The technique is a modification of Lillie's. As soon as possible after the urchins were collected, the eggs were removed, either by inverting each washed and partly dried individual or by removing the ovaries into separate dishes. The eggs were washed two or three times, allowed to settle by gravity in graduates, and finally enough fresh sea water was added to make a given concentration. The dry sperm was collected from each individual separately. From this sperm any required concentration was made immediately before fertilizing the eggs. The time required to make the different suspensions varied from 30 minutes to 2 hours.
In a typical experiment 5 drops of a standard egg suspension was added to 10 c.c. of filtered sea-water and fertilized with 1 drop (0.05 c.c.) of standard sperm suspension.
The following characters were studied in some detail: (1) Size of eggs. (2) Shape of eggs. (3) Number of eggs with and without jelly layer. (4) Rate of membrane formation. (5) Number and rate of cleaving eggs.
The first three are matters of direct observation; the last two require a word of explanation. for I have found that such matters of detail are significant in these studies. The eggs were shaken and several fields were oounted, totaling 100 to 200 or more eggs. At least two counts were made, the first when moat of the eggs were in the 2-cell stage—i. e., 1 hour—and the second when most eggs were in the 4- or 8-cell stage— e., 2 hours. Preliminary experiments showed that there was little or no increase in total cleavage after the second hour in Toxopneustes and Arbacia. Therefore the number of eggs that had cleaved in 2 hours was taken as the maximum. In many instances a count was made 40 minutes after fertilization, when the eggs first began to cleave. These three counts afforded the bases for computing the rate of early cleavage and the total cleavage, and also served as a check upon a possible error in the previous count.
Most of the experiments are referred to by date. Thus 7/5 means that the experiment was made on July 5, 1916.