"60 Special Procedures I (chap. 22) 



Calcium chloride, M/10: 



calcium chloride 11.0 gm. 



distilled water lOUO.O ml. 



..'Rocedure: 



1. Smears are fixed by Papanicolaou method, ether-95% alcohol: at 

 least 15 minutes. 



2. Hydrate to water: few seconds in each solution. 



3. Rinse briefly in acetic acid, 1% (1 ml./99 ml. water), followed by 

 wash in distilled water: few minutes. 



4. Stain in acridine orange: 3 minvues. 



5. Destain in phosphate buffer: 1 minute. 



6. Differentiate in calcium chloride: 30 seconds. Wash with buffer. 



7. Repeat step 6. If smear is very thick, a longer differentiation may 

 be necessary. Nuclei should be clearly defined. 



8. Wash thoroughly with phosphate buffer. Calcium chloride must 

 be removed. 



9. Blot carefully and mount with a drop of buffer. 



Frozen Sections, Method I. 



1. Wash out ether-alcohol, freeze, and cut. 



2. Omit step 2 and proceed as usual. Use small shallow dishes, mount- 

 ing sections on slide after staining. 



Frozen Sections, Method II. 



1. Section immediately after biopsy or wrap tissue in saline-soaked 

 gauze and wax paper surrounded by ice. Keep in refrigerator, 4°C 

 until processed. Cut, transfer to slides, and fix in alcohol or Car- 

 noy's fluid. 



2. Hydrate and stain. 



results: 



DNA of chromatin of nucleus — gTeen from whitish to yellowish hues 



connective tissue fibrils, cornified epithelia; structures — greenish 



mucus — dull green 



leukocytes — bright green 



RNA of basophilic structures of cytoplasm, main portion of nucleoli 



— red and orange fluorescence 

 sites of strongly acidic groups — deep carmine 

 malignant cells under low power — brilliant flaming red-orange 

 proliferating malignant cells under high power — intense fluorescence 

 cytoplasm — flaming red-orange containing reddish granules, 

 patches, or fine granules 



