The Relation to Nitrogen 247 



tion of a drop or two of the culture solution will give in the 

 presence of nitrates (no nitrites being present to give the same 

 reaction) a blue-violet color. At the close of the experiment 

 test thoroughly the control in order to determine if the ammo- 

 nium salt has remained unchanged. 



Denitrification. To about 100 cc. of prepared nutrient 

 bouillon (consult any bacteriology) add 3 grams of sodium ni- 

 trate, and pour about 10 cc. of the solution into each of several 

 test-tubes. Inoculate the tubes from a pure culture of some 

 denitrifying organism, such as Bacillus denitrificans, or each 

 tube with about .5 gram of fresh cow manure. Place the tubes 

 at a temperature of from 30 to 35 C. Note all the changes in 

 appearance of the culture and test occasionally for nitrates as in 

 the preceding experiment. Discuss the results. 



Root tubercles. Study the root tubercles of several legumi- 

 nous plants, such as vetch, red clover, and alfalfa, with special 

 reference to the form and distribution of the nodules. 



Examine prepared slides to determine the distribution of the 

 organism within the tissues. If prepared slides are not at hand, 

 make sections, stain in gentian violet, counterstain with orange 

 G., locate the band-like colonies of the organism, and note the 

 general conditions of the tissue modifications. 



Crush a bit of tissue from the inner portion of the nodule on a 

 clean cover glass, dry, and stain several hours in dilute gentian 

 violet ; then rinse the cover glass, dry, and mount in balsam. 

 This affords a satisfactory preparation for an examination of the 

 organism (Fig. 62). 



If facilities are at hand, determine the necessity of the bacteria 

 for tubercle production and contrast the growth of certain leg- 

 umes in inoculated and uninoculated soil. The experiment can 

 only be relative unless much care is taken with sterilization 

 precautions. The materials needed are six pots of fairly poor 

 soil, a pure culture of the root tubercle bacteria, seed of the legume 

 host plant, and a .2 per cent solution of formaldehyde. Steam 

 the pots of soil 2 hours, disinfect the seed by soaking 1 hour in 

 the formaldehyde solution, then sow a few seed in each pot. 

 Inoculate three of the pots with the pure culture and leave three 



