94 THE AMERICAN ARBACIA 



immediately in the refrigerator at about 8 °C. They keep perfectly for several (4-5) 

 days and give nearly 100% fertilizations. 



To Kill Sperm 



Formalin, o.i to i % (Tyler). Distilled water kills Arbacia sperm almost immedia- 

 tely (E. B. H.). Keep at 40 °C. for 20 minutes (E. B. H.). Rothschild and Swann 

 (1951) use for Ps. miliaris, hypotonic sea water, 45 % sea water with distilled water, 

 to kill spermatozoa in presence of eggs and prevent fertilization without harmful 

 effect on the eggs. 



To Prepare Sperm Suspensions for Fertilization 



One drop (o. i cc.) of concentrated ("dry") sperm to 100 cc. sea water, then one 

 drop of this to 10 cc. sea water which contains two drops of concentrated eggs; this 

 is the amount for a Syracuse watch glass (E. B. H.). Just (1939a) recommends one 

 drop of "dry" sperm to 10 cc. sea water, then two drops of this to inseminate eggs 

 in 250 cc. sea water. 



The dilute suspensions of sperm last only a short time after preparation, owing to 

 their activity, so that fresh dilutions must be made frequently. 



A simple and convenient method of fertilizing a small dish of eggs is to take a small 

 amount of sperm on a toothpick and agitate the eggs with it. With experience, this 

 becomes fairly accurate (E. B. H.). 



Determination 0/ Number 0/ Sperm in a Suspension 



Barron (personal communication) obtains a constant suspension by measuring 

 the turbidity with a Coleman Junior spectrophotometer, and checks this against 

 the dry weight. Mazia (personal communication) counts the number of sperm in a 

 dilute suspension, fixed in o. i % formalin, with a haemocytometer. 



There are 5 cc. of concentrated sperm in one Arbacia, obtained by 

 allowing the sperm from the five ruptured testes to settle. Tyler found 

 2 X 10^® spermatozoa in i cc, by haemocytometer measurements (per- 

 sonal communication, July 1954). This is the same number as he had 

 obtained from S. purpuratus and Lytechinus (Tyler and Rothschild, 1951 ; 

 Tyler, 1953). The complete number of sperm in one Arbacia then, is 

 lo^^, approximately a million million; the number of eggs in one Ar- 

 bacia is about 8,000,000. One sperm has been calculated to be about 

 I /30,ooo the volume of the egg. 



The various methods of counting spermatozoa have been discussed 

 and evaluated by Rothschild (1950 a), who advocates a photoelectric 

 absorptionometer. 



Other Species (Additional) and General References 



Bernstein and Mazia, 1953 a. S. purpuratus, DNA. 



Fuchs, 1914c. Paracentrotus lividus, A. lixula, etc., fertilizing power increased by egg secretions. 



Gray, 1931. Experimental Cytology p. 408-421. References to older literature. 



Morgan, 1927. Experimental Embryology p. 15-93. Chemotaxis, activity etc. 



Rothschild, 1951a. Review of recent literature, with good bibliography, with titles. 



Runnstrdm, 1949 a. Many references to sperm in comprehensive review The Mechanism of 



Fertilization. 

 Southwick, 1939. Echinometra subangularis, activity of sperm. 

 Tyler, 1948. Review. 



