3i6 Journal of Agricultural Research voi. xvi, No. la 



One of this group, Pseudomonas fiuorescens , is known to be an ammonifier. 

 This, together wdth the fact that the non-spore formers have been found 

 to be especially abundant in freshly manured soil suggests that they 

 may be among the important soil ammonifiers. The present work 

 was planned to test whether this assumption is correct, and, if so, to 

 obtain as rigid proof as possible of the ammonifying agency of the non- 

 spore-forming organisms. 



TECHNIC 



The soil used throughout the series of experiments was Dunkirk 

 silty clay loam ^ obtained from a plot on the station grounds. This soil 

 was mixed with fresh horse manure or fresh cow manure, always in 

 the proportion of 20 parts of soil to i part of manure. 



All samples were plated according to the usual methods, with at 

 least two dilutions. The degree of dilution depended upon the char- 

 acter of the samples to be plated. Four plates were made from each 

 dilution used and the average count of the four plates was taken to 

 represent the count for that dilution. Whenever possible, the count 

 was based upon the dilution averaging between 30 and 150 colonies per 

 plate. In some cases, "^however, it was necessary to take into account 

 plates which varied from these limits. In a few cases where plates were 

 lost on account of contamination or liquefaction, the count represents 

 an average of three instead of four plates. 



The medium used in all the plating was "tap-water gelatin" made by 

 dissolving 200 gm. of "gold-label" gelatin in i liter of tap water, adjust- 

 ing the reaction to about Ph = 6.8, with bromthymol blue as the indi- 

 cator, and clarifying with white of &gg. 



Nearly all of the plate counts were checked by direct microscopic 

 examination of the soil according to the method described by Conn 

 {12). An infusion of the soil to be examined was made by shaking up 

 I gm. of the soil in 9.5 cc. of a fixative prepared by dissolving 0.15 gm. 

 of gelatin in 1,000 cc. of hot water. Of this infusion o.oi cc. was meas- 

 ured out with a capillary pipette and smeared evenly over an area of i 

 square centimeter on a glass slide. This smear was then dried and 

 stained with hot rose Bengal for i minute. 



For all pure culture studies the manured soil was placed in small 

 Erlenmeyer flasks, 150 gm. per flask. These were then plugged with 

 cotton and sterilized in the autoclave at 15 pounds' pressure for two 

 hours. Subsequent platings proved that in this way all organisms and 

 spores were killed. The infusion for inoculating the soil was prepared 

 as follows: A freshly streaked culture of the organism was suspended in 

 sterile water, and the number of organisms per cubic centimeter of this 



' Described according to the system of the Bureau of Soils of the U. S. Dept. of Agriculture. (Marbut, 

 Curtis F., Bennett, Hugh H., Lapham, J. E., and Lapham, M. H. Soils of the United States. U. S. 

 Dept. Agr. Bur. Soils Bui. 96, 791 p., 1913. Carr, M. Earl, Lee, Ora, jr., Maynadier, Gustavus B., HaI/- 

 I.OCK, D. J., and Frost, V. J. Soil Survey of Ontario County, New York. U. S. Dept. Agr. Bur. 

 Soils, Adv. Sheets, Field Oper. 1910, 55 p., i fig., i map. 1912.) 



