250 Cytoplasmic Elements (chap. 18) 



7. Rinse in several changes of distilled water. 



8. Tone in oold chloride: 10 minutes. 



9. Rinse in distilled water. 



10. Fix in sodium thiosulfate, 5% (5 gm./lOO ml. water): 3 minutes. 



11. Wash in running water: 5 minutes. 



12. Coimterstain in safranin (or other red nuclear stain). 



13. Rinse in 70% alcohol: few seconds. 



14. Dehydrate, clear, and mount. 



results: 



argentaffin granules — black 



melanin — black 



other tissue elements — reds and pinks 



grayish to blackish background — slide is overstained 



comments: 



Chromaffin material is found only in the adrenal medulla and the 

 gastrointestinal tract, and received its name because of its reaction 

 with chromium salts and certain other metals to produce a yellowish 

 to brown color, a brownish chromiinn oxide. A dichromate fixative 

 should be used to preserve this material, or post-chromating with po- 

 tassium dichromate, page 23. Chromaffin is also argentaffin and is 

 blackened in the above method. Melanin, also brownish, is not a 

 chromaffin substance. 



Diazo-safranin Method (lillie et al., 1953) 



fixation: 10% formalin buffered with 2% of calcium acetate: 1-3 days. 



solution: 



Diazotized safranin: 



Stock solutions: 

 Acid safranin: 



safranin O, C.I. 50240 3.6 gm. 



distilled water 60.0 ml. 



N HCl 30.0 ml. 



Stable for several weeks. 



N sodium nitrite: 



sodium nitrite, NaNOo 6.9 gm. 



distilled water 100.0 ml. 



Keep in refrigerator, stable for 3 months. 



