240 PRINCIPLES OP SOIL MICROBIOLOGY 



used. When fruit extracts, like raisin or plum extracts, are used as a base for 

 the medium, no acid is required, since the natural acidity of the fruit is sufficient 

 to prevent the development of bacteria. After the organism has been isolated, it 

 is often necessary to obtain a single spore culture; especially when the organism 

 is wanted for the study of hereditary characteristics or for physiological in- 

 vestigations. 



For the demonstration of fungi present in the soil in the form of 

 vegetative mycelium, the direct inoculation method 26 and the direct 

 microscopic method 27 are available. According to the first method, 

 lumps of soil, about 1 cc. in diameter, are placed, with a sterile forceps, 

 into a sterile plate containing 10 cc. of sterile solidified agar medium. 

 The plates are allowed to incubate for 24 hours at 22°C. This period of 

 time is not sufficient for spores of the majority of soil fungi to germinate 

 and form a mycelium visible to the naked eye, whereas the organisms 

 actually living in the soil and forming a mycelium develop at once from 

 the soil, so that the mycelium becomes visible earlier. After 24 hours' 

 incubation the mycelium is transferred from the plates into sterile 

 slants of fresh agar medium (Czapek's), care being taken to isolate the 

 mycelium, which has grown away from the soil. The organisms thus 

 isolated can now be cultivated, purified if necessary, and identified. 



According to the second method, a small crumb (10 mgm. or less) 

 of soil is placed upon a microscopic slide and mixed with two or three 

 drops of water. A drop of methylene blue solution (saturated aqueous 

 or Loeffler solution) is then added by means of a glass rod, well mixed 

 with the soil suspension and covered with a cover slip. The preparation 

 appears blue to the naked eye. If too much stain has been added, it is 

 diluted by a drop of water. Examination is made with a dry lens and a 

 highpower eye piece. By this method, fungus filaments can be demon- 

 strated in all the soils examined. Some soils contain only 4 to 5 fila- 

 ments in a preparation (comprising 5 to 10 mgm. of soil), while others, 

 especially soils rich in undecomposed organic matter, contain fungus 

 mycelium in great abundance. 



The microscopic method, however, gives no means of identifying the 

 particular species of fungi present in the soil as vegetative mycelium. 

 This would be rather difficult, since the very nature of the growth of 



26 Waksman, S. A. Do fungi actually live in the soil and produce mycelium? 

 Science, N. S., 44: 320-322. 1916; The growth of fungi in the soil. Soil Sci., 

 14: 153-158. 1922. 



27 Conn, H. J. A microscopic method for demonstrating fungi and actin- 

 omycetes in soil. Soil Sci., 14: 149-152. 1922. 



