Division of the Generative Nucleus in Pines . 195 
or cut into quarters longitudinally. Very soon the individual 
scales were removed from the receptacle before fixing, and, 
when the scales were of sufficient size to admit of such 
manipulation, all superfluous parts were cut away, leaving the 
two tiny ovules still united by a small portion of the scale. 
In the spring the ovules were removed from the scales and, 
as soon as it was feasible, a portion pf the* integument was 
cut away from two or more sides of each ovule. For later 
stages the endosperms were frequently removed, but such 
material did not prove to be as satisfactory as that in which 
the nucellar cap and a small portion of the integument were 
left in connexion with the prothallium. Throughout the 
entire mechanical process of preparing material for the fixer, 
the most extreme care was used, as it was found that a very 
slight pressure was sufficient to cause distortions and thus 
render the material worthless for cytological studies. 
The methods used in fixing and staining do not differ 
materially from those generally employed in cytological work. 
Several fixing fluids were tried, but the Flemming chrom- 
osmo-acetic acid solution gave by far the best results, and 
the other fixers were entirely discarded. The material was 
embedded in paraffin which had a melting point of 54 0 and 
was cut on a Minot-Zimmermann revolving microtome. Some 
sections were cut four, others thirteen and one-third, microns 
thick ; but by far the greater part of the material was cut 
six and two-thirds microns thick. 
Various stains were tried, among which might be mentioned 
Rosen’s (’92) fuchsin and methylene-blue method; the Ehrlich- 
Biondi-Heidenhain mixture, as prepared by Dr. G. Griibler ; 
Guignard’s combination of methyl green, acid fuchsin, and 
orange G (Guignard has not given the formula which he 
uses and hence solutions of various strengths were tried) ; 
Flemming’s safranin, gentian-violet and orange combination ; 
and Heidenhain’s iron-haematoxylin. The last two proved 
the most satisfactory. Flemming’s triple stain was often used 
without the safranin with excellent results. The iron-haemat- 
oxylin was followed by orange G, or if it was desirable to 
