The success of an instrumental insemination can be measured by the number of sperm reaching the spermatheca. To dissect out the sperma-theca for examination, the last visible segment of the abdomen is torn off by grasping the last ventral plate with forceps. The spermatheca is usually found embedded in tissue inside this removed segment. It is a sphere about 1 mm. in diameter and appears rough and white because of the net work of tracheae which covers it completely. The spermatheca is lifted onto a finger and the tracheae removed by rolling with the forceps. The spermatheca itself is smooth and transparent in a virgin queen; and in mated queens it varies from translucent through milky-white to cloudy and a dark cream color depending on the number of sperm present. With up to 0. 5 million sperm it ranges from clear to opaque milky. When the number reaches 0. 8 to 1. 0 million it becomes slightly cloudy. At 1. 5 million it is cloudy and light cream colored. As the number increases it becomes dark-cream colored.

A more accurate estimate can be made by counting the number of sperm in a sample taken from the diluted contents in the following manner.

Equipment needed includes a counting-chamber slide, a 10-cc. pipette, a medicine-dropper pipette, a small dish holding a little more than 10 cc., a pair of forceps, and a dissecting needle.

The spermatheca is placed in the dish, and 1 cc. of 0. 9 percent sodium chloride solution added. Then the spermatheca is broken with the forceps and needle, the sperm teased out, and the empty skin removed. The sperm is dispersed by alternately drawing the saline solution into and expelling it from the medicine-dropper pipette until all lumps have disappeared. Then 9 cc. of tapwater is added to make a total of 10 cc. and the sperm again thoroughly dispersed with the medicine-dropper pipette. A drop of this mixture is quickly placed in the counting chamber.

In the saline solution the sperm appear as headless filaments about 0. 25 mm. long and are easily dispersed. The tapwater kills them, but before they die most of them coil up into various shapes such as small circles or configurations resembling the numerals 6 and 8. In these forms they are more easily counted than when relaxed. They are most easily seen and counted under a phase-contrast microscope or one with a dark field. The number in 10 cc. is calculated from the number counted in a given volume.

The number of sperm in the seminal vesicles of drones can be estimated by the same method. A window is carefully cut into the dorsal wall of the abdomen and the seminal vesicles very gently cut off where they join the mucous glands. If this is not done very carefully, the muscles of the seminal vesicles will contract prematurely and some of the sperm will be lost. After being placed in the saline, the muscles are made to contract by pricking and mashing, and what sperm is not forced out in this way is released by tearing the vesicles to pieces.