190 ISAO IIJIMA. 



the tissues to swell in water; but for accurate measurements 

 or sections, leeches were opened in salt solution. The fresh 

 ovaries were carefully taken out with a pair of forceps, put 

 in dilute glycerine or osmic acid (^ per cent.), and dissected 

 with needles under a dissecting microscope. 



S. For sections of an entire leech, it was found best to 

 make transverse cuts at convenient places, and to harden in 

 chromic acid (-'- per cent.) three or four hours. In order to 

 remove as much of the acid as possible, the parts were next 

 placed in water for several hours, the water being renewed 

 two or three times, and then the hardening completed in 

 alcohol, using first weak, then strong, and finally absolute 

 alcohol. Dilute Beale^s carmine was used for staining 

 (twenty-four to thirty hours). After colouring, they are 

 again to be passed through weak, strong, and absolute 

 alcohol. 



3. For hardening ovaries and egg-strings the above 

 method was sometimes used ; but the object was exposed 

 for a shorter time (one hour) to the action of a weaker acid 

 solution (ca. -f per cent,). 



A more convenient method, with about equally good 

 results, was to harden in Kleinenberg's picro-sulphuric acid 

 (two to three hours), succeeded by TO per cent, (two to 

 three hours) and 90 per cent, (two to three hours) alcohol. 

 Coloured in Ranvier's picro-carmine, washed, and again 

 hardened in alcohol as before. 



4. For section-cutting, specimens taken from absolute 

 alcohol were thoroughly soaked in clove oil or bergamot oil. 

 Embedded in paraffine, to which a little pig's lard had been 

 added. Cut by means of a microtome. Paraffine removed 

 from sections by benzine, and clarified with creosote or clove 

 oil. Or, paraffine removed and sections clarified with a 

 mixture of four parts of essence of turpentine and one part 

 of creosote. Mounted in Canada balsam. 



5. For surface views of the ovary- wall, the ovary was first 

 split open along one side with needles, then stretched flat on 

 a slide, and hardened and coloured as in the case of ovaries 

 and egg-strings. 



I have also obtained very good preparations by hardening 

 in ,'; per cent, osmic acid (ten minutes), coloured with picro- 

 carmine, and mounted in glycerine. 



6. For the examination of early changes in mature eggs, 

 I have adopted the method used by O. Hertwig {' Morph. 

 Jahrb,,' B, iii, p, 9) with very slight modifications. It is as 

 follows: — Treated with acetic acid (1 or 1| per cent.) for 

 about ten minutes, the acetic acid replaced by absolute 



