FIXATION OF NITROGEN BY BACTERIA 205 



Now isolate and prepare pure culture of the nodule organisms 

 from beans, peas, red clover, and other leguminous plants. 



Dig up the plant and wash the roots. Remove a clean nodule 

 with forceps, taking care not to squeeze it too much, and im- 

 merse it for one minute in mercuric chloride solution : 

 Mercuric chloride . ... . .25 gr. 



Hydrochloric acid, sp. g. r.2 , . .5 c.c. 



Water ...... 250 c.c.* 



Take it out with sterile forceps, and place it on blotting-paper 

 to absorb the solution. Cut it open with a sterile knife : dig 

 out with the flamed point a small portion of the bacteroidal tissue 

 from the centre of the nodule, and transfer it to a heated watch- 

 glass containing a drop or two of sterile water. 



Inoculate a tube of melted agar with a loopful of the liquid on 

 the watch-glass, pour into a Petri dish, and incubate at 20 C. 



In two or three days note the white paraffin-like shining 

 colonies ; examine and stain organisms from them. 



Make streaks on agar slants. 



Ex. 100. Prepare wood-ash-maltose solution (Harrison and 

 Barlow). 



Take 8 grams of good, well-burnt wood ashes, and put them in 

 a flask with 500 c.c. of distilled water : boil for a minute, and 

 then filter. 



To 400 c.c. of the filtrate, add 4 grams of maltose, and boil. 



Then pour about 10 c.c. into a number of sterile tubes, and 

 sterilize in the usual way. 



In these tubes grow the nodule organisms obtained from 

 nodules in the manner described in the previous experiment. 



