CHEMICAL CRITERIA OF ANAEROBIOSIS 25 



In summary, the difference between plant and animal tissues 

 and inert substances such as sand are the differences between 

 more or less soluble organic substances and insoluble inorganic 

 substances. No doubt, plant and animal tissues may serve the 

 same mechanical purposes as sand; in addition they may supply 

 nutrients to the medium, buffer substances and possibly even 

 "vitamins." With plant and animal tissues in media, otherwise 

 lacking in reducing substances, these may be of supplementary 

 importance in the cultivation of obligate anaerobes. Finally, 

 unless we are willing to concede some importance to adsorbtion 

 as a factor in anaerobiosis, sand and other inert porous substances 

 may serve only as a means of maintenance of reduced oxygen 

 tension, i.e., as seals, whereas tissues may serve not only this 

 purpose but may actually aid in the reduction of oxygen tension 

 in addition to the nutritive functions they fulfill. We may 

 emphasize the importance of heat in this connection since the 

 existence of a true self active reducing agent as distinguished 

 from the phenomenon of adsorbtion seems as yet unproved. 



As to physical reduction, i.e., ebullition, the data already 

 presented bear testimony to the efficacy of boiling. In these 

 tests, as in the actual culture of anaerobes, boiling is often an 

 essential preliminary procedure in the test. The use of both 

 liquid and solid deep media so decolorized shows the first return 

 of color at the top and proves the importance in the case of 

 Hquid solutions, of such factors as narrowness of bore in the tube 

 volume of solution, the effect of diffusion currents, etc. With 

 solid media these are not so important. 



In 1, 2 and 3 per cent agar, with n/100 NaOH and 1:100,000 

 methylene blue, decolorized by boiling in standard culture tubes 

 of 1.5 cm. bore, the depth of the blue band at the top of the 

 agar at various intervals appeared as in table 4. 



Apparently variation of agar content, within the limits of 

 1 to 3 per cent makes only a little difference in the rate or depth 

 of recoloration. We may point out that the depth of blue color 

 at the top of the agar column corresponds roughly to that in a 

 deep glucose agar stab or shake culture which is free from growth 

 though there are doubtless variations according to species, and 



