236 DR. W. N. F. WOODLAND ON THE 



Appendix A. 



The Sources and Modes of Preparation of Material. 



A great part of my material was obtained by me during my occu- 

 pation of the British Association Table at the Naples Zoological 

 Station during April, 1907 ; additional specimens were sub- 

 sequently sent to me from Naples and the remainder I obtained 

 from the Plymouth Marine Biological Station. As regards 

 methods of preparation, I may remark, first of all, that it is 

 important to fix the lining-epithelium of the bladder and its 

 special development, the gas gland, in a distended condition, and 

 this is best effected by puncturing the bladder (in situ) posteriorly 

 and immediately filling it with the fixative used, also pouring 

 fixative on the outside of the bladder. By this means all shrinkage 

 of the bladder wall is avoided on removal from the body. Another 

 advisable precaution is to separate the lining-epithelium of the 

 bladder, after it has been well fixed, from the outer layers of the 

 bladder wall, since I found that in many cases the tougb muscle 

 find connective tissue composing these latter cause difiiculty in 

 obtaining thin sections across the gas gland. The fixatives which 

 I employed chiefly were Zenker's Fluid, Corrosive Acetic, and 

 Mann's Fluid (Distilled Water 100 c.c, Corros. Sub. 2-5 gm., Picric 

 Acid 1 gm.. 40 % Formol 10-25 c.c. — a fixative which, in conjunc- 

 tion with Borax Carmine and Picro-indigo-carmine, gave me most 

 beautiful results). Some of my specimens were simply fixed in 

 10 °/o Formol — a fixative which did not yield such good results as 

 the others, though good enough for most purposes. I left objects 

 in Zenker and Mann overnight, but in Cori'osive Acetic only for 

 an hour or so. In many cases I fixed duplicate specimens in dif- 

 ferent fixatives and was thus able to compare results. | Objects 

 fixed with Zenker were well washed for several hours with Distilled 

 Water and then, like the remaining objects fixed with other fix- 

 atives, graded slowly up to 70 7o Alcohol (the pei-centages being 

 in all cases made up with Absolute Alcohol and Distilled Water). 

 Objects fixed with fluids containing Corrosive Sublimate were 

 treated with Iodine at this stage. In most cases I subsequently 

 stained in bulk with Grenacher's Borax Carmine, leaving the 

 objects in for at least twelve hours, and toithout differentiation with 

 Acid Alcohol, dehjAvKiQc]^ cleared, and embedded in Paraffin. The 

 sections (both longitudinal and tra,nsverse in many cases), well 

 stained with Borax Carmine, were then stained on the slide with 

 Picro-indigo-carmine *. This stain is made up by adding one part 

 of a satiirated solution of Picric Acid in 90 °/^ Alcohol (sat. sol. 

 = circa 4'5 "/^ ) to two parts of a saturated solution of Grubler''s 

 Indig-carmin in 70 7^ Alcohol (sat. sol. = circa 1 7^), and it is well 

 to dilute this stain so obtained with twice its bulk of 70 7o Alcohol. 

 The sections are placed in this stain for a short time, varying 

 according to the thickness of the sections and depth of staining 

 with Borax Carmine, from a few minutes to over half an hour. 



* I am iiidobted to Mr. A. D. Davbishire for recoinmendiiiff me this stain. 



