PROTOPLASM THROUGH THE WALLS OE VEGETABLE CELLS. 
823 
dehydrating agents a further shrinking of the protoplasm may be induced. The 
second and more important objection is, that the narrowing of the pit, on account 
of the swelling, imprisons and firmly embraces the protoplasm in the pit cavity. In 
answer to this, it may be urged that the shrinking of the protoplasm takes place 
more quickly than the swelling of the wall, and that the protoplasm projecting into 
the pit would have time to withdraw before being imprisoned. In deep pits of small 
diameter, it is indeed possible that the narrowing of the cavity does play some definite 
part, but whether this be so or not, experiment proves that the protoplasm also 
adheres to pits which are shallow, and moreover possess sloping sides. In this case, 
any such explanation could hardly be brought forward. Lastly, it must be remem¬ 
bered that the action of the sulphuric acid is carefully regulated, and is not carried 
to an extreme limit, and that the results obtained with this reagent have been fully 
confirmed with Chlor. Zinc Iod. All the preceding remarks as to the action of 
sulphuric acid apply only to the cases in which fresh material is used, since here the 
protoplasm has not been rendered brittle by any preliminary treatment with reagents, 
and consequently has undergone as little alteration as possible, and will not break 
when any slight tension is set up. 
After treatment with sulphuric acid, and washing out with water, the section may 
be stained with iodine, as in the usual process; but I used in preference, and with 
greater success, analine dyes, especially the violet and blue. 
In my earlier experiments I used Hofmann’s violet (Trimethyl rosanilin ) as the 
staining reagent. In the first place, Hofmann’s violet is a dye which, of all others, 
is extremely rapid in its action, quickly and thoroughly permeating the tissues. 
Again, it works extremely well with sulphuric acid, being soluble in, and hardly 
affected by this reagent, as far as all its staining properties are concerned. Thus one 
need not take such care to wash out the acid before staining ; for, although when the 
proportion of acid is large the Hofmann’s violet is temporarily turned green, yet on 
subsequently washing with water before mounting in glycerine, the violet colour is 
restored. The whole process may, indeed, be done in one operation, for the solid dye 
may be dissolved in strong sulphuric acid; the mixture furnishes a dark brown-yellow 
solution. The section is now simply treated with the mixture, and then washed well 
with water. The above method gives extremely satisfactory results with sieve-tube 
preparations; and, moreover, any lignified tissue which happens to be present is 
coloured gold yellow, as in the ordinary aniline sulphate reaction. 
To the use of Hofmann’s violet there is, however, the great objection that the 
whole of the tissue—protoplasm, cell-wall, middle lamella, and pit-membrane—is 
stained. If, however, the stained section be treated for some long time (three to four 
days), with dilute glycerine the dye in the cell-wall, middle lamella, and pit-membrane 
dissolves out, whereas that staining the protoplasm remains but little acted upon. 
This lengthy manipulation is an obvious objection, but nevertheless Hofmann’s 
violet often gives extremely satisfactory preparations, and by mounting the section in 
5 n 2 
