BACTERIA OF THE SOIL 67 



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

 poisonous to bacteria and is easily reduced to the colorless leuco 

 compound. 



1. Add 20 ec. of standard methylene blue solution (1 : 1000) 

 to each 100 ce. of Giltay and Aberson's solution. Fill several 

 test tubes and fermentation tubes. Sterilize. 



2. Inoculate the tubes with B. denitrificans, B. Hartlehii, B. 

 pyocyaneus, and B. fluorescens Uquefaeiens. In order to exclude 

 oxvgen 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 methj-lene 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 



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



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

 each 25 cc. 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° C. 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 smaU flecks of this surface fihn to a sHde. 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 Hammek. 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. 



