64 BASIC METHODS FOR EXPERIMENTS 



side is obvious. I have made very careful comparisons between 

 eggs of Nereis limbata, Chaetopterus, Echinarachnius and of 

 Arbacia washed during 24 hours and those washed for one hour by 

 the method which I shall now give and have been unable to 

 detect any difference as the result of the prolonged washing. 

 Eggs washed for an hour are stained as beautifully as those 

 washed in running water for 24 hours. 



The eggs are washed for one hour by the simple expedient of 

 changing the water as often as the eggs settle, gently inverting 

 the vial after each change; the eggs settle quickly and when this 

 has taken place, the water is withdrawn and replaced. 



Dehydration 



Following washing the eggs are dehydrated by being changed 

 to grades of alcohol of increasing strength. In each grade the 

 eggs remain for i hour. The grades used are 35, 50, 70 and 80 

 per cent. Some workers omit the lower grades; this I think is a 

 bad practice. On the other hand, some begin the dehydration 

 with 15 per cent alcohol, going from it to 25, then to 35 and 

 further as given above. I have been unable to detect any differ- 

 ence between eggs the dehydration of which was begun at 15 and 

 those whose dehydration was begun at 35 per cent alcohol. 



In 80 per cent alcohol the eggs may remain until the worker 

 is ready to imbed them. I would very strongly urge that they be 

 not kept in the 80 per cent alcohol any longer than necessary, 

 I always endeavor to imbed my eggs as quickly as possible after 

 they have been fixed. The maximum time that I allow them to 

 remain in 80 per cent alcohol is two weeks, although in excep- 

 tional cases I have not imbedded the eggs until return from the 

 sea-side to an inland laboratory, about four weeks later. In 

 addition to the practical advantage of transporting eggs in 

 paraffin blocks rather than in alcohol is one far more important 

 yet to the experimental embryologist: by rapidly completing the 

 procedure of fixation and staining, he can learn enough from the 

 sections to orient him in making additional experiments during 

 the current season. 



From 80 per cent, alcohol the eggs are carried through final 

 stages preparatory to imbedding in parafhn. These stages will 

 vary depending upon the method to be used in the staining of the 



