12 PRINCIPLES OF SOIL MICROBIOLOGY 



acid solution and both are well mixed. The mixture is then distributed into 

 Petri dishes and these are allowed to rest over night. A firm gel is obtained. 

 The uncovered dishes containing the gel are then placed in flowing water for at 

 least twenty-four hours, until no reaction is given with methyl red or brom cresol 

 purple and with AgN0 3 . A solution containing the minerals and specific sub- 

 stance, either in solution or as an insoluble suspension, may then be poured 

 over the surface of the gel and the dishes placed in an oven at 60° to 65°C, until 

 the excess moisture has dried off. The gel in the dish is inoculated with small 

 particles of soil, the dish is covered and placed in an incubator. After a few days, 

 the specific organism, if present in the soil, will develop on the gel surrounding 

 the particles of soil. By this method the presence of Azotobacter in soil can be 

 readily demonstrated, provided mannite and CaC0 3 are employed in addition to 

 the minerals. Nitrite-forming bacteria will develop in an ammonium salt 

 medium, nitrate-forming organisms in a nitrite medium, etc. 14 



However, the cultural methods, largely the enrichment media de- 

 veloped by Winogradsky and Beijerinck, and the common gelatin and 

 agar plate have been used most extensively for establishing the presence 

 and abundance of specific organisms in the soil. 



Cultural methods for the determination of numbers of microorganisms 

 in the soil. The earliest investigations in soil bacteriology were car- 

 ried out, 15 - 16 . 17 by the use of methods developed in medical bacteriology. 

 Soils were diluted with sterile soil, then plated out with gelatin and 

 numbers determined after a certain incubation period. Later, sterile 

 water was used for making the dilutions. In some cases small quantities 

 of soil were weighed directly for the preparation of the plates. The 

 method itself was imperfect and the results unrepresentative, and no 

 relation was established between numbers and soil productivity. 

 Hiltner and Stormer 18 suggested the use of the dilution method, with the 

 hope of doing away with the plate method, but here again the hetero- 

 trophic bacteria were determined by their growth on agar or gelatin 



14 The gel may also be prepared by methods described elsewhere (p. 196). 

 16 Koch, R. Zur Untersuchung von pathogenen Organismen: Bodenunter- 

 suchung. Mitt. K. Gesundheitsamt. 1: 34-36. 1881. 



16 Proskauer, B. Uber die hygienische und bautechnische Untersuchung des 

 Bodens auf dem Grundstucke der Charite und des sogen. "Alten Charitd- 

 Kirchhofes." Bakteriologisches Verhalten des Bodens. Ztschr.Hyg.il: 22-24. 

 1882. 



17 Frankel, C. Untersuchungen iiber das Vorkommen von Mikroorganismen 

 in verschiedenen Bodenschichten. Ztschr. Hyg. 2: 521-582. 1887. 



18 Hiltner, L., and Stormer, K. Studien fiber die Bakterienflora des Acker- 

 bodens,mit besonderer Beriicksichtigung ihres Verhaltens nach einer Behandlung 

 mit Schwefelkohlenstoff und nach Brache. Arb. Biol. Abt. Land. r. Forstw., K. 

 Gesundheitsamt. 3: 445-545. 1903. 



