204 FIXATION OR ASSIMILATION OF NITROGEN 



Ex. 97. Cut transversely across a nodule of a bean root : dig 

 out a small portion from the central part exposed, and crush it 

 in a few drops of distilled water. Then take a small drop of the 

 latter, and spread it over the surface of a clean cover-slip. 



When dry float it, bacteria side downwards, on a very weak 

 solution of gentian violet, and leave to stain all night. 



Excellent stained specimens of the nodule organisms are 

 obtained in this way. 



Ex. 98. Prepare the following solution : 



Mono-potassium phosphate (KH 2 PO 4 ) i.o gr. 

 Magnesium phosphate . . . .05 gr. 

 Distilled water . ... . 500 c.c. 

 To one 100 c.c. add .5 gr. sodium succinate. 

 another 100 c.c. i gr. glycerine. 

 100 c.c. i gr. peptone. 

 ,, 100 c.c. ,, 2 gr. mannite. 



Heat each to boiling, and filter: then pour 10 to 15 c.c. 

 of each into sterile tubes, and sterilize on three successive days. 



Inoculate a tube of each with organisms taken from the interior 

 of nodules of pea, runner bean, red clover, and broad bean, with 

 a sterilized knife and needle. 



Incubate for fourteen to twenty-one days, and then examine a 

 drop of the culture for " bacteroids " in a fresh state and stain 

 with dilute gentian violet as in the previous experiment. 



In which medium do the organisms grow least satisfactorily ? 

 Ex. 99. Grow the nodule organisms on Harrison and Barlow's 

 wood-ashes-maltose agar. 



Add 8 gr. of well-burnt wood ashes to 500 c.c. of distilled 

 water, and boil for one minute : filter through two sheets of 

 paper. 



To 400 c.c. of the filtrate add : 



4 gr. of agar, 

 and 4 gr. of maltose. 



Heat until dissolved: then filter, tube, and sterilize in the 

 usual way, leaving some to solidify for agar slants. 



