8 BOTANICAL GAZETTE [JULY 
MATERIAL AND METHODS. 
Woody tissues of the following species were studied: Preris 
aquilina L., Pinus silvestris L., Nerium Oleander L., Kosa sp., 
Tilia Americana 1.., and [lex opaca Ait. Of these, Mangin had 
investigated tissues of Pinus and Ilex. 
Free-hand sections were cut, usually both transverse and 
longitudinal. In many cases, before staining, they were treated 
for twenty-four hours,as suggested by Mangin (5), with a mixture 
of one part of concentrated hydrochloric acid and four or five 
of absolute alcohol, then washed in distilled water. The stains 
characteristic of pectic acid, especially ruthenium red, are taken 
up, as a rule, more freely after this treatment, and almost invari- 
ably the coloration is more clear-cut and distinctive. 
For staining, Griibler’s preparations were used. Unless other- 
wise stated, they were in neutral saturated solution. When an 
alkaline solution was used, it was prepared by adding to the 
neutral solution about % per cent. of concentrated ammonia 
solution. Acidulated solutions were prepared by adding a fe 
like proportion of acetic acid. In some cases where saturated ae 
solutions, ¢. g., of methylene blue and methyl violet B, were 
too strong for good results, they were diluted to such a strength 
that a few seconds’ exposure gave a differential stain. Aniline- | 
water-safranin was prepared as directed by Zimmermann (16). | 
An aqueous solution of ruthenium red of a strength of about 
one five-thousandth was used. An increase in strength above 
this did not seem to make any noticeable difference in the depth 
of the stain or in the time of exposure required. This substance 
is reduced by the action of light in the presence of water, hence 
the solution must be kept in the dark. It was tried in neutral, 
alkaline, and acidulated solutions, the alkaline and acidulated 
solutions being prepared as described for the other coloring 
matters, substituting hydrochloric for acetic acid. In all cases, 
the alkaline solution was found to give the deepest and most 
distinctive colorings, and it is this preparation of ruthenium red 
that is referred to, hereafter. But a few minutes’ exposure to 
the stain is required. 
