94 BULLETIN OF THE 
may be successfully used without the removal of the egg-shell ; but in 
the use of osmic acid it is necessary to remove the shell, in order to 
enable the reagent to penetrate with sufficient rapidity, 
In staining, I have attained my best results from the use of Kleinen- 
berg’s hematoxylin in toto for 20 to 24 hours, and decoloring with 70% 
acidulated alcohol for about 2 to 4 hours. This statement applies to ma- 
terial preserved with Perenyi’s fluid and Kleinenberg’s mixture. With 
osmic material the best results in staining have been attained by the use 
of Czokor’s cochineal for 10 to 12 hours. The embryos were removed 
from the yolk under the dissecting microscope, dehydrated, penetrated 
with clove or cedar oil, followed with paraffin at a temperature of about 
55° C. imbedded in paraffin in the usual way, and sectioned with a 
Thoma or a Cambridge rocking microtome. 
(c) Variation in the Development of Fundulus. — In Fundulus I have 
noted a very considerable individual variation in the progress of embry- 
onic development. Inasmuch as the ova of several individuals were 
placed together in the same aquarium for fertilization, this variation 
could not be owing to differences of surroundings, but may be due to 
a difference in the stages of maturity of the ova from the different 
females. I do not believe that a delay im the fertilization of some of 
the eggs could be sufficient to account for the existing variation in 
development. The period of vitality of the free teleostean spermato- 
zoón İs not very accurately known, but it probably does not exceed a 
Jew hours. It seems to me quite certain that no possible delay in the 
fertilization of any of the ova could agcount for the great difference in 
the hatching period, which has been found to vary as much as forty- 
eight hours; and since this variation also exists at the point of hatch- 
ing, it cannot be assumed that all the retarded embryos below the 
hatching period are imperfect and do not reach maturity. 
It is very probable that ova which are removed from the ovary by 
the process of “stripping” may not have reached the highest degree of 
maturation, and it may be possible that such ova are yet capable of fer- 
tilization, but that when thus artificially fertilized the development is 
not as rapid as in case of fully matured ova, 
Owing to this variation, the embryos preserved at the same time do 
not all show the same stage of development ; hence, to ascertain the 
relative advancement of the individual embryos of the same age requires 
considerable study of certain structures, — e. g. the condition of the 
optic and auditory vesicles, the nephrostome, gill-elefts, ete., — which 
may be selected as a safe index to exact stages of development. It has 
