1528 Journal of Applied Microscopy 



Staining Bacteria in the Root-tubercles of Leguminous Plants. 



In a paper presently to be published in the Proceedings of the California 

 Academy of Sciences, Third Series, Botany, I have discussed some of the rela- 

 tions of the bacteria which cause the formation of the root-tubercles of legumi- 

 nous plants to the cells in which they occur. One or two matters of technique 

 developed in the course of the study, and reported in the paper above referred 

 to, may be of some interest to the readers of the Journal. 



When I once casually made some hand-sections of the root-tubercles of Bur 

 Clover {Medicago denticulata IV/lld.), the plant which I have especially studied, 

 these objects seemed to me unusually favorable for treatment with paraffin and 

 the microtome. I tried fixing them in a concentrated 35 per cent, alcoholic 

 solution of corrosive sublimate, and found that they were easily penetrated by 

 paraffin from xylol solution, embedded, and sectioned. They stain readily by 

 the usual anilin stains. I was particularly interested in demonstrating the man- 

 ner of infection of the cells of the root, and of the tubercle subsequently formed, 

 and in order to produce the best conditions for cytological study, I fixed a fresh 

 lot of young and growing tubercles in dilute FJemming's chrom-osmic-acetic 

 mixture. This visibly browns the tubercles of any considerable size, blackens the 

 oldest, and darkens all. Of this change in color I took no active notice until 

 just before staining the sections on the slide. Then I bleached by immersing 

 the slide for half an hour in a solution of one part Marchand's hydrogen per- 

 oxide in twenty parts 80 per cent, alcohol. 



Since the tubercles are composed almost exclusively of soft tissues, paraffin 

 melting at 54°C. is hard enough for embedding and sectioning, provided of 

 course that the room temperature is suitable. I cut very thin sections. 1 f.i in 

 some cases, with perfect success. 



The staining method was fundamentally that described by Hof^. For mak- 

 ing up the stains I used the proportions given in Humphrey's translation of 

 Zimmermann's Botanical Microtechnique, anilin safranin, anilin gentian-violet, 

 orange G. The sections were attached to the slide by albumen fixative. Hof's 

 directions for staining, followed without modification, give excellent preparations, 

 showing the degeneration of the nucleus and cytoplasm as the bacteria multiply 

 in the cells. This method, however, does not show the infection threads by 

 means of which the root-hairs, root, and new tubercle cells are infected, for it 

 does not clearly differentiate the bacteria from the cytoplasm. This can be 

 readily done by treating the slide, washed with water as it comes from the anilin- 

 gentian-violet solution, with Gramm's iodine solution, for a half hour or longer, 

 before staining with orange (i. 



This method consists simply in applying to bacteria in tissues the well known 

 bacteriological method used in differentiating cover-glass preparations stained by 

 Ehrlich's anilin-gentian-violet. By this means the infection threads running 

 from older infected cells toward and into the daughter cells of the tubercle 



^ Hof, A. C. Histologische Studien an Vegetationspunkten. Botan. Centralb., Bd. 76, No. 

 3. 1898. 



