-17- 
removed, the spermatheca is fovuad to be smooth and transparent in a 
virgin queen and milky-white to cloudy-cream in mated queens, depending 
on the degree of insemination. 
The spermatheca is placed in the dish suad 1 cc# of tap water added 
from the burette* Then the spermatheca is broken with the sharp-pointed 
forceps and needle, the sperm teased out, and the empty skin removed. The 
sperm is dispersed by alternately dravring the water into, and expelling 
it from, the pipette about twenty times, or until all lumps have disappeared, 
Then 9 cc« of water is added to make a total of 10 cc. and the sperm again 
thoroughly dispersed with the pipette. A drop of this mixture is quickly 
placed in the co(jntiag chambero ^'hen the sperm number seems to be small, 
a dilution to only 5 cc« or less c&n be used. The sperms are counted 
under a compound microscope with a magnification of 60 diameters, and 
against a dark field, which makes the sperms stand out as white filaments. 
From the number counted in a certain volume the nvimber in 10 cc. is calcu- 
lated. 
The sperms usually appear as almost headless, slightly curved, fila- 
ments about 0.27 mm. long, but they may be coiled or looped into various 
shapes, su%h as small circles, spheres, or like the numerals 6 and 8. 
It is important that the dispersion be continued no longer than is 
absolutely necessary and that the drop be placed in the counting chamber 
quickly before any great amount of settling or adhesion of sperm can take 
place. It is also important that the pipette be kept clean to prevent 
lumps of sperm from sticking to its walls. In some localities a physiolog- 
ical salt solution may be more satisfactory than tap water. Distilled water 
is not recommended. 
There is, of course, considerable chance of error in making counts 
from such a small sample. Nevertheless, this method is certainly more 
accurate than judging by appearance of the spermatheca and the viscosity of 
the contents, as has been done by previous investigators. Greater accuracy 
can be obtained by counting a larger sample. All sperm counts given in 
this manual were made by diluting individual matings to 5 oc. and other 
matings to 10 oo* and counting the sperms in 0«8 mm*^ 
The number of sperms in the seminal vesicles of drones can be estimated 
by the same method. A window is carefully out into the dorsal wall of the 
abdomen eind the seminal vesicles are very gently cut off where they join 
the mucus glands. If this is not done very carefully, the muscles of the 
seminal vesicles will contract and some of the sperm will be lost. After 
being placed in water, the muscles are made to contract by pricking and 
mashing, and what sperm is not forced out in this vmy is released by 
tearing the seminal vesicle to pieces. 
THE USE OF CARBON DIOXIDE 
Carbon dioxide not only serves as an anesthetic, but also stimulates 
queens to begin laying (Mackensen 4^) . Proper treatment with this gas 
causes virgin as well as inseminated queens to begin laying practically as 
soon as naturally mated queens, whereas without such treatment only about 
20 percent start earlier than 30 days after emergence. 
