Lapis ISSN 2398-2969

Staining technique: Papanicolaou

Synonym(s): Pap

Contributor(s): Molly Varga, Sarah Pellett


  • Papanicolaou was originally associated with cervical screening, staining cervical cells in three stages with competing dyes.
  • The Papanicolaou stain is also used for clinical material and is normally used to stain bodily secretions, fine needle aspirates and touch preparations.
  • Two dyes are used that differ in their affinity for various sites within a cell, ie nucleus and cytoplasm.
  • Different to Romanowsky stains in that there is no chemical interaction between nuclear and cytoplasmic solutions. They bind electrostatically, resulting in salt formation.
  • Similar to hematoxylin and eosin, except that eosin is replaced with two cytoplasmic counterstains. This highlights keratinization in squamous epithelium.
  • The important elements of Papanicolaou staining methods are nuclear staining followed by orange and polychromic cytoplasmic staining (hematoxylin stain + orange stain + polychromic stain = Papanicolaou stain).
  • Using this method, cells are highly transparent allowing diagnosis, even when cells are overlapping and when mucus or inflammatory cells are present.
  • The staining method can be applied either manually with the aid of cuvettes, or mechanically in automated staining systems.


  • Used in special diagnostic situations, especially where neoplasia is suspected.
  • Provides architectural information for cytopathologists and allows nuclear detail to be visualized even in thick smears and tissue fragments.


  • Provides excellent detail of nuclear and nucleolar changes.
  • Unlike other stains, it highlights cytoplasmic keratin; stain appears orange to orange-red.
  • Wet fixation causes red cell lysis, improving visual examination of clumps of cells; useful for smears heavily contaminated in blood.


  • Compared with Romanowsky stains, cytoplasmic detail and organisms, including bacteria, are not well defined.
  • Cells must be immediately wet fixed in alcohol or by using a spray fixative, unless use modified Papanicolaou where smears are rehydrated.
  • Slides must not be allowed to air-dry between steps or between staining and coverslip mounting.
  • Adhesive-coated slides (poly-l-lysine, gelatin) may aid cell retention.
  • Time consuming; not very practical for a veterinary practice laboratory.


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Further Reading


Refereed papers

  • Recent references fromPubMedandVetMedResource.
  • Cray C & Zaias J (2004)Laboratory procedures.Vet Clin Exotic Anim Pract7(2), 487-518PubMed.
  • Jorundsson E, Lumsden J H & Jacobs R M (1999)Rapid staining techniques in cytopathology: A review and comparison of modified protocols for haematoxylin and eosin, papanicolaou and romanowsky stain.Vet Clin Pathol281(3), 100-107PubMed.

Other sources of information

  • Merck KGaA (2013)Cytodiagnosis Staining Methods.Merck Millipore Manual.
  • Dunn J K & Gerber K (2005)Diagnostic Cytology. In:BSAVA Clinical and Feline Clinical Pathology. 2nd edn. Eds: Villiers E & Blackwood L. BSAVA, Gloucester. pp 305-339.
  • Dacia Sir J V & Lewis S M (2001)Practical Haematology. 6th edn. Longman Singapore Publishers Pte Ltd. pp 50-61.


  • Mary Pinborough, Pinmoore Animal Laboratory Services Ltd, The Coach House, Town House Barn, Clotton, Cheshire CW6 0EG, UK. Tel: +44 (0)1829 781855; Email: ; Website: .