the Tracheae in the Ferns . 
519 
I was able to satisfy myself that these holes are real and normal and 
are not due to any accident or preservation, because they can be observed 
in sections taken from fresh and living material. They are, of course, 
exceedingly narrow, but at the same time they are distinctly visible under 
a Zeiss D objective in stained sections of sufficient thinness. 
The actual formation of these holes can be followed in sections cut at 
different levels just below the meristem of the apex, in a region where the 
xylem is still more or less imperfectly lignified. The sections should be treated 
with some dye that has an affinity for the pectic substances of which the 
young cell-walls are chiefly composed. The best results were obtained 
with ruthenium red ; a dye especially recommended for this purpose by 
Mangin 1 and used with success by Allen in his work on the middle lamella. 2 3 
It is made up in watery solution ; about *02 gr. of the dye to 100 c.c. 
of water. The solution should then be rendered alkaline by the addition 
of about *5 c.c. of concentrated ammonia. It should be kept in a blackened 
bottle, for it is gradually decomposed by the action of light. In any case, 
its action becomes much less intense if kept for over a month. Sections 
stained by ruthenium red can be brought up into Canada balsam. Allen 
states that the stain gradually fades away, but after several months my 
preparations hardly show any deterioration. A very pretty effect may be 
obtained by counterstaining with methylene blue, which has an affinity for 
lignin. Partly lignified walls show gradations in the intensity of the blue 
stain. Pectic substances also stain well with methanil violet and Hoffman’s 
violet. 
When a transverse section of a partially differentiated xylem strand is 
treated with ruthenium red, the primary walls of the youngest tracheae are 
stained a deep red. The dye is taken up by the whole thickness of the 
wall, but at the same time an extremely thin middle lamella is just per- 
ceptible as a very fine, line more deeply stained than the rest. The primary 
wall remains comparatively thin except at the angles, which later on become 
especially thickened (Fig. 5). These angular thickenings stain somewhat 
differently to the rest of the wall, although they still give a pectic reaction. 
In a slightly older wall the thick secondary layers will have been deposited 
on the thin primary wall, over all its surface except those regions destined 
to become pits. When first deposited these secondary layers also consist 
of pectic substances and take up the stain almost as readily as do the 
primary walls (Figs. 6 and 7). At this stage a fair amount of protoplasm 
is still present in the young trachea, but the nucleus seems to disappear 
during the deposition of the secondary layers or even earlier. Longitudinal 
1 Mangin, ‘Sur l’emploi du rouge de ruthenium en anatomie vegetale,’ Comptes-Rendus de 
l’Acad. des Sc. Nat. de France, t. 1 1 6, p. 653, 1893. 
3 Allen, ‘On the Origin and Structure of the Middle Lamella,’ Bot. Gaz., vol. xxxii, No. 1, 
p. 1, 1901. 
