Conjugation in the hétérogamie geuus Zygorhynchus 
243 
difference in size is always distinct, though in some cases less marked 
than in others. The contents of the two gametes become united through 
the disappearance of the intervening wall, and the zygote here formed 
(figs. 7, 13, 22), by the gradual enlargement of the two cells thus united, 
assumes the shape of a mature zygospore (figs. 8, 15, 24). The supply 
of nutrient for this ripening process comes almost entirely by way of the 
more vigorous zygophoric branch, and, although the stretched wall of the 
larger gamete makes up the greater part of the outline of the zygospore, 
still the stretched wall of the smaller contributes to it. Although it may 
show a certain tendency in this direction, the condition here is thus not 
comparable to an oogamous fertilization where the male gamete furnishes 
protoplasm to, but forms itself no essential part of, the mature oospore. 4 . 4 
It does not seem necessary at the present writing to change the 
account of conjugation already given. 
In the usual method of obtaining stages of development by taking 
material from a young culture and examining it stained under the micro¬ 
scope, it is extremely difficult to find the earlier stages. Attempts to 
follow the development in living material in the ordinary 1 inch yanTieghem 
cell culture are difficult from the small amount of nutrient in the hanging 
drops of agar. Moist chambers made of Syracuse watch-glasses, 2 inches 
in diameter, and PETRi-dishes, 4 inches in diameter covered with number 
2 covers cut from thin sheets of glass, give much better results. Sufficiently 
numerous drops of the nutrient agar are placed on the under side of the 
cover to counteract the tendency shown by the aerial hyphae to dry up 
despite the presence of water in the bottom of the culture dish. The 
mycelium grows rapidly to the edge of the nutrient drop and out for a 
short distance upon the glass itself where it produces zygospores in 
abundance. Many will be found so near the surface that their development 
can be followed under the 4 mm objective. 
In practice, a considerable number of the earliest stages are marked 
on the glass and camera lucida drawings made of each at various times 
throughout their development. Some become covered over by other 
hyphae, while some dry up or touch the drops of condensed moisture on 
the glass or abort for other reasons If, however, a sufficient number 
are watched at the same time, there is no great difficulty in securing a 
complete series of stages for several conjugations from the same culture. 
Figures 1—8 and 9—15 were thus obtained and show the development 
respectively of Z. heterogamus and Z. Moelieri. It will be noted that the 
process is relatively rapid at first and this fact probably accounts for the 
difficulty most observers have experienced in finding the early stages of 
development. 
It is needless to say that camera drawings of hyphae in the air at 
some distance from the cover glass cannot be expected to be as accurate 
in outline as those made from mounted material. They do, however, 
show with fidelity the succession of the stages figured. 
In living material again it is difficult to be certain of more than 
surface markings. Mounted material is therefore necessary to determine 
the completeness of the cross walls, The cover of a large moist chamber 
culture which shows stages in conjugation may be conveniently inverted 
and used directly as an object slide. If a mixture of equal parts alcohol 
and glycerine colored with eosin be added, the protoplasm will be strongly 
16 * 
