296 CALIFORNIA ACADEMY OF SCIENCES. [Proc. 3D Ser. 



order, if possible, to ascertain the real relations of the 

 tubercle organism to the cells in which it is found. This I 

 believe I have done. The bacteria are parasites, not bene- 

 fiting but injuring, if not finally killing, the cells in which 

 they occur. Whether the association of these bacteria with 

 a leguminous plant benefits the plant as a whole is another 

 question, answerable not from microscopic examination but 

 solely by experimental cultures. According to Frank (1890, 

 p. 109), the LeguminoscB are not all similarly affected by the 

 bacteria. Some are greatly benefited, stimulated to in- 

 creased growth and other activities, while in others the 

 bacteria are ordinary parasites, not benefiting the host in 

 any way in return for the food derived from it. Without 

 implying whether the results of infection benefit or injure 

 the plant as a whole, one may speak of the roots or cells of 

 leguminous plants being infected by tubercle bacteria. I 

 shall later, however, take occasion to discuss whether the 

 presence of the tubercle organism is really beneficial. 



The material studied was either fresh, growing out of 

 doors wild or sown in boxes in the laboratory, or alcoholic. 

 The latter was fixed in Flemming's chrom-osmic-acetic 

 mixture, dilute, and after washing for twelve to twenty-four 

 hours in running water was dehydrated and ke^t in 90 per 

 cent, alcohol. These tubercles, which were of different sizes, 

 ages and conditions, according to the season, were imbed- 

 ded in paraffin melting at 54° C, sectioned and mounted in 

 the usual way. The youngest tubercles scarcely turn brown 

 in the fixing fluid, but older ones become brown or almost 

 black. In any case I transferred the slides, after the paraf- 

 fin had been removed by turpentine, to a solution contain- 

 ing one part commercial peroxide of hydrogen in twenty 

 parts 80 per cent, alcohol. In this solution they remained a 

 half hour, or until the sections were no longer in the least 

 brown, and were then run down into water for staining. 



The method of staining which I found most useful is a 

 combination of Flemming's well known and now very popu- 

 lar triple stain — anilin safranin, anilin gentian violet, and 

 orange G. — with Ehrlick's method of staining cover-glass 



