2 37 
of Pilularia globulifera , L, 
neutralized, and the preparation washed until every trace of it 
is removed. Before the potash is applied the spores must 
be thoroughly hardened in alcohol, or better a chromic acid 
mixture, either a i per cent, watery solution, or Flemming’s 
mixture of chromic, acetic, and osmic acids, a thorough wash- 
ing being requisite after use of either of the latter. After 
addition of the potash the slide is heated as before. If the 
prothallium is not completely set free, this can generally 
be accomplished by gently rubbing the cover-glass to and 
fro, but the potash should previously be as far as possible 
drawn off by means of blotting paper, and pure water run 
under the cover-glass. After a final washing, the preparation 
is neutralized with acetic or hydrochloric acid, and may then 
be stained with haematoxylin or some anilin color. Haema- 
toxylin is preferable, as the preparation can then be preserved 
in dilute glycerine, which extracts anilin color at once or 
after a short time. Care must be taken with haematoxylin 
not to overstain, as the color deepens very much after the 
spores have lain in the glycerine for a short time. 
According to Arcangeli 1 there are but two primary coats 
to the spore, the outer one showing, however, a division into 
three layers. The outermost of these is composed of numerous 
fine papillae of irregular form, and more or less anastomosing 
so as to form an irregular network. Within this is a layer to 
which these papillae are attached, and lastly the exosporium 
proper, which he describes as ‘ soltilissimo ,’ ‘ very delicate,’ 
while in reality it is of appreciable thickness and decidedly 
firm and resistent. All of these three layers react like cuti- 
cularized membranes, while the endosporium proper shows the 
reaction of cellulose. Besides these there is often to be seen, 
at least in chromic acid preparations, what appears to be a 
loosely-fitting, nearly transparent but well-defined membrane 
outside, the episporium. Arcangeli assumes that all the mem- 
branes are derived from the plasma of the mother-cell, but it 
is more than likely that, as in the spores of Marsilia 2 and in 
A 1. c., p. 327. 
2 Strasburger, Bau und Wachsthum der Zellhaute. 
