NUMBERS OF MICROORGANISMS 7 



great majority of other soil microbiologists. Artificial culture media are 

 employed, or at least artificial conditions are created. In many in- 

 stances, therefore, no direct evidence is furnished as to what is actually 

 taking place in the soil, under natural conditions. The results obtained 

 under laboratory conditions often have to be interpreted as to their 

 bearing upon actual field results. 



The information obtained from the study of soil microbiology by the 

 use of the different methods can throw light upon three groups of 

 phenomena: (1) the numbers and kinds of microorganisms occurring 

 in the soil; (2) the activities of soil microorganisms; (3) the bearing of 

 these activities upon soil fertility. 



Direct microscopic method. 



The method consists in preparing; a suspension of soil in a dilute fixative solu- 

 tion, then spreading one or two drops of the suspension upon a clean slide, drying 

 and staining with an acid dye. For qualitative purposes, about 0.5 to 1 gram of 

 soil is placed 4 in a test tube; 6 to 8 cc. of a fixing solution, consisting of 0.04 per 

 cent sterile gelatin in water, are then added and the mixture well shaken. Two 

 loopfuls of the suspension are placed upon clean slides; after drying, the slides 

 are stained with a 1 per cent solution of rose bengal in 5 per cent phenol-water 

 mixture. The preparation is heated on a steam bath until most of the liquid has 

 evaporated and the excess of stain is removed by dipping the slide in water. 

 The preparation is then dried on the steam bath and examined microscopically. 

 The gelatin fixative can be omitted 5 and the films fixed to the slide by flooding, 

 after drying, with a very dilute solution of collodion in ether and alcohol. 



The method was modified and improved by Winogradsky, 6 who found that the 

 presence of large yellow grains of inorganic soil material hinders the proper 

 examination of the field under the microscope. The soil samples are well mixed 

 and powdered. One gram of the soil (on a dry basis) is then added to 4 cc. of 

 distilled water and shaken vigorously for five minutes. After allowing to rest 

 30 seconds the suspension covering the large sedimented inorganic particles is 

 poured off into a small tube of a hand centrifuge. Two 3-cc. portions of distilled 

 water are then added to the residue, shaking each time one minute, allowing to 

 rest 30 seconds and then pouring into the same tube of the centrifuge. Ten 

 units of water are thus used for one unit of soil. After these three washings the 



4 Conn, H. J. The microscopic study of bacteria and fungi in soil. N. Y. 

 Agr. Exp. Sta. Tech. Bui. 64. 1918; An improved stain for bacteria in soil. 

 Stain Technol. 1: 126-128. 1926. 



5 Whittles, C. L. The determination of the number of bacteria in soil. Jour. 

 Agr. Sci. 13: 18-48. 1923; 14: 346 369. 1924. 



8 Winogradsky, S. Sur l'etude microscopique du sol. Compt. Rend. Acad. 

 Sci. 179: 367-371. 1924; Etudes sur la microbiologic du sol. 1. Sur la methode. 

 Ann. Inst. Past. 39: 299-354. 1925. 



