PAPANICOLAOU TECHNIQUES 



257 



PAPANICOLAOU TECHNIQUES 



Method II. This is the technique 

 described by Papanicolaou in Science, 

 1942, 95, 438-9, with a few minor modifi- 

 cations. It is recommended for stain- 

 ing smears of the female genital tract 

 which are to be used for either cancer 

 diagnosis or endocrine studies. 

 L Transfer slides, without drying, 

 from alcohol-ether fixative to 80% 

 alcohol and run down through 70% 

 and 50% alcohols to aq. dest., leaving 

 in each solution long enough to 

 clear. 



2. Stain in Harris Hematoxylin (1) 

 6 min. 



3. Rinse in aq. dest. 



4. Dip in 0.25% aq. hydrochloric acid 

 6 times. 



5. Place in running tap water 6 min. 



6. Rinse in aq. dest. and run up 

 through 50%, 70%, 80%, and 95% 

 alcohols, leaving in each long 

 enough to clear. 



7. Stain in 0G6 (2) for U minutes. 



8. Rinse in 95% ale, two changes. 



9. Stain in EA36 (3) for H min. 



10. Rinse in 95% alcohol, 3 changes. 



11. Dehydrate and clear by running 

 through absolute ale, absolute 

 alcohol and xylol (equal parts) to 

 xylol, leaving in each solution long 

 enough for the smears to be thor- 

 oughly penetrated and dehydrated. 



12. Mount, without drying, with a 

 coverslip, using permount, gum 

 damar, Canada balsam or another 

 neutral mounting medium. 



Nuclei — dark purple; non-cornified 

 cells — pale greenish-blue; cornified cells 

 — variations from pink to orange, de- 

 pending on the degree of cornification; 

 keratinized cells — intense orange. 



Method III. A modification of 

 method II. Recommended for smears 

 of sputum, prostatic secretion and all 

 sediments except urine. 



1-8. Same as steps 1-8 or method II. 

 9. Stain in EA65 (4) 1| min. 

 10-12. Same as steps 10-12 of method II. 



Note: Rectal and colonic washings 

 are washed in running tap water (step 

 5) 15 min. instead of 6 min. 



Method IV. A modification of 

 method III. In this procedure blueing 

 of the nuclei is accomplished by the 

 use of ammonium hydroxide rather 

 than running tap water, as the latter 

 tends to loosen cells from the slide. 

 It is therefore recommended for smears 

 of urine sediment which is the least 

 adhesive of the sediments. It is also 

 shorter than method II and may be 

 used for other types of smears for that 

 reason. However, in thick smears, it 

 has a disadvantage in that the cyto- 

 plasm retains some hematoxylin and 



therefore does not take a clear cyto- 

 plasmic stain. 



1. Transfer slides from alcohol-ether 

 to 80% alcohol and run down through 

 70% and 50% alcohols to aq. dest. 



2. Stain in Harris hematoxylin (1) 2 

 min. 



3. Rinse in distilled water, then 50% 

 ale. 



4. Place in a solution of 1.5% ammo- 

 nium hydroxide made up in 70% 

 alcohol for 1 min. 



5. Rinse in 70% alcohol, 2 changes. 



6. Run up through 80% and 95% ale. 



7. Stain in 0G6 (2) for U min. 



8. Rinse in 95% alcohol, 2 changes. 



9. Stain in EA65 (4) for U min. 

 10-12. Same as steps 10 to 12 in method 



II. 

 Method V. For sections. 



1. Remove paraffin in xylol and run 

 down through descending alcohols 

 to aq. dest. in the usual way. 



2. Stain in Harris hemato.xylin (1) 2^ 

 min. 



3. Rinse in aq. dest. (2 changes), and 

 50% and 70% alcohols. 



4. Place in 3% ammonium hydroxide 

 made up in 70% ale. for 1 min. (or 

 until sections turn blue). 



5. Rinse in 70% alcohol (2 changes), 

 80% and 95% ale. 



6. Stain in 0G6 (2) for If min. 



7. Rinse in 95% alcohol (2 changes). 



8. Stain in EA65 (4) or EA36 (3) for 

 1^ min. 



9. Rinse in 95% alcohol (3 changes), 

 dehydrate, clear and mount in the 

 usual way. 



Formulae for Stains. 



(1) Harris hematoxylin is prepared from the 

 standard formula, using ammonium alum- 

 inum sulphate, but omitting the glacial ace- 

 tic acid. It is diluted with an equal volume 

 of distilled water before using, and filtered 

 into a dark bottle for storage when not in use. 

 It should be reinforced by the addition of a 

 small amount of fresh undiluted stock solu- 

 tion fairly often in order to maintain uniform 

 staining results. 



(2) 0G6: 



Orange G— 0. 5% in 95% alcohol . 100 cc. 

 Acid phosphotungstic 0.015 gm. 



(3) EA36: 



Light Green SF (yellowish) — 

 0. 1% in 95% alcohol* 45 cc. 



Bismarck Brown Y— 0.5% in 

 alcohol 10 cc. 



Eosin yellowish— 0.5% in 95% 

 alcohol 45 cc. 



•Original formula for EA36 calls 

 for 0.5% light green solution 

 but 0. 1% is now used 



