132 E. W. MACBRIDE. 



1 per cent, acting for five minutes only ; they were then rinsed 

 with water and transferred to Miiller's fluid for eighteen to 

 twenty hours, then put at once into alcohol of 30 per cent., 

 and brought slowly up into alcohol of 90 per cent. In the 

 latter they were hardened for a night ; then two or three drops 

 of nitric acid were added to some fresh alcohol of 90 per cent., 

 so as to give roughly a solution of from ^ to 1 per cent, of 

 acid, and the animals were immersed in this till decalcifica- 

 tion was complete, a process which occupied not more than 

 twenty hours. 



I found that Midler's fluid itself has a slow decalcifying 

 action, and in some cases the acid alcohol was unnecessary. 

 For this reason, were I doing the work again, I should prefer 

 to stop the action of the osmic acid with ammonium picro-car- 

 minate instead of Miiller's fluid, so as to avoid even the slight 

 and easily recognisable " artefacts " produced by the latter. 

 Glacial acetic acid gave fair results, and I used it to confirm 

 results obtained by osmic acid. Strange to say, it has little or 

 no decalcifying efi'ect, and the suddenness of its action and its 

 penetrating power are unsurpassed. But it is apt to cause 

 swelling of the tissues, and of course on transferring the ani- 

 mals to alcohol one gets a solution of acetic acid, which then 

 decalcifies before the tissues are hardened. 



II. Stains. — Double staining was used in order to be 

 certain about the boundaries of sinuses, since the ordinary 

 plasma of Echinoderms stains with great difficulty. 



As a nuclear stain I used Dr. Mayer's paracarmine. The 

 great advantages about this are that it acts rapidly, and that all 

 superfluous stain can be extracted by 70 per cent, of alcohol, 

 which can be allowed to act for an indefinite time. I allowed 

 my earlier stages about twenty minutes in the stain, adults 

 and later stages an hour or two. An hour or two suffices to 

 remove all surplus stain, provided the alcohol be changed 

 several times. The plasma stain was applied on the slide. I 

 used two, both of which gave good results, viz. solution of 

 picric acid in turpentine and Dr. Mayer's oxidised hajmatoxylin 

 or " haimateiu." The advantage of the former is that it can 



