PROTOPLASM THROUGH THE WALLS OF VEGETABLE CELLS. 
829 
If, however, the section be first treated with the acid, then washed, and stained with 
methylene blue, only the closing membrane and the sides of the pits will be stained (see 
Plate 68, fig. 8), unless the action of the sulphuric acid be forced. Both the protoplasm 
and the rest of the cell-wall undergo scarcely any coloration. * * * § Thus methylene blue, apart 
from its great value as a stain for cell-wall, becomes by this modification a reagent for 
pit membrane. Naturally Hofmann’s blue stains neither the cell-wall nor the substance 
of the pit membrane. The whole results of my investigation appear to point to the 
conclusion that the staining of this reagent is specially confined to the protoplasm. 
In the case of many palm endosperms, where after the action of Chlor. Zinc Iod. and 
picric-HoFMANN’s-blue threads can be observed going through the closing membrane 
of the pits, it is the threads which are specially stained, and are in consequence 
defined from the substance of the pit membrane. Indeed, so characteristic is the 
staining of Hofmann’s blue, that experiment seems to point to the conclusion that in 
those cases where a staining of the pit membrane occurs, such staining points to the 
presence of protoplasm. 
There is, however, another special structure which is also stained by Hofmann’s 
blue, and which can be distinguished from protoplasm by its solubility in strong sul¬ 
phuric acid, viz.: the callus of sieve-tubes. It was Russowt who first used aniline blue 
as a reagent for callus, and even combined it with Chlor. Zinc Iod. As I did not know 
what particular blue Bussow used, I made a number of experiments with the various 
blues I had at my disposal,J with the result that the special staining of the callus 
was confined to two of them, viz. : Hofmann’s blue and water or soluble blue, one of 
which it is pretty certain that Bussow employed. Water blue is only second to 
Hofmann’s blue in that it also especially stains protoplasmic structures. Now the 
properties of callus are somewhat peculiar.§ Wilhelm showed that it was soluble in 
sulphuric acid, and insoluble in ammoniacal oxide of copper. In the former respect it 
resembles cellulose, and indeed its mode of formation—arising as symmetrical warts on 
either side of the cell wall—as described by Janczewski,|| and confirmed by Stras- 
BURGERjt certainly give some colour to this idea.** On the other hand, unlike cellulose it 
* After keeping the section for a long time in dilute glycerine, staining of the protoplasm does take 
place since the glycerine dissolves the dye. This solution ultimately stains the protoplasm. 
f Russow, ‘ Sitzber. Dorpater Nat. Ges.,’ 1881, April 23, and ‘ Bot. Ztg.,’ 39, 1881, p. 723. 
X Hofman’s blue stains protoplasm and callus. Soluble or water blue, ditto. Benzyl blue, proto¬ 
plasm cell-wall and callus, like rest of cell wall. Insoluble aniline blue, i.e., solution in spirit, as benzyl 
blue. Neither of the latter appear to stain the pits. Methylene blue, stains cell-wall and pit membrane 
Phenylene blue resembles methylene blue. Both water blue, methylene blue, and Hofmann’s violet 
may be obtained at Martindale’s, New Cavendish Street, Portland Place, London. The rest of the dyes 
that I used were obtained from the Actien Gesellshaft fur Anilin Fabrication, Berlin. 
§ ‘ Beitrage zur Kentniss des Siebrohrenapparatus Dicotyler Pflanzen.’ Leipzic, 1880. 
|| ‘ Etudes comparees sur les tubes cribreux.’ Cherbourg, 1881. 
IT ‘ Ban und Wachsthum,’ p. 56, et seq. 
** This was first noted by Wilhelm. Loc. cit., p. 16. 
MDCCCLXXXIII. 5 O 
