Papanicolaou stains : Principle ,Procedure and Interpretation

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Papanicolaou stain : Principle ,Procedure and Result Interpretation

Papanicolaou stains are used to differentiate cells in smear preparations of various body secretions. Pap stain is a very reliable technique. As such, it is used for cervical cancer screening.Malignant cells are generally characterized by relatively large nuclei with an abnormal chromatin pattern and small amounts of cytoplasm. They are characterized by the presence of prominent nucleoli, a marked variation in size, shape and staining properties and are usually hyperchromatic and by atypical or irregular mitosis.Cancer cells can be found in the imprints of puncture biopsy material and cervical cell smears, vaginal secretion, prostatic secretion, urine, gastric contents, bronchial aspirations, cavity fluids and sputum.A diagnosis of malignancy from stained smears should be considered tentative and tissue sectioning should be checked.

Papanicolaou- EA is Eosin Azure, consisting of three dyes; the number indicates the proportion of the dyes, e.g.EA-36, EA-50 ( use for routine diagnostic cytology to aid in the identification and classification of exfoliative cells.) and EA-65 .This group of reagents provides excellent cytoplasmic stain for gynecological and non- gynecological samples.The wide range of formulations available allows the end user to choose from different color intensities and colours.

Principle of Papanicolaou stains EA-36

Papanicolaou Stains are used in conjunction with Hematoxylin nuclear stains to diagnose malignant cytological diseases.In conjunction with OG-6 ( a multichromatic cytological stain), EA-36 and EA-50 are used for gynecological staining.EA-65 is used for non- gynecological staining with OG-6. When properly performed, the stained specimen should display hues from the entire spectrum: red, orange, yellow, green, blue and violet.The chromatin patterns are visible, the cells from borderline lesions are easier to interpret and the photomicrographs are better.

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The staining results in very transparent cells, so that even thicker specimens with overlapping cells can be interpreted. The cell nuclei are crisp blue to black on a well prepared specimen.Cells with a high keratin content are also yellow, and glycogen stains are yellow.Superficial cells are orange to pink, and intermediate and parabasal cells are turquoise to blue.Metaplastic cells often stain green and pink at once.

Composition of Papanicolaou stains EA-36

Light green45.0 gm
Bismark brown10.0 gm
Eosin Y 45.0 gm
Phosphotungstic acid0.20gm
Lithium carbonate, saturated aqueous solution1drop

Composition of Papanicolaou OG6 stains

Orange G-6 Certified 0.3 gm
Phosphotungstic acid0.015gm
Denatured alcohol 100.0ml

Composition of Papanicolaou E 50 stains

Eosin Y Certified 0.23gm
Bismark Brown Certified0.05gm
Fast green FCF Certified 0.08gm
Phosphotungstic acid0.2gm
Denatured alcohol 100.0ml

Appearance of various Papanicolaou dyes

Papanicolaou E 50 stainsAppear dirty green coloured solution without any precipitate
Papanicolaou E-50 stainsGreen coloured solution with reddish purple tinge. May contain fine particles
Papanicolaou OG6 stains
It has an orange coloured solution without any precipitate
papanicolaou stains
papanicolaou stains

Papanicolaou staining Procedure

Fixation: Do not allow smears to dry and immediately fix in 95 percent alcohol for 5- 15 minutes. If necessary, the smear may be left in the fixative for 3 days, but prolonged fixation affects the staining reaction.

  1. Rinse in 70 percent alcohol, 50 percent alcohol and distilled water.
  2. Stain in Harris haematoxylin (without acetic acid) for 5 -10 minutes.
  3. Rinse in distilled water
  4. Rinse 3 or 4 times in 0,5% aqueous hydrochloric acid solution.
  5. Thoroughly rinse in water.
  6. Leave in a weak solution of lithium, carbonate for 1 minute( 3drops saturated aqueous solution / 100 ml of water).Rinse thoroughly in water.
  7. Rinse in distilled water, 50% alcohol, 70% alcohol, 80% alcohol and 95% alcohol.
  8. Stain in the Papanicolau Orange G-6 solution for 1 minute.
  9. Rinse 5- 10 times in 2 jars each containing 95 percent alcohol.
  10. Stain for 2 minutes in Papanicolau EA36 OR in Papanicolau EA50
  11. Rinse 5- 10 times in each of the three jar containing,95 percent alcohol (not the same alcohol used after G-6 orange solution).
  12. Rinse in absolute alcohol, then in a mixture of equal parts of absolute alcohol and xylene, then in xylene.
  13. Mount in any satisfactory neutral medium and observe under microscope
Also read  Properties and Principles of Hematoxylin & Eosin staining technique

Microscopic examination

Staining is performed and the staining characteristics of the organisms are observed under a microscope using an oil immersion lens.


When stained with Papanicolaou EA-50, cytoplasm appears with various shades of blue, pink, orange, red and green. Younger cell appear with green shades.

When stained with Papanicolaou EA-36, cytoplasm appears pink to pale pink while nuclei appears blue

Nuclei blue
Cytoplasmpink to pale pink
pap smear
pap smear


  • Carson, Freida L; Hladik, Christa (2009). Histotechnology: A Self-Instructional Text (3 ed.). Hong Kong: American Society for Clinical Pathology Press. pp. 361–3363. ISBN 978-0-89189-581-7

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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