SELMAN A. WAKSMAN 311 



the determination of the numbers of protozoa, algae, and specific physiological groups of 

 bacteria, the dilution method has to be employed. The nematodes and other worms can be 

 counted directly in soil, by using a magnifying glass and special methods of suspension. The 

 study of biochemical activities can be carried out either in the soil itself or by the use of 

 pure and mixed cultures of organisms, which have been separated from the soil, under con- 

 trolled laboratory conditions. 



Most of the bacteria live in or upon the surface of the colloidal material which surrounds 

 the inorganic soil particles. It is frequently very difficult to remove the cells from the dead 

 organic and inorganic matter and separate them from one another. As a result of this and 

 also because many of the soil bacteria do not develop upon ordinary agar media, the determi- 

 nation of the number of soil bacteria by the plate method is far from sufficient to give an 

 idea of the abundance of the soil population. The direct microscopic method is more accurate 

 for the determination of the abundance of micro-organisms in the soil. This method consists 

 in spreading a definite amount of a dilute suspension of soil in water upon a definite area of 

 a glass slide, drying, covering with a dilute solution of agar or gelatin, fixing in alcohol and 

 staining with a solution of rose bengal or erythrosine in 5 per cent phenol solution. The 

 bacteria are stained red, while the inorganic particles and the dead organic matter is either 

 not stained at all or is stained only faintly. Only the bacteria, the spores of fungi, and proto- 

 zoan cysts can thus be counted. The living protozoa, as well as some of the mycelium of 

 fungi and actinomyces, are largely destroyed in the process of staining. It is also somewhat 

 difficult to distinguish, by this method, the living from the dead bacteria, the active from 

 the inactive forms. This method, having the great advantage of giving the absolute number 

 of bacteria present in the soil, has the disadvantage that it does not lend itself to determina- 

 tions, except within certain broad groups, of the specificity of the relative groups of organisms 

 in the soil. The presence and abundance of fungi in the soil can be determined by adding a 

 drop of methylene blue to a suspension of soil in water placed upon a slide and examined 

 directly. 



The cultural methods which are used for the determination of the numbers of micro- 

 organisms in the soil are divided into two categories: (i) the plate method, and (2) the dilu- 

 tion method. 



1. The plate method has been used most commonly. It consists in diluting the soil with 

 different amounts of sterile tap water, then plating out i-cc. portions of the final dilutions, 

 using an appropriate agar or gelatin medium. The plates are incubated for a period of time 

 ranging from two to fourteen days, then the number of colonies developing on the plate is 

 determined. This method has a number of very serious disadvantages, which make it almost 

 worthless as far as accurate information concerning the actual abundance of soil organisms 

 is concerned. There is no single medium yet devised which allows the development of 

 all soil organisms. The anaerobic bacteria do not develop at all or only to a very limited 

 extent. The autotrophic bacteria, including the nitrite- and nitrate-forming, sulphur-oxi- 

 dizing organisms, etc., do not develop at all. Many of the cellulose-decomposing bacteria do 

 not grow on common media; the nitrogen-fixing forms may grow only to a limited extent, 

 etc. However, as far as the organisms that are capable of developing on the particular 

 agar medium are concerned, the plate count gives just as accurate results as the direct mi- 

 croscopic examination. The justification for the use of the plate method is that it permits 

 determination of at least the relative abundance of those organisms which are capable of 

 developing upon the particular medium. 



2. The dilution method consists in diluting the soil with sterile water, then adding i-cc. 

 portions of the final dilutions to a medium (liquid or solid) which contains a particular 

 pabulum favorable for the development of the specific organisms. Thus the number of urea- 



