240 FIELD. [Vol. XI. 



acid (1% or 2%)). The heads of the spermatozoa become 

 slightly distorted by swelling (Fig. 21). The osmic vapor 

 appears to blacken the parts of the spermatozoon unequally, 

 acting quickest upon the mitosome and centrosome. 



a. Add methyl-green in solution in sea-water ; after 2-3 

 minutes remove the surplus with filter-paper ; add a drop of 

 glycerine diluted with a weak aqueous solution of dahlia : the 

 nucleus stains a delicate green ; mitosome and centrosome 

 violet. 



b. If treated with very dilute Delafield's haematoxylin the 

 nucleus stains deeply ; the mitosome and centrosome slightly 

 tinged. 



c. Stain with very dilute aqueous solution of gentian-violet, 

 mixed with glycerine : after a short time the mitosome and 

 centrosome are darkly stained, the nucleus but slightly. 



B. Prolonged Fixation : Teased Material. 



This method is valuable for studying the details of sperma- 

 togenesis from the spermatogone to the mature spermatozoon. 

 A piece of the testis is teased in a small quantity of water. 

 Fix in Flemming's chrom-osm-acetic (strong formula), Her- 

 mann's fluid, or platinum chloride (0.3%) for 24 hours. Wash 

 for 24 hours or more in distilled water, frequently changed 

 and shaken. Treat with some aqueous stain. Mount in 

 dilute glycerine or in damar. Dissociate cells by tapping 

 cover-glass gently with the point of a needle. 



7. Fix in Flemming's chrom-osm-acetic (strong formula) for 

 24 hours. 



a. Stain with aqueous solution of safranin 10-20 minutes : 

 only nucleus stained (Fig. 17). Add very dilute aqueous 

 solution of dahlia : nucleus violet-red, mitosome and centro- 

 some violet, 



b. Run methyl-green under cover-glass : nucleus darkly, 

 mitosome and centrosome slightly stained. 



c. Stain in dilute aqueous solution of dahlia ; add dilute 

 methyl-green : nucleus green, centrosome and mitosome violet 

 (Fig. 16). 



