358 Special Procedures I (chap. 22) 



Note: EA6'y is the same, except for the light green content; 0.25% in 

 95% alcohol is used. This gives a lighter, more transparent stain, 

 which sometimes is desirable in smears containing much mucus. Dif- 

 ferentiation between acidophilic and basophilic cells is better, how- 

 ever, ^vith EA 36 (EA 50) and therefore is preferable for vaginal, en- 

 docervical, and endometrial smears. 



procedure: 



1. Out of fixative, hydrate slides to water. Rinse in distilled Avater. 

 A few seconds in each solution is adequate, or dip slides up and 

 down until their surface has a homogeneous appearance. 



2. Stain in Harris hematoxylin, either of following methods: 



(a) Papanicolaou dilutes Harris with an equal amount of dis- 

 tilled water: 8 minutes. 

 (h) Harris hematoxylin without acetic acid: 4 mintites. 



3. Wash in tap water, running water if flowing only slightly; do not 

 Avash off or loosen parts of smear: 3-5 minutes. 



4. Differentiate nuclei in 0.5% hydrochloric acid in 70% alcohol 

 (0.5 ml./lOO ml.) tnitil nuclei are sharp against a pale bltie cyto- 

 plasm. 



5. Wash in slightly running tap water: 5 minutes or until nuclei are 



a clear blue. 



6. Rinse in distilled water. 



7. Transfer through 70%, 80%, and 95% alcohol: few seconds in 

 each or until surface appears homogeneous. 



8. Stain in OG 6: 1-2 miniUes. 



9. Rinse in 95% alcohol, 3 changes: few seconds each. 



10. Stain in EA 36 (or EA 50): 2-3 minutes. 



11. Rinse in 95% alcohol, 3 changes: few seconds in each. 



12. Dehydrate in absoliUe alcohol, 2 changes: 1 minute each. 



13. Clear and mount. 



results: 



nuclei — blue 



acidophilic cells — red to orange 

 basophilic cells — green or blue green 



cells or fragments of tissue penetrated by blood — orange or orange 

 green 



comments: 



Papanicolaou cautions against agitating slides excessively, or crowd- 

 ing them while staining. If parts should float off from a positive slide 

 onto a negative one, a false positive can occur. 



