164 PRINCIPLES OF SOIL MICROBIOLOGY 



determining the end point of free oxygen. 13 However, various difficul- 

 ties are found in an attempt to use indicators, such as methylene blue, 

 as criteria in anaerobiosis. 



For the existence of even obligate anaerobes in the soil we need not 

 imagine a soil atmosphere free from atmospheric oxygen, but simply 

 that anaerobic conditions, favorable for the activities of these organisms, 

 are produced due to the active utilization of the oxygen and production 

 of CO2 by aerobic organisms, which results in a reduction of the oxygen 

 tension. This can be imitated artificially in the laboratory, when 

 anaerobes are grown readily under ordinary conditions, in the pres- 

 ence of rapidly growing aerobic bacteria, like Bac. sabtilis. Another 

 illustration of this phenomenon is the growth of the two nitrogen- 

 fixing organisms, the anaerobic Bac. amylobacter and the aerobic, 

 rapidly growing Azotobacter. Exposure to oxygen has, however, an 

 injurious effect upon anaerobic organisms, vegetative cells being de- 

 stroyed in 10 minutes and spores in 8 days; 14 in the case of Bac. amylo- 

 bacter; the injurious effect of air exposure upon the vegetative cells 

 sets in only after 40 minutes, while the spores are not injured even after 

 3 hour exposure. ^ 5 



Methods of isolation of anaerobic bacteria from the soil. There are a 

 number of methods available for the isolation of anaerobic bacteria. 16 

 These bacteria have to be separated not only from aerobic organisms, 

 but often also from other facultative or obligate anaerobic bacteria. 

 The anaerobes, just as the aerobic bacteria, vary greatly in their food 

 requirements and manner of growth, and the methods of isolation have 

 to be adapted to the particular organism in question. There is a large 

 number of species of anaerobes in the soil and it is insufficient to depend 

 on microscopic examinations alone for demonstrating the existence of 

 specific forms. In all cases, the isolation and demonstration of the 

 different species must be undertaken. In case the nature of an organ- 

 ism that is looked for is known, the development of a proper culture 



13 Hall, I. C. Chemical criteria of anaerobiosis with special reference to 

 methylene blue. Jour. Bact. 6: 1-42. 1921. Kadisch, E. Centrbl. Bakt. I, 

 Orig. 90:462-468. 1923. Clark, W. M., Cohen, B., and Gibbs, H. D. Studies on 

 oxidation-reduction. VIII. Methylene blue. U. S. Publ. Health Serv. Publ. 

 Health Repts. Repr. no. 1017. 1925. 



14 Bachmann, F. Beitrag zur Kenntnis obligat anaerober Bakterien. Cen- 

 trbl. Bakt. II, 36: 1-41. 1912. 



15 Dorner, 1924 (p. 165). 



16 Heller, H. H. Principles concerning the isolation of anaerobes. Jour. Bact. 

 6: 445-470. 1921. 



