METHODS OF RESEARCH: STAINING METHODS 117 



The proper degree of stain for sections should be determined 

 by inspection under the microscope during the process of stain- 

 ing. The slides must be shaken or jarred as little as possible 

 during these transfers, and, after staining, the shifting through 

 the graded alcohols must be swift, so that the stain shall not be 

 all removed. In my sections I prefer a good strong stain. 



Many of the slides put up in this laboratory and formerly 

 very good are now nearly or quite worthless because they have 

 faded. This has been due partly to use of aniline dyes which are 

 fugitive even to diffused light, but more often I think to overwash- 

 ing or to the presence of unsuspected traces of acid in the Canada 

 balsam. If you would avoid much future disappointment use 

 the utmost care in the selection of balsam for mounting pur- 

 poses and do not overwash. Valuable sections which have 

 faded may be restored by dissolving off the cover-glass in xylol 

 and restraining. No invariable or general rule can be laid down 

 for staining bacteria in situ since plants and parasites both 

 vary in their reaction toward stains. Some bacteria also diffuse 

 out of the sections vexatiously. Under the various heads in 

 Part III, special stains are mentioned when such have been found 

 particularly useful. 



Gram's stain varied by the substitution of amylic alcohol 

 for ethyl alcohol (generally referred to later as amyl Gram) 

 has been found to give a clean sharp picture of the bacteria in 

 a number of tissues, e.g., olive tubercle, angular leaf-spot of 

 cotton, pear blight. 



Carbol fuchsin may also be mentioned here as a widely 

 applicable permanent bacterial stain. Its chief objection is a 

 tendency to over-stain the tissues of certain plants, e.g., those 

 of the mulberry. 



Two other very good stains are iron-haematoxylin and 

 Flemming's triple stain. Nigrosin (soluble in water) also some- 

 times gives good results. 



Long since (1894) the writer resorted to methyl violet, 

 preceded by a bath in tannin water to reduce the excessive stain 

 of the host tissues in stems of cucumber attacked by Bacillus 

 tracheiphilus, and the bacterial masses in the vessels still hold 

 their massive blue stain on a pale background (1915) but the 



