CULTURES OF SPERMATOZOA 383 
III. OBSERVATIONS ON LIVING MATERIAL 
When the spermatozoa of the fowl are observed in a hang- 
ing drop of white of egg, kept at about 40° C., the first change is 
seen after fifty or sixty minutes. It consists in the collection of 
a small amount of some substance having a low refractive index 
about the middle pieces of some of the spermatozoa. In favor- 
able cases as many as 60 per cent of the spermatozoa may undergo 
this change. At this time many of these spermatozoa are still 
swimming. During the course of the next few hours these lowly 
refractive areas increase in size until they are about half as long 
as the sperm head and acquire a fairly distinct ellipsoidal outline. 
Then in many cases the sperm head can be seen to be bent in a 
horse shoe or spiral shape, and to be included in the wall of the 
vesicle, which has now become spherical, while the tail of the sper- 
matozo6n still remains unchanged or has disappeared without 
taking any part in the transformation. The next change is an 
increasing indistinctness in the sperm head, and an increasing 
refractive power of the whole vesicle so that it can hardly be dis- 
criminated at all in the albumen. It is not possible to follow the 
process farther in unstained material. 
In some cases these vesicles instead of being spherical stretch 
out along the whole side of the sperm head, or may become en- 
tirely disconnected from the spermatozoon. 
If yolk is used as a culture medium for the sperm essentially 
the same phenomena occur; and in the various Ringer solutions 
vesicles containing the sperm heads are also formed, but in the 
Ringer solution, as a rule, the steps in the formation of these ves- 
icles could not be seen without staining. 
IV. OBSERVATIONS ON PRESERVED MATERIAL 
When the hanging drops are fixed in Flemming’s fluid and 
stained and examined in Herla’s vesuvin and malachite-green 
mixture, it can be seen that in its early stages the vesicle has dis- 
tinct walls and a homogeneous unstained fluid of a low refractive 
index in its interior. This fluid is possibly water and this would 
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 12, NO. 3 
