14 EFFECT OF BLACK ROT ON TURNIPS. ^ 



jdelded a prompt and very abundant growth of the same organism 

 with which the plant had been inoculated fifty-two days before, and 

 no other organism appeared in the tubes. The root remained in strong 

 alcohol for more than four years — i. e., until an opportunity was found 

 for making sections. Portions of it were then placed successively in 

 absolute alcohol, alcohol and chloroform, pure chloroform, cold chloro- 

 form containing paraffin, warm chloroform with more paraffin, and 

 tinallv pure melted paraffin. When thoroughly infiltrated with par- 

 affin they were suitably embedded and cut on the Reinhold-Giltay 

 microtome with a very sharp knife. The sections were floated out 

 and cemented to clean glass slides by means of sterile, distilled water 

 containing one-half per cent of gelatin, freshly prepared. Mild heat 

 was used in straio-htenino- out the wrinkles in the sections and the excess 

 of water was removed by setting the slides on end. When dry, the 

 slides were gently warmed until the paraffin was melted, and were 

 then placed in turpentine or xjdene until the paraffin had been removed. 

 They were then passed in succession, gently, into Coplin staining jars 

 containing a mixture of turpentine or xylene and absolute alcohol, pure 

 alcohol, graded mixtures of alcohol and water, and finall}" Ziehl's car- 

 bol fuchsin, in which they were allowed to remain from three to five 

 minutes. The excess of stain was removed by leaving the slides in a 

 mixture of equal parts of alcohol and water until the proper differen- 

 tiation in color had been secured. They were then passed rapidly 

 through graded alcohols into absolute alcohol, and from that into a mix- 

 ture of alcohol and xylene, into pure xylene, and finally into Canada 

 balsam or Dammar balsam dissolved in xvlene. In a verv few of the 

 sections here shown, viz, PI. I, fig. 2, and Pis. II and IV, nigrosin was 

 substituted for carbol fuchsin. A series of sections (cross and longi- 

 tudinal) from this root were prepared, stained, and studied, and the 

 results obtained are illustrated by means of the accompanying photo- 

 micrographs. 



The fixing of the root in strong alcohol was considered necessary in 

 order to prevent the bacteria from diffusing out into the fluid. Even 

 95 per cent alcohol does not entirely prevent diffusion in case the bac- 

 terial cavities are large, but does so quite satisfactorilj^ in case of single 

 vessels or small cavities. The knife used for making the sections was 

 •very sharp, and did not shove or tear them to any extent, but the 

 strong alcohol in which the material was fixed did, of course, cause 

 more or less twisting and shrinking of the delicate parenchymatous 

 tissues. During the process of hydrating, staining, bleaching, deh}^- 

 drating, and mounting the cell walls were also occasionallv broken and 

 displaced. Observations on the fresh root left no doubt that the vessels 

 were the primary seat of the disease, but the exact limits of the bacterial 

 occupation remained to be determined from properh' infiltrated material. 

 This has now been accomplished. The manner of fixing, of infiltrating, 



