202 Barratt . — A Contribution to our Knowledge of the 
method, described below, has been employed to determine the relations 
existing between integral parts of the vascular system. 
The species which has been most carefully worked through is 
E. arvense , owing to the ease with which the spores of this species could be 
obtained and the young plants raised under cultivation. But sporelings of 
E. maximum and E. limosum have also been employed. 
The spores were sown on light soil immediately after gathering and the 
pots were kept in a frame at the ordinary temperature out of doors. 
The prothallia appeared in the course of two or three days after sowing 
and the first young sporophytes were visible two months later. The prothallia 
were carefully removed from the soil, washed and pickled in 75 per cent, 
spirit. An abundance of material was thus available. 
Plants of different sizes, varying from those still buried within the 
prothallial lappets to those showing three or four shoots, were detached 
from their prothallia and treated for twenty- four hours with a solution of 
eau de Javelle in the cold. At the end of that time the whole sporelings 
were transparent and extremely fragile. They were then washed in water 
and stained with ammoniacal fuchsin. 1 
The material was allowed to remain in the staining fluid for 24 hours 
or longer ; it was then washed with alcohol several times and during this 
process the red colour appeared in the lignified tissues. The specimens 
were then dehydrated, cleared with oil of cloves, and mounted in Canada 
balsam. For some purposes it was found convenient to mount the stained 
specimens in glycerine jelly or euparal, the lower refractive index render- 1 
ing the cellulose walls of the parenchymatous cells more visible. 
Thin slides were employed for mounting, so that when desired the 
specimens could be examined from either side. 
The result of this treatment is to render the whole sporeling transparent, 
and the lignified tissue, being vividly stained, stands out as a complete 
internal skeleton ; it was possible to follow the course not only of the 
vascular strands but of their individual components, and thus to correct or | 
confirm conclusions drawn from the examination of serial sections. Since 
the fuchsin also stains cuticularized membranes, the characteristic bands on 
the radial walls of the endodermal cells appear as a connected network 
(Plate VII, Fig. 1). 
The same method of clearing and staining was applied to the apices 
and mature shoots of adult plants with equal success. Before clearing the 
material each apex or shoot was split longitudinally into two halves and so 
mounted that the interior of the stem was uppermost. These thick speci- 
mens were mounted in Canada balsam in cells constructed for the purpose. 
1 This stain is prepared for use by adding 5 per cent, solution of basic fuchsin in alcohol to strong 
ammonia o*88o, so long as the liquid remains colourless ; refer Zimmerman, Botanical Microtech- 
nique, § 271. 
