SPERMATOGENESIS OF AMERICAN CRAYFISH 589 



3. Continue washing for twenty-four hours in 2 grams potas- 

 sium bichromate dissolved in 100 cc. water. 

 »4. Then wash for twenty-four hours in water and run up 

 through alcohols to xylol. Infiltrate with paraffin and imbed. 



5. Cut sections 5^ thick, mount on slides and then run down to 

 distilled water. Place into a 4 per cent iron-alum solution (at 

 room temperature) for twenty-four hours. 



6. Wash thoroughly in water until all trace of the iron-alum 

 has been removed and then transfer to a solution of sulfoali- 

 zarinsauren natron (made by taking one part of a saturated 

 aqueous solution of the stain to from 80 to 100 parts of distilled 

 water) for twenty-four hours. 



7. Rinse in distilled water and place slide into a krystalviolet- 

 anilin water solution [consisting of equal parts of a 3 per cent 

 alcoholic solution of krystalviolet (3 grams to 100 cc. 95 per cent 

 alcohol) , and anilin water] , and warm until the solution steams, 

 keeping it here for about three minutes. 



8. Wash in distilled water and transfer to 30 per cent acetic 

 acid for one or two minutes. Then rinse in distilled water which 

 removes the stain very rapidly. Continue washing in running 

 water from five to ten minutes so as to remove every trace of 

 acid. The alizarin stain appears reddish. 



9. Dry the slide with filter paper and dip for a moment into 

 absolute alcohol. Next place into bergamot oil until cleared, 

 and thence into xylol. Then mount in Canada-balsam. 



By this method the chromatic or nuclear elements are stained 

 a beautiful purple with the krystalviolet, while the cytoplasm is 

 stained a light reddish color with the sulfoalizarinsauren natron. 



Living testicular cells were also studied under the oil immersion. 

 This was accomplished by teasing out the cells in Ringer's solu- 

 tion and preparing hanging drop cultm-es of the same. Methy- 

 lin blue and lichtgriin were used as intravitam stains for study- 

 ing the details of these live cells. 



Smear preparations were also made. They were fixed in 

 either Bouin's, Carnoy's or Gilson's fluids and stained with 

 Heidenhain's iron-hematoxylin and acid fuchsin. Bouin's fluid 



