the Echinoderm Egg during Fertilisation. 



217 



after being washed several times in clean sea-water, were placed in large 

 open-mouthed bottles suspended in the water of the thermostat. They were 

 taken from these bottles as required for an experiment and were therefore 

 always at the same temperature as the bath water. The pipettes used to 

 mqasure out the eggs and sperm were always kept in separate bottles of sea- 

 water when not in actual use, these bottles being also suspended in the 

 water of the bath. The manometers, finally, were always kept in position on 

 the thermostat when not in use, so that when required for an experiment 

 •they were already thoroughly cooled to the temperature of the bath. As 

 already mentioned, in addition to these precautions 20 minutes was always 

 allowed after the eggs and sperm were placed in the manometer chamber 

 before the stop-cocks were closed and the first reading taken. It was found 

 from preliminary experiments in which plain sea-water was used instead of 

 eggs and sperm, that when these conditions were observed in performing an 

 experiment no cooling effect could be produced, and that, under these 

 conditions, the first reading on the manometer was perfectly accurate and 

 satisfactory. In revolving the chambers on the manometers the stop-cocks 

 of the apparatus were opened and immediately closed a moment later. It 

 was possible that, in the operation of revolving the chambers, they might be 

 slightly loosened on their respective stoppers and so the oil drawn round, and 

 a faulty reading obtained in this way. This was avoided if the stop-cocks of 

 the instrument were open while the chambers were revolved. The chambers 

 of my manometers were so accurately ground on to their respective instruments 

 that I was unable, as a matter of fact, to produce any such error experi- 

 mentally when I turned the chambers while the instruments were closed. 

 An error of this kind could only be produced by actually forcing the chambers 

 completely loose from their manometers. Observing the foregoing precautions 

 then, at the end of 20 minutes the hands of the manometer stop-cocks could be 

 closed and readings commenced on the unfertilised eggs. The same eggs 

 could then be fertilised by turning the chambers and allowing the sperm to 

 run down on the eggs. There was a small error in this reading due to the 

 presence of the drop of sperm in the small tube in the manometer stopper, 

 but care was always taken to use very dilute sperm so that this error could 

 be neglected. To rotate the chambers, when they were attached to their 

 instruments and the manometers themselves in position submerged in the 

 water of the thermostat, wooden handles were wired on the necks of each 

 chamber. These were long enough to project well above the surface of the 

 water, so that the warm hand or fingers never came in close contact or 

 touched the manometer chamber when they were rotated. Separate mano- 

 meters were used for the oxygen and CO2 measurements, the output of C0 2 



VOL. XCIIL— B. R 



