388 Special Procedures II (chap. 23) 



Kessel (1925) and Chen (1944A) Smears (modified) 



fixation: Schaudinn's (page 21), 40°C: 10-15 minutes or more. 



solutions: 

 Iron alum: 



ferric ammonium sulfate 4.0 gm. 



distilled water 100.0 ml. 



Hematoxylin. 

 Stock solution: 



hematoxylin 1.0 gm. 



absolute ethyl alcohol 10.0 ml. 



Allow to ripen several months or hasten process (page 128). 



Working solution: 



hematoxylin stock solution 0.5 ml. 



distilled water 99.5 ml. 



Add 3 drops of saturated aqueous lithium carbonate. If the hematoxy- 

 lin working solution looks rusty or muddy brown, it is unsatisfactory 

 and will not stain efficiently. 

 procedure: 



1. Transfer slides into 70% alcohol (from fixative): 2-3 minutes. 



2. Treat with Lugol's solution: 2-3 minutes. 



3. Wash in running water: 3 minutes. 



4. Decolorize with 5% sodium thiosulfate: 2 minutes. 



5. Wash in running water: 3-5 minutes. 



6. Mordant in iron alum, 40°C: 15 minutes. 



7. Wash in running water: 5 minutes. 



8. Stain in hematoxylin, 40°C: 15 minutes. 



9. Wash in running water: 5 minutes. 



10. Destain in iron alum 2% (dilute stock 4% with distilled water, 

 1:1) until nuclei and chromatoidal bodies are sharp against color- 

 less cytoplasm. Check under high dry objective. 



11. Wash thoroughly in running water: 15-30 minutes. 



12. Dehydrate, clear, and mount. 



results: 



nuclei, chromatoidal bodies — sharp blue black 



comments: 



Saturated aqueous picric acid may be used for destaining. Follow it 

 with a rinse in dilute ammonia (2-3 drops/ 100 ml. water) and thor- 

 ough washing in water. 



