METHODS AND MATERIALS USED. 15 
dant at Miami, and almost an unlimited number of cones can be secured 
any season. The following statement of dates at which time impor- 
tant changes take place in the developing organs of Zama floridana 
may be of service in guiding investigators in the securing of important 
stages for investigation. It must be remembered, however, that dif- 
ferent plants vary considerably in their stages of development,-and the 
dates are thus only approximate. 
(1) Pollination takes place the last of December and first of January. 
(2) Germination of pollen and growth of prothallial apparatus from January 1 to 
June 1. 
(3) The division of the second prothallial cell, giving rise to the stalk cell and 
central cell, occurs February 15 to March 10. 
(4) The blepharoplasts first appear about March 1 to 20. 
(5) The gradual development of the central cell blepharoplasts and _ prothallial 
apparatus continues from March 1 to May 30. 
(6) The prophase of division of the central cell appears about May 20 to 25. 
(7) Spermatozoids mature mainly between June 1 and 15. 
(8) Fecundation takes place mainly between June 1 and 15. 
In Zamia pumila the date of maturing of the spermatozoids and of 
fecundation in 1897 was fully three weeks later than in Z. floridana, 
and it is probable that this species is ordinarily considerably later. 
On the other hand, the date of maturity of the male cones, the polli- 
nation, and the first appearance of the blepharoplasts in the two 
species was found to be about the same. | 
When the cones were received the seeds were cut out and a portion 
of the apex of the nucellus 3 to 4 mm. in diameter, which contained 
the developing pollen tubes, was transferred as quickly as possible to 
the fixing solutions. In preparing the archegonia for study, cylinders 
about 5 mm. long and 23 or 3 mm. in diameter were cut out of the 
apical portion of the prothallus containing the archegonia, and trans- 
ferred to the fixing solution. Quite large portions of tissue must be 
used in this case from necessity, as the egg cells if cut into are 
destroyed for study, the protoplasm flowing out. In some cases por- 
tions of the seed were cut out and prepared, with the nucellus and pro- 
thallus in connection, to show the apparatus 77 stu, but this method is 
not satisfactory for the study of the finer cytological details of 
structure. 
Various fixing agents were used in the course of the work, including 
Flemming’s chromic-aceto-osmic acid solution, weak and strong, Her- 
mann’s platino-aceto-osmic acid solution, Van Rath’s solutions II, LI, 
and IV, chromic acid one-half per cent and 1 per cent, etc. Flemming’s 
strong solution was used more than any of the other fixatives, and gave 
in general the best results. Its time of action was varied considerably, 
and in probably the majority of cases it was used diluted somewhat 
with water in the ratio of one part of the strong solution to two, four, 
nine, or nineteen parts of water. In general, a solution of one part to 
