Methods of Stud)ing the Soil Popiihitioii 45 



ing organized contents. Such hyphae are stained purple in contrast 

 with the deep blue coloration of those filled with protoplasm. This 

 was confirmed by inoculating sterilized soil with fungal mycelium, 

 allowing it to incubate for several days, and making films from a 

 sample of this soil; on these films only deeply stained fungal frag- 

 ments were seen. In normal soils, mycelium is scanty, and because 

 of its filamentous nature and very variable length is not amenable to 

 accurate statistics, though useful comparative results may be ob- 

 tained. There were significantly fewer pieces of mycelium present 

 in the soils receiving mineral fertilizer than in those receiving 

 farmyard manure. 



Lengths of well-stained mycelium frequently have organic matter 

 adherent to their walls, probably through secreted mucilage. This 

 may have an important bearing on the formation of soil crumbs. 

 Few fungal spores are seen. Fibers may be distinguished from hy- 

 phae by their lack of staining and their polarization colors under 

 crossed nicols. Other plant tissue absorbs but little dye and, at most, 

 has a greenish hue. Stained nematodes are sometimes seen, and 

 what are thought to be earthworm setae can be distinguished from 

 fragments of mycelium by their tapering apices. 



Plate Culture Methods. The gelatin plate followed by the agar 

 plate was the first method used for the enumeration of soil organisms. 

 It still remains the one most commonly employed. The method con- 

 sists in suspending a given portion of soil in a given volume of sterile 

 tap water, and making a series of dilutions, such as 1 : 100, 1 : 1,000, 

 to 1:10,000,000. The final dilutions of soil are prepared in such a 

 manner as to allow 40-200 colonies to develop on each plate. One- 

 milliliter portions of the final dilutions ai'e placed in plates to which 

 suitable agar media are then added; the contents of the plates are 

 carefully mixed; the plates are then incubated at 28-30° C, and the 

 colonies counted after varying periods of time. 



The plate method is very convenient, but its chief limitation lies 

 in the fact that it allows the development of only the heterotrophic 

 aerobic bacteria, certain yeasts, molds, and actinomycetes. Fre- 

 quently, special media are used for the enumeration of fungi, 

 whereby bacterial development is suppressed either by acidifying 

 the medium or by the addition of antibacterial agents. This makes 

 possible the use of much lower dilutions of soil than those required 

 for the enumeration of the larger numbers of bacteria. 



The plate method, supplemented with other methods, has made 

 it possible to establish that different soil types possess characteristic 



