232 Campbell \ — Contributions to the 
male prothallium, and Kienitz-Gerloff 1 has corrected some of 
Hofmeister’s errors in regard to the embryo. 
There are serious difficulties in the study of the germinating 
macrospore and young embryo ; indeed, so great are these 
that without the aid of the modern histological methods, they 
are quite insurmountable. Free-hand sections of the fresh 
macrospore and young embryo are impossible, and even in 
the later stages very difficult. Having successfully imbedded 
in paraffine and sectioned the macrospores of Pilularia 2 and 
Marsilia 3 , the study of the development of the female pro- 
thallium of Isoetes was taken up, in the hope that by the same 
methods the early stages might be successfully made out. 
Although it proved a difficult task, it was at last possible to 
trace out the whole development of both the prothallium and 
the embryo. A brief summary of the most important points 
in the development of the former has already appeared 4 . 
Owing to the thick and impervious exospore, great care is 
necessary in imbedding to prevent shrinkage, and even with 
every precaution, many preparations proved perfectly worth- 
less. This was especially true with young prothallia after 
the formation of the cell-walls, but before the exospore was 
ruptured. 
On the whole, material fixed with one per cent, aqueous 
solution of chromic acid proved most satisfactory. After 
washing thoroughly and dehydrating, the specimens were 
gradually brought into turpentine, and after remaining several 
hours in a saturated solution of paraffine in turpentine, were 
placed in melted paraffine for from six to eight hours, and then 
imbedded for sectioning. At first chloroform was used in- 
stead of turpentine, but it almost invariably caused shrinkage, 
and so was finally abandoned. When chloroform is used, a 
much shorter time (two to three hours) is usually sufficient for 
a thorough saturation by the melted paraffine. 
1 Kienitz-Gerloff, Entwicklung des Embryos v. Isoetes lacustris\ Bot. Zeit. 1881. 
2 Campbell, on Pilularia globulifera ; Annals of Botany, vol. II., no. VII. 
3 Campbell, on Marsilia Aegyptiaca ; Ber. der Deutschen Bot. Gesellschaft, 
1888, VIII. 
4 Berichte der Deutschen Bot. Gesellschaft, 1890, No. III. 
