolorized by boiling, the former may be seen to regain 

 its color even before the latter. 



These experiments lend little support to the use of oil as a 

 means of anaerobiosis and limit the technic where it is used to 

 layering on of freshl}^ boiled oil quickly cooled nearly to 100°C., 

 but even in this case it is less efficacious than the marble seal. 



In none of the experiments with alkaline glucose methylene 

 blue solution has there been any evidence of absorption of the 

 dye by the oil. Methylene blue is insoluble in oil. A bluish 

 tinge sometimes observed in the oil layer is reall}^ due to the 

 dye dissolved in a film of water which separates the oil from the 

 glass wall as I have mentioned elsewhere (1917) or, in the case 

 of oil-dye solutions actively boiled over the free flame or in a 

 strong salt solution bath, to emulsified water holding the dye in 

 suspension. A suggestion that the dye might be absorbed in 

 the form of the colorless leuco-base was proven erroneous by 

 pipetting off the oil from the tube of decolorized dye solution 

 into a tube of distilled water; on exposure to air the color returned 

 at once to the original dye solution whereas the water and the 

 oil overlying it remained quite colorless. 



Experiments analogous to some of those with the liquid solu- 

 tion have been performed with 2 per cent neutral agar made 

 alkaline by the addition of 1 cc. n/1 NaOH per 100 and colored 

 with 1 part methylene blue per 100,000 as offering a roughly 

 quantitative measure of the rate of air absorption which is indi- 

 cated by the thickness of the blue band that appears at the top 

 and deepens as exposure continues. Another advantage of this 

 means of test is that disproportionate volumes do not introduce 

 time differences into the observations of recoloration as they do 

 with a liquid test solution, yet in both cases the volumes and 

 areas exposed in different tubes have been kept identical for 

 comparative purposes except where otherwise noted. 



