Journal of Applied Microscopy. 



ing the late spring and summer months. 

 Karyokinesis may be found well into 

 August, though not abundantly. The 

 males are easily distinguished by the 

 greater size of the hind legs and the 

 broad caudal fin-fold. The portion of 

 the testis containing cells is a translu- 

 cent gray, while that in which the lobules 

 are filled with ripe spermatozoa is an 

 opaque white or yellow-white, as Flem- 

 ming pointed out in Salamandra, there 

 being in neither form pigment to ob- 

 scure the effect. 



Amblystoma (punctatum in the East, 

 tigrinum in the central portions) also 

 mates in the early spring (March or 

 April), and presumably in the stages of 

 spermatogenesis it corresponds in gen- 

 eral to Diemyctylus. Cell-multiplication 

 is still going on in early August, how- 

 ever. Amblystoma is not as easily ob- 

 tained as Diemyctylus, secluding itself 

 under logs and stones except at the 

 breeding season, when it is valueless for 

 karyokinetic purposes. 



Necturus (the mud-puppy or water- 

 dog of the vernacular) presumably must 

 likewise be taken in the early summer, 

 since individuals taken from early fall 

 to late winter show the testis filled with 

 ripe spermatozoa. It has been difficult 

 to obtain it here during late spring or 

 early summer. 



In addition to these salamanders, 

 which are available, and contrasting 

 with them, we have as occupants of the 

 brooks in the eastern United States, two 

 other genera of salamanders, Desmog- 

 nathus, the dusky salamander, and Spe- 

 lerpes. Little is known of the breeding 

 habits of these genera, but all evidence 

 that we have points to the fact that the 

 breeding season, instead of coming in 

 early spring, comes in late summer or 

 even late in the fall, eggs having been 

 found in October. tt At all events, lobules 

 in stages of karyokinesis are found in 

 specimens taken in early fall to mid- 

 winter, and probably, in the case of Des- 

 mognathus at least, up to April. In both 

 of these the testis are densely pigmented 

 and the regions of cells and spermatozoa 

 cannot be as easily distinguished as in 

 Salamandra or Diemyctylus. Sperma- 

 togenesis, however, seems to proceed 

 from the cephalic toward the caudal end, 

 causing an enlargement of the testis and 

 a diminution of the amount of pigment, 

 and it is in the narrower region, or where 

 the larger and smaller portions meet, 

 that cell-division is generally found. In 

 Desmognathus, there are sometimes (not 

 always) found two, rarely three, di- 

 visions of the testis, corresponding ap- 



ttSherwood, W. L. The Salamanders 

 found in the vicinity of New York City, 

 with notes on extra-limltal or allied 

 species. Proc. Linnaean Soc. of N. Y., No. 

 7, 1895; pp. 21-37. 



parently to two centers of spermatozoa 

 formation. 



Testes of Diemyctylus and Ambly- 

 stoma, then, are suitable for karyoki- 

 nesis during spring and early summer 

 (May, June, July); Desmognathus and 

 Spelerpes, on the other hand, may be 

 employed during fall and winter. Doubt- 

 less other genera belonging to the same 

 families will likewise be found equally 

 serviceable at the same seasons. 



The fixing fluids that experience has 

 shown to be most suitable are: Her- 

 mann's platino-aceto-osmic, (Formula: 

 Platinum bichlorid ten per cent. aq. sol. 

 three parts, one per cent, osmic acid sol. 

 sixteen parts, glacial acetic acid two 

 parts, water nineteen parts; or, take 

 platinum bichloride one per cent. sol. 

 fifteen parts, two per cent, osmic acid 

 four parts, glacial acetic acid one part); 

 Flemming's chromo-aceto-osmic mix- 

 ture (strong formula) is the same as Her- 

 mann's, save that chromic acid is sub- 

 stituted for the platinum bichloride), and 

 also picric acid. The method of their 

 use is as follows: It will be found best 

 to cut the testis through the middle of 

 the enlarged portion. Place both pieces 

 for twenty-four hours in an abundance 

 (15-20 cc. per testis) of either Hermann's 

 or Flemming's fluid; wash in running 

 water six hours or over night, and 

 harden in alcohols of fifty, seventy, and 

 eighty-two per cent, strengths. The 

 superficial layers of cells will be found 

 to be over-nxed and detail partly or 

 entirely lost; deeper cells will, however, 

 be satisfactory. A thorough washing out 

 of the fixer is important, that there be 

 no subsequent blackening of or precipi- 

 tate in the tissue. Sometimes, neverthe- 

 less, a precipitate occurs which may be 

 removed by bleaching for a few minutes 

 before staining, with a mixture of one 

 cc. of hydrogen dioxide solution in ten 

 or twenty cc. of seventy per cent, alco- 

 hol. Parafflne should be employed for 

 imbedding; the sections should be 

 between five and ten/^th in thickness 

 and be made longitudinally of the testis. 

 For staining, most serviceable will be 

 found Heidenhain's Iron Hematoxylin 

 with or without after-staining in orange 

 G, or safranin with or without light 

 green as a counter-stain. For the iron 

 hematoxylin (a) mordant for one hour 

 in a four per cent, aq. sol. of ferric alum 

 (iron-ammonium-persulphate), rinse well 

 in water one or two minutes, (b) stain one 

 to three hours or until black in a four- 

 tenths per cent. aq. hematoxylin (may be 

 conveniently made up by taking three 

 cc. of a sixteen per cent, alcoholic stock 

 solution of hematoxylin in one hundred 

 cc. of water.) Any aqueous hematoxylin 

 may be taken, however, the time of 

 staining being longer for weak formulas. 



