n.UTKRIA OF THK SOIL r,7 



to the solutions. Methylene blue serves well because it is non- 

 poisonous to bacteria and is easily reduced to the colorless leuco 

 compound. 



1. Add 20 cc. of standard methyleiie blue solution (1 : 1000) 

 to each 100 cc. of Giltay and Abersoifs solution. Fill several 

 test tubes and fermentation tubes. Sterilize. 



2. Inoculate the tubes with B. Jenitrificam, B. Hartklu, B. 

 pyoci/aMuZi and B. fluorescens liquefaciens. In order to exclude 

 oxygen pour in paraffin oil to a depth of about 2 cm. in the 

 test tubes. Keep the tubes in the incubator for three to seven 

 days, or until the solutions become colorless. So long as nitrates 

 are present the bacteria do not attack the methylene blue to any 

 great extent. 



3. When a tube has lost its color, test for nitrates ; shake the 

 solution or pass air through it. What happens ? Explain. 



Exercise 97. Nonsymbiotic Bacteria which Fix Atmospheric Nitrogen. 

 Isolation and Study of Azotobacter t 



1. Prepare Ashby's solution (see Exercise 49). 



2. Clean three small flasks or salt-mouthed bottles. Put into 

 each 25cc.of Ashby's solution and sterilize in the Arnold sterilizer. 



3. Inoculate each with 1 g. of soil. It will be well to use soil 

 samples collected from different places, for Azotobacter is not 

 necessarily most abundant in the most productive soils. Incubate 

 the flasks at 30 0. for three days ; at the end of that time pick 

 out flasks which have a greasy film on the surface of the liquid. 



4. Transfer small flecks of this surface film to a slide. Ex- 

 amine with the microscope. Add a drop of Gram's iodine solution 

 to the preparation. Azotobacter cells are stained golden yellow. 



5. Make a cover-glass preparation and stain with aqueous 

 methylene blue. 



Exercise 98. Growth of Azotobacter in Pure Cultures 

 HOFFMAN and HAMMER. Centralbl. f. Bakt., 2te Abt., 24: 181. 1909. 



1. Prepare nutrient agar by adding 2 per cent of agar to 

 Ashby's solution. Sterilize in the Arnold sterilizer. 



