54 
Walter Gardiher. 
sap, somo modifications mighl be required for vegelable lissues '). Gold Chlo¬ 
ride and Silver nitrate wcre also employed. The experiments pointed to 
the fact that treatment of small pieces of fresh tissue with a saturated watery 
solution of Picric acid gave the best results although these resulls wcre far 
from being perfectly satisfactory. Wlieu small pieces of the tissue were 
treated for some time (*/i hr. to 2 hrs.) with this reagent, and subsequently 
with dilute (50 p. c.) and progressively stronger aleohol, the close relation 
existing botween the protoplasm and cell-wall was fairly maintained, the 
latter having undergone but little shrinking. Still the amount of shrinking 
was sufficient to modify considerably the normal relations botween proto¬ 
plasm and cell-wall, and moreover the protoplasm was found to have become 
rigid and briltle. In consequence of these objections I regarded it as most 
satisfactory to give up the attempt to use preserved material; hence all the 
observalions deseribed bclow were ruade upon fresh material alone. -) 
1 tlieu proceeded to make a number of very careful observations upon 
the slaiuing propcrties of as many dyes as l could obtaiu. Most of thern 
were not remarkablc for any well-defined selective staining power, but in 
the end I succceded in findiug two very oxcellcnt reagenls for botanieal 
1 ) From my moro recent Experiments 1 find that I had been working with too 
streng Solutions of Osmic and Cliromio acid. In the casc of young tissue where the cells 
are full of protoplasm, exactly the same treatment may be resorted to, as in animal 
structures, Itul with l'ully grown and muoh vacuolated cells, rauch moro care is required 
all rapid difl'usion must be avoidod, and the strength of the solution with which such 
tissue is treated, must beonly slowly and progressively iucroased. The staining solution 
must also be appro.xitnalely of llie same specific gravity as that of the fluid in which the 
tissue is, at the time. 1 found that as regards Osmic and Chromic acid a 1% solution 
gave good results. The tissue is placed in such a solution for 12 or 24 honrs. It may 
tlieu be rcmoved to 30% aleohol for a day, and into 30%, 70%, 90% and absolute as 
required. II it is to be mounled in glyecrine il must in Ibe same way be gradually 
brnugbl into water: into very dilute glycerine (I Giycerine to 10 waler), and so on. In 
all cases small pieces of tissue must be used. The black staining produced by Osmic 
acid may be removed by treating the sections with Cddot'ine-water or by suspending 
them in aleohol tbrougb which Ghlorine is allowed to hubble. The aleohol must be kept 
cool by means of a current of cold water, tt is best not to aet on large pieces of tissue, 
both bccauso the bleacking Ilion takes a long time, and because it is not well in a doli— 
cate investigation to exposo a tissue to the prolonged action of cfdorino. The method is 
a sligld modification of that first proposed by Mayer (Müll. Arcb. 1 874. p. 321). As 
regards Picric acid, the saturated solution may have to be diluted with 3 times its bulk 
of water. For sea-weeds, Floridene &c the following method gives good results. Make 
ii saturated solution of Picric acid in sea-water, add to it 3 or 4 times its Volume of 
sea-waler, The material is treated for % liour to 1 hr and tlieu placed in 30% aleohol&c. 
Fiually for any ordinary investigation there is no doubl that absolute aleohol is the most 
useful reagent. 
2) Aleohol material may be used but it must be recotlected that it will always cause 
the protoplasm to slirink from Ibe cell-wall, and tlie relation botween the pit-mombranes 
and the protoplasm will no longer be maintained. 
