172 PRINCIPLES OF SOIL MICROBIOLOGY 



upon the specific organisms. Certain special methods may also be used. Among 

 these, gelatin and milk have played an important part. By inoculating milk 

 with a small quantity of soil, a certain type of butyric acid bacteria can be readily 

 demonstrated. This is not a medium for enrichment of anaerobes, as for differ- 

 ential purposes. Various protein (egg-albumin) and glucose media can be used. 

 The medical bacteriologists have made extensive use of brain and blood agar 

 media. To demonstrate the presence of certain organisms, specific media may 

 have to be used. To demonstrate the presence of Bac. amylobacter, nitrogen-free 

 g'.ucose (2 per cent) agar placed in a deep tube is inoculated with a soil sus- 

 pension; if quantitative results are wanted, various dilutions are employed (the 

 soil suspension may be previously heated, in a water bath, at 80° for 10 minutes, 

 whereby only the number of spores is obtained). The tubes are closed with rubber 

 stoppers (when the culture is to be isolated, a surface layer of sublimate agar is 

 used) and incubated at 30°. Gas formation will take place on the second day, 

 breaking up the medium. This and the production of butyric acid will indicate 

 the presence of the organism ; the colonies are lens-shaped. Microscopic examina- 

 tion of the culture can be made by staining with Lugol's reagent. The method of 

 Burri can be used for determining the number of anaerobic bacteria in the soil, 

 not only by establishing the presence of growth in the final dilution, but by 

 actually counting the colonies in the deep tube. 



Classification of soil anaerobes. Various systems for the classification 

 of anaerobic bacteria and their relation to aerobes have been proposed 

 at different times. 54,55 But even at the present time, a proper classifica- 

 tion of anaerobes, especially of soil forms is lacking. The idea that 

 anaeobic bacteria vary greatly has served further to increase the exist- 

 ing confusion. This led to various exaggerations, such as the existence 

 of only a few anaerobic forms which change into one another, or the 

 making of new genera on the basis of minor physiological differences. 56 



The following system of classification of soil anaerobes may be sug- 

 gested here merely as a tentative working basis : 



I. Bacteria acting primarily upon carbohydrates: 



1. Bacteria utilizing largely simple carbohydrates and starches as sources 

 of energy, often referred to as saccharolytic. Here belong the buty- 

 ric acid bacteria, often classified as one species, Bac. amylobacter 

 A. M. et Bred. These decompose sugars with the formation of 

 butyric acid and gas : 

 (a) Nitrogen-fixing bacteria — ■Clostridium pastorianum Winogradsky 

 (Bac. amylobacter von Tieghem, Bac. amylocyme Perdrix, 

 Bac. butyricus Botkin, Granulobacler saccarobutyricum Beij., 

 Bac. orthobutylicus Grimbert, Clostridium americanum Pring- 



" Hibler, 1908 (p. 167); Bredemann, 1909 (p. 109). 

 65 Jungano, M., and Distaso, A. Les anaerobies. Paris. 1910. 

 "Heller, H. H. Classification of the anaerobic bacteria. Bot. Gaz. 73: 

 70-79. 1922; Jour. Bact. 7: 1-38. 1922. 



