Papanicolaou Staining

• Modifications of the papanicolaou staining

• Haematoxylin
• Papnicolaou technique I: uses Harris hematoxylin
regressively.
• In this technique the cells are intentionally over stained &
excess hematoxylin is removed by differential extracation
in HCl
Staining
• Papanicolaou staining technique II :
Mainly used for urinary & gastric preparations .
It uses haematoxylin progreesively .in this technique
differential extraction in HCl is ont necessory
Why HCl differential extraction is not
necessory?
• Reduction Staining time prevents over staining of
the cytoplasm.

• Mayers hematoxylin & gills hematoxylin

rarely over stain the smear regardless of the time
so they are used in progressive staining
technique
Progressive staining is recommended for cell
samples that do not adhere well to glass slide
Staining
• Eosin & alcohol Stain:EA-36,EA-50 mainly used.
• Each contain twice the amount of light green used in EA-
65
• The increased amount of LG in EA-36 & 50 tend to stain
the back ground of thick non gynecological smear to
intensely & for such smear EA- 65 is preferred.

• OG-6 counterstain. The Orange G is used to stain keratin.

Its original role was to stain the small cells of
keratinizing squamous cell carcinoma present in sputum.
Staining
• Bluing:Ammonium hydroxide in 70% alcohol
• Lithium carbnate
• Scott’s tap water
• Hydration :Series of graded alcohol
• It is to prevent cell disortion
Papnicolaou stainingProcdure
• The slides are removed from the Fixative(95%ehyl
alcohol). Then rehydrated by successively transferring
them to
• 80% Ethylalcohol-10 dips
• 70% Ethyl alcohol- 10 dips
• 50% Ethyl alchohol-10 dips
• Nuclear staining :
Harris haematoxylin : 6minutes
Rinse in tap water/ 2 Distilled water rinse -10 dips each
Staining
• Differential Extraction:
• 0.5% aqueous solution of HCl.-6dips
• Bluing : Running tap water-6minutes
• Hydration:
• 50% ethyl alcohol:10 dips
• 70% ethyl alcohol:10 dips
• 80% ethyl alcohol:10 dips
• 95% ethyl alcohol: 10 dips
• Orange G:
• 11/2 minutes
• Cytoplasmic stain:
• EA 36, 50, 65-11/2 minutes
• Rinse
• 95% ethyl alcohol-10 dips
• 95% ethyl alcohol- 10 dips
• 95% ethyl alcohol -10 dips
• De hydration:
• Absolute ethyl alcohol
• Absolute ethyl alcohol- 10 dips each
• Equal parts of absolute ethyl alcohol& xylol-10 dips
• Clearing:
• Xylol 5-2 minutes
• Xylol 5-2 minutes
• If xylol turns milky , the slides have not been throughly
dehydrated, return to absolute alcohol.
• Mount in mounting medium
RAPID-PAP stain kit is most useful at times when a cytology decision is
demanded instantaneously while at a surgical operative procedure or on mass
cytology screening camps., which requires minimum skill and laboratory aids to
achieve best staining result.

Rapid Papanicolaou Staining

• 1 95% Alcohol 1 minute • 12 EA-50 1 minute

• 2 95% Alcohol 10 dips • 13 95% Alcohol 10 dips
• 3 Distilled Water 10 dips • 14 95% Alcohol 10 dips 15
• 4 Hematoxylin Gill 2 1 minute 100% Alcohol 10 dips 16 100%
• 5 Distilled Water 10 dips
Alcohol 10 dips 17 Xylene 10
dips 18 Xylene 1 minute
• 6 Bluing Reagent 1 minute
• 7 Distilled Water 10 dips
• 8 95% Alcohol 10 dips
• 9 Orange-G-6 1 minute
• 10 95% Alcohol 10 dips
• 11 95% Alcohol 10 dips
Staining Interpretation

• Nuclei Blue
• High Keratin Cells Orange
• Superficial Cells Pink
• Erythrocytes Dark Pink
• Para basal Cells Blue/Green
• Intermediate Cells Blue/Green
• Metaplastic Cells May contain both Blue/Green and Pink
• Control Tissue: Gynecological Smear; any Superficial Cell
Smear