192 FREDERICK G. E. PAUTARD 



mere, Westmorland. In November and December, the spawning 

 months for the brown trout, ripe fish and regular supplies of milt were 

 made freely available by the Welham Park Fish Hatchery, Malton, 

 Yorkshire. From time to time, other fresh-water fish, such as the pike 

 (Esox lucius) and the char (Salvelinus willoughbii), became available. 



Ripe fish were stripped of milt by carefully wiping dry the skin sur- 

 rounding the urogenital papilla and cloaca and then expressing the 

 milt by gentle pressure along the line of the testes. Drops of water ac- 

 cidentally introduced into the milt tended to produce motility in the 

 sperm, while fecal contamination usually resulted in the cells becom- 

 ing inactive. Both these hazards were avoided as much as possible, and 

 the viability of the sperm was checked at frequent intervals under the 

 optical microscope. 



Several procedures were adopted for the separation of tails from 

 heads. In the case of the unicellular algae, careful shaking was essen- 

 tial to keep cell rupture down to a minimum, but in fish sperm the 

 heads seemed to remain intact even after vigorous shaking, although 

 the flagella themselves were disorganized depending on the way in 

 which they were prepared. In earlier preparations, the spermatozoa 

 were either allowed to swim in distilled water or immobilized by di- 

 luting the milt with phosphate buffers (/ = 0.17) at pH 6.5. The sus- 

 pended cells were then usually centrifuged at about 3000 X g for 15 

 min, redispersed in a suitable (about 50 vol) amount of distilled water 

 and shaken sharply for a minute or so. The heads were separated from 

 the tails by several spinnings of the supernatant (containing the lighter 

 tail fraction) at 3000 X g for periods up to 15 min. When the super- 

 natant was free of heads, the flagella were sedimented at about 10,000 

 X g for 10 min and washed once or twice with distilled water by dis- 

 persion in suitable volumes of liquid, followed by centrifugation. Fla- 

 gella prepared in this way (Fig. 2a) were usually frayed and frag- 

 mented but nevertheless useful for extraction. 



Later, after it was found that distilled water removed into solution 

 a proportion of the tail material, 0.03M KC1, buffered to pH 6.8 with 

 phosphates (0.0 1M K 2 HP0 4 , 0.005M KH 2 P0 4 ), was used to stabilize 

 the flagella. In these preparations the milt was first centrifuged at 

 10,000 X g for 5 min and the supernatant seminal fluid was carefully 

 removed with a pipet. The densely packed cells were then suspended 

 in 20 vol cold buffered KC1 solution (the sperm were not motile) and 



