APPENDIX 355 



Method. 1. Add -the above chemicals to 1000 c.c. 

 distilled water. 



2. Titrate using phenolphthalein and neutralize using 

 normal NaOH. 



Do not filter. The presence of CaCOs offers an additional 

 means of isolating Azotobacter, as these organisms are found 

 in soil in much greater numbers around the particles of 

 calcium carbonate. 



3. Sterilize at 120 C. (autoclav), for ten minutes. 

 Mannit agar is prepared by adding 1.5% washed agar 



to the above solution, boiling until the agar is wholly dis- 

 solved and sterilizing as above. Do not filter. 



Nitrogen-free ash agar for cultivation of Ps. radicicola. 



1. Stir 5 gins, of wood ashes (beech, elm, maple) into 

 1000 c.c. distilled water for two to three minutes only. 

 Filter. 



2. Add 1% washed agar. 



3. Heat in steam for thirty minutes. 



4. Then add 1% commercial saccharose. 



5. Boil five minutes over a free flame. 



6. Strain while hot through several thicknesses of clean 

 cheese-cloth. This may be filtered if desired. 



7. For Exercise 9, Soil Microbiology, tube, placing 

 about 6 cm. of agar in the large test tubes with foot, the 

 rest in ordinary test tubes. Sterilize. (Tyndall method.) 



Nitrogen-free solution may be prepared as above, 

 omitting the agar. 



Congo-red agar for differentiating Ps. radicicola from Bact. 

 tumefaciens: 



Distilled water 1000.00 c.c. 



Saccharose 10.0 gms. ' 



K 2 HP0 4 1.0 gm. 



MgSO 4 0.2gm. 



Washed agar 15.0 gms. 



Congo-red 0.1 gm. 



