PROTOPLASM THROUGH THE WALLS OF VEGETABLE CELLS. 
821 
Method of preparation .—Experiments have shown that in order to demonstrate in 
the most satisfactory manner the perforation of the cell wall by protoplasmic threads, 
it is usually necessary that the wall should he either swollen or dissolved. 
Both these methods have already been successfully made use of in the case of sieve 
tubes by Sachs, who employed, as his reagent, strong sulphuric acid; and by 
Hanstein, who used Chlor. Zinc Iod. In both cases iodine served as a stain for the 
protoplasm. In investigating the subject of protoplasmic continuity, I have made 
use of both these methods, but with important modifications. Sulphuric acid is 
naturally by far the more powerful: strongly swelling or dissolving the cell wall, and 
laying bare, as it were, the protoplasm to the action of staining reagents, while 
Chlor. Zinc Tod., on the other hand, when possible, is always preferable, on account of 
its less vigorous action, attended with less distortion of relative arrangement. 
The method used by Sachs for demonstrating the actual perforation of the sieve-plate 
is essentially based upon the difference of reaction of strong sulphuric acid towards the 
cell-wall and the protoplasm. The former is partially dissolved, or excessively swmllen, 
while the latter remains but little acted upon, and can be readily stained and 
examined. The usual plan has been to mount a thin section of tissue in dilute iodine 
solution, and when sufficiently stained, strong sulphuric acid was run in, and the 
observation was made. Or the section was first stained with iodine, and then mounted 
in sulphuric acid. But there are some objections to this method. First, the sulphuric 
acid is run in, once for all, and thus its action cannot be regulated. Secondly, the 
iodine from its very colour is not a sufficiently deep stain. Further, the cellulose blue 
produced ; the precipitation of the iodine ; and the rapid disintegration of the tissue 
due to the powerful action of the acid ; cause the method to he only satisfactory in 
such cases as sieve-tubes, where the continuity is pronounced and the material 
favourable, for here the cell-walls easily dissolve, and the middle lamella is but little 
developed. 
The modification I have adopted has been to divide the process into two parts, and 
to substitute aniline colours for the iodine. I propose to give a detailed account of 
the whole process. 
A thin section of fresh material is taken up on a platinum spatula; the water is 
removed with blotting-paper, and a drop of strong sulphuric acid is dropped upon it 
by means of a glass rod. When the acid has been allowed to act for a determinate 
time (some seconds), depending on the nature of the tissue and the extent to which 
the action is required to be carried, the section is rapidly washed by immersing the 
spatula in a quantity of water contained in a large watch-glass, at the same time 
stirring, so as to wash out the acid as quickly as possible, and stop its action. Thus 
the sulphuric acid can be kept entirely under control. After about two changes of 
water the section may be at once stained, or put into alcohol for future use. 
The length of time that the acid requires to act naturally varies with the nature of 
the material used. Thick-walled tissue requires longer treatment than thin-walled, 
MDCCCLXXXIH. 5 N 
