458 INVERTEBKATA CHAP. 



data derived from it, with some confidence, in filling", in the gaps in 

 our story of asteroid development. 



Before entering on this subject, however, there are certain 

 practical questions to be considered. Loeb, in his work Die chemische 

 Entwicklung des tierischen Eies (1910), states that the eggs of 

 Asterias are not ripe when laid, but ripen after lying in sea-water for 

 five or six hours. This seems to be a most misleading statement. 

 The eggs when shaken out of the excised ovary, or when made to 

 exude through the genital openings by the application of pressure, 

 are certainly unripe. They are surrounded by a glassy choriou 

 which disappears in a few hours and renders them fit to receive 

 the spermatozoa. But if perfectly ripe males and females be selected, 

 they will often emit their genital products spontaneously, and then 

 every egg which is emitted is capable of instant fertilization. 



As to methods of preservation and of preparation a few words 

 may be said here. Goto used corrosive sublimate, with a certain pro- 

 portion of glycerine and of acetic acid, to preserve the larvae, but it is 

 exceedingly doubtful whether his histological results are at all reliable. 

 Further, this preserving fluid has one strong disadvantage, it is strongly 

 acid, and, as a developing Echinoderm contains numerous calcareous 

 deposits, the sudden solution of these is apt to generate quantities of 

 carbonic acid gas, which distend and tear the tissues. The cavities so 

 produced, which even so great an authority as Ludwig (1882) has 

 regarded as existing in the living animal, are nevertheless only 

 artefacts. 



Faced with this difficulty, we ourselves, many years ago, 

 adopted a method of preservation which, although it has numerous 

 drawbacks, still yields better results with Echinoderm larvae than 

 any other. This method consists in adding a half per cent or even a 

 quarter per cent solution of osmium tetroxide to the watch-glass 

 containing the eggs and larvae, and leaving them in the mixture till 

 they are thoroughly impregnated with it and have assumed an 

 almost black colour. They are then transferred to a bottle containing 

 Mtiller's fluid (which is a mixture of the solutions of bichromate 

 of potash and sulphate of soda), in which they remain for twenty-four 

 hours at least, though several months' immersion in this fluid does not 

 hurt them. After treatment with Miiller's fluid they are rinsed 

 in distilled water, and then dehydrated by being passed successively 

 through various grades of alcohol. They are embedded in celloidin 

 and then in paraffin, according to the method described in Chapter II. 

 They may or may not be stained in borax-carmine before being 

 embedded, for the effect of this is merely to darken their colour and 

 render them more easy to orientate before finally making up the 

 blocks of paraffin. In all cases the sections are finally stained on 

 the slide in Grenadier's haeruatoxylin. 



The penetration with osmium tetroxide gives a black stain of a 

 diffuse kind ; this is, in fact, due to a deposit of the metal osmium in the 

 cells, and it is difficult under these circumstances to get the ordinary 



