OrR—LEAF GLANDS oF D1osCOREA MACROURA. 67 
‘couversely by its absence from the latter, of some factor capable 
of bringing about this increase in the amount of combined nitro- 
gen in the tissues of the acumen. Bearing in mind the proved 
proteid character of the glands, an explanation for the increase 
was sought for in the activities of the bacterium associated with 
them. 
A necessary preliminary to a study of the organism and its 
vital processes was its isolation from the plant, and this was 
accomplished by adopting, with some modifications, the methods 
employed by Harrison and Barlow,* and others, to isolate the 
bacterium from root-nodules. The acumen of a living leaf was 
first sterilised by dipping it into a fluid containing small pro- 
portions of hydrochloric acid and mercuric chloride. After 
washing with distilled water, it was cut across with a sterilised 
scalpel, and by means of a platinum loop, a small quantity of 
the gland content, with the included bacteria, was then trans- 
ferred from the cut surface to a test tube containing a liquid 
culture medium. From this, other tubes were inoculated, and 
pure cultures of the bacterium were obtained. The usual pre- 
cautionary measures, to guard against contamination, were taken 
at each step in the process, and controls were set up for all 
subsequent cultures, and these remained perfectly sterile. 
Various culture media, both liquid and solid, were used for 
the cultivation of the bacterium, but it was found that liquid 
media gave the best results in the first transference from living 
leaf to artificial conditions. ‘The following culture fluid, which 
has been used in the investigation of root-nodule bacteria, proved 
very successful in the initial stages of isolation. 
saccharose, I.0 grm. 
acid potassium phosphate, 0.5 grm. 
magnesium sulphate, 0.02 grm. 
distilled water, roo ce. 
Substitution of other sugars for saccharose did not produce the 
Same amount of growth, and glucose was particularly unsatis- 
factory, but excellent results were obtained by using a sterilised 
decoction of the leaf tissue. Only neutral, or slightly alkaline 
solutions, were employed throughout the experiments. 
After incubation for a period of 36 hours, at a temperature of 
25° C, the inoculated fluids become distinctly turbid, and at the 
end of 3 days, the cloudiness took the form of a dirty-white 
growth of a somewhat ropy nature, which collected at the bottom 
of the tube. Microscopic examination of an air-dried drop of 
this milky liquid, after staining with carbol-fuchsin, or gentian- 
* Harrison and Barlow in Proc, Roy. Soc. Canada, 1go6. 
