104 LABORATORY MANUAL OF MICROBIOLOGY 



Before drawing the sample for counts mix the contents of the 

 tumblers thoroughly. This may be done with a sterile spatula. 

 In the case of treated soils plate from the dilutions 1 '- 100,000 and 

 1:1,000,000 for bacteria, and 1:10,000 and 1:100,000 for fungi. 



Exercise 14 



Dilution Method for Determining the Number of Specific 

 Physiological Groups of Bacteria 



This method is based upon the growth of different groups of 

 organisms upon specific substrates. 



Prepare a series of dilutions of soil in sterile tap water, as 1 : 10, 

 1:100, 1:10,000, etc., and add two 1-cubic centimeter portions 

 of the final dilutions to flasks or tubes containing the desired 

 liquid medium or to plates containing the desired agar, gelatin 

 or silica-gel medium. 



For example: For the determination of the number of Azoto- 

 bacter in soil, a medium free from nitrogen, containing mannitol 

 as a source of energy and of a pH 7.0 to 9.0 is used. For the 

 determination of the number of urea bacteria, a medium contain- 

 ing 2 to 5 per cent of urea as the only source of energy and nitro- 

 gen is used. To determine the number of nitrite forming 

 bacteria, a medium containing an ammonium salt and CaCOs 

 or MgCOs is employed. 



Exercise 15 



Direct Microscopic Examination of Soil^ 



Make a suspension of soil in nine times its weight of a 0.015 

 per cent solution of gelatin. 



Smear a drop of this in a thin film on a slide and dry. (Both 

 the amount used and the size of the smear must be measured if 

 the number of bacteria is to be counted.) 



Immerse for 1 to 3 minutes in a 40 per cent solution of acetic 

 acid or a O.liV solution of hydrochloric acid. Wash off the excess 

 acid quickly, and dry on a flat surface over a boiling water bath. 



While still on the water bath cover the fllm with a 1 per cent 

 aqueous solution of Rose Bengal, or Erythrosin, and allow it to 

 stain for about 1 minute. 



1 Conn, H. J., " Stain Technology," 1: 125, 1926; Soil Sci., 26: 257, 1928. 



