Sept. 4. 1916 Influence of Barnyard Manure on Soil Bacteria 895 



METHOD OF SAMPLING THE SOIL 



All possible precautions against the contamination of one sample by 

 another were taken in collecting them. The surface soil to a depth 

 of half an inch was scraped off by means of a sterile spade. A hole 12 

 inches deep was dug, and a slice of soil to this depth was taken from the 

 side of the hole and placed in a sterile mixing pan. This process was 

 repeated from four or five places in the field and then the contents of the 

 pan carefully mixed by means of a sterile spatula. From this composite 

 sample a representative portion, about 5 pounds of soil, was placed in a 

 sterile ore sack and conveyed to the laboratory for analysis. 



Before each sampling, the spade, mixing pan, and spatula were all 



carefully sterilized by heat from a plumber's torch, thus preventing the 



transfer of organisms from one soil to another. The samples were 



immediately transferred to the laboratory, partly air-dried in the dark, 



and then ground in a sterile mortar, all coarse rock being removed. The 



analysis was begun in all cases within 24 hours of the time of taking 



samples. 



METHODS OF SOIL ANALYSIS 



The number of organisms were determined by growing on modified 

 synthetic agar having the following composition: 



1,000 c. c. of distilled water. 



10 gm. of dextrose. 



0.5 gm. of dipotassium phosphate (K 2 HP0 4 ). 



0.2 gm. of magnesium sulphate (MgS0 4 ). 



2 gm. of powdered agar per 100 c. c. of media. 



After the samples of soil had been carefully mixed by shaking 100 gm. 

 were weighed on a sterile watch glass, using a small sterile spatula. This 

 soil was transferred to 200 c. c. of sterile water and shaken for one minute, 

 1 c. c. of this suspension transferred to 99 c. c. of sterile water, and the 

 dilution continued with 9 c. c. of sterile water. The plates were made 

 so as to give a dilution of 1 to 20,000 and 1 to 200,000. They were 

 incubated at 2 8° C. for four days and then counted. No attempt was 

 made to differentiate between bacteria and molds, but all were listed 

 together as total numbers of colonies. 



The ammonifying power of the soil was determined by weighing 100- 

 gm. portions of the soil and 2 gm. of dried blood into sterile tumblers and 

 covering them with Petri dishes. The dried blood was thoroughly 

 mixed with the soil by means of a sterile spatula and the water content 

 made up to 18 per cent with sterile water. The samples were incubated 

 at 28 to 30 C. for four days and the ammonia determined by transferring 

 to Kjeldahl flasks with 250 c. c. of distilled water, adding 2 gm. of mag- 

 nesium oxid and distilling into N/10 sulphuric acid. The determinations 

 were all made in duplicates and compared with sterile blanks. 



The nitrifying power of the soils was determined in tumblers, like 

 the ammonifying power, except that they were incubated for 21 days. 

 The moisture content was made up weekly to the initial 18 per cent. 



