KOFOID : DEVELOPMENT OF LIMAX. 39 



man's Merkel; Fol's modification of Flemming's chrom-osmic-acetic, 

 either alone or followed by Whitman's Merkel. Uy far the most .satis- 

 factory results were obtained by subjecting the eggs to the action of 

 Fol's modification of Flemming's mixture for one minute and transfer- 

 ring them at once to Orth's picro-carminate of lithium. The eggs were 

 allowed to remain in the stain twelve to twenty-four hours, and were 

 then decolorized with acidulated alcohol until the cytoplasm retained 

 but a slight tinge of red. Itapid decolorization with ninety per cent 

 alcohol plus five per cent hydrochloric acid gave very good results. 

 The eggs when properly decolorized have cell boundaries and nuclear 

 membranes sharply marked, and the chromatic elements of the nucleus 

 remain a deep red. Asters show plainly, but centrosomes are not 

 stained. Eggs killed in Flemming's fluid and afterwards bleached by 

 chlorine, or those killed in Merkel's fluid, are satisfactorily stained in 

 Mayer's HCl-carmine. These also must be thoroughly decolorized. 

 Eggs killed in corrosive sublimate were stained in alum-carmine or 

 Czokor's cochineal, but the best results after this killing agent were 

 obtained by the addition of a drop of Delafeld's hematoxylin to slightly 

 acidulated water in which the eggs had been placed after hardening in 

 alcohol (Conklin, '92). This is especially valuable for the demonstra- 

 tion of astroccels in the early stages of cleavage. Satisfactory results 

 were not obtained on whole preparations with Heidenhain's iron-hsema- 

 toxylin or Henneguy's method with permanganate of potash and safra- 

 nin. The first, however, gives very good results with sections. 



The processes of killing, hardening, staining, and clearing were carried 

 on in watch-glasses. Capillary glass tubing was found to be very con- 

 venient for transferring individual eggs when such transfer was necessary. 

 Turpentine, xylol, or cedar oil was used as a clearing agent. Eggs can 

 be kept without harm for a long time in turpentine evaporated down to 

 a waxy consistency, or in xylol to which soft parafine has been added. 

 If the xylol is allowed to evaporate, it leaves the eggs embedded in the 

 soft parafine, which can be redissolved by fresh xylol without harm even 

 to these very delicate objects. 



The eggs were studied in the clearing agent under a cover-glass placed 

 on glass rollers made of bits of capillary tubing. This allows the use of 

 high-power objectives and the orientation of the embryo in any desired 

 position for a camera drawing. When permanent preparations were 

 desired, they were mounted in xylol-balsam or a solution of dammar in 

 cedar oil. By the addition of a drop of xylol to the margin of the cover- 

 glass, the mounting medium is sufficiently softened to allow the cover- 



