No. 2.] THE ECHINODERM SPERMATOZOON. 239 



2. Tincture of iodine ; a very weak solution in sea-water (it 

 must be very carefully filtered) preserves the shape and size 

 very well. It stains the mitosome and centrosome a dark yel- 

 low or brown, the nucleus and tail a light yellow (Fig. 15), 



3. Methyl-green in dilute aqueous solution drawn under the 

 cover-glass by aid of filter-paper, stains nucleus darkly ; but 

 after a few minutes a swelling of the heads of the spermatozoa, 

 probably due to osmosis, begins, and the nucleus finally bursts. 



4. Chloride of manganese (10% solution), add concentrated 

 aqueous solution of dahlia ; filter. In a drop of this tease the 

 fresh material on the slide. Examine immediately. For after 

 a short time the heads of the spermatozoa become swollen. 

 This reagent was particularly recommended by Pictet (18). I 

 found it remarkably good for use in studying the mode of 

 formation of the tail of the spermatozoon, and fairly good for 

 the mitosome and centrosome (Fig. 24), but not so good as 

 some other methods, notably i and 2. 



5. a. Acetic acid (0.1% to 3.0%) combined with dahlia 

 and methyl-green gave fair results. In many instances, par- 

 ticularly with the stronger solutions, the centrosome very 

 gradually swelled, and slipped itself out of the cap-shaped 

 depression in the anterior wall of the nucleus, into which it had 

 fitted (Fig. 20). In some cases it separated completely (Fig. 22). 



b. Acetic acid (33%), while preserving very well the shape 

 of the nucleus, distorted beyond recognition the mitosome and 

 centrosome (Fig. 23). 



c. Schneider's acetic carmine. Stain for ten minutes ; de- 

 colorize in 33% acetic acid; wash in distilled water. Examine 

 in dilute glycerine. The size and shape of the nucleus well- 

 preserved. There occurs a great distortion by swelling in case 

 of the mitosome and centrosome, but this swelling is in a large 

 measure reduced by the action of the glycerine. The nucleus 

 is stained carmine ; the chromosomes darker. Mitosome and 

 centrosome unstained. 



6. Osmic acid is very useful for demonstrating centrosome 

 and mitosome, particularly the former, making it very refringent. 



Fix in osmic vapor, by inverting a drop of sea-water contain- 

 ing the teased material over the mouth of a bottle of osmic 



