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Staining techniques: Perl's Prussian Blue – Safranin reaction

ISSN 2398-2977


Synonym(s): Perl's, Prussian Blue

Podcast: Staining techniques: Perl's Prussian Blue / Safranin reaction

Introduction

Rationale
  • Hemosiderin combines with potassium ferrocyanide to form a colored pigment potassium ferric ferrocyanide.
  • Iron - positive material stains blue, rest of cell and background stain dark pink (contrast).

Uses

  • Hemosiderin - in macrophages in tracheal wash/BAL.

Cons

  • 1% acidic potassium ferrocyanide solution only has a shelf-life of a few hours, therefore must always be made fresh.
  • Biohazard since need to store strong acids in clinic/laboratory.

Work time

Preparation

  • 5 min.
  • 2% HCl = 500 ml distilled H2O + 28 ml concentrated HCl.
  • 0.1% Safranin solution (1% acidified potassium ferrocyanide) = 2% HCl + 2% K ferrocyanide.

Procedure

  • 40 min stain (overnight + 40 min).
  • 2 min microscope identification.

Requirements

People competence
  • Veterinary: high.
  • Nursing: high.

Instrumentation

  • Equipment: minimum - Coplin Jars; 100 X oil immersion objective.

Consumables

  • Minimum: 28% HCl + 2% potassium ferrocyanide (keep in brown bottle in the dark) to make 1% acidified potassium ferrocyanide solution and 1% Safranin aqueous solution (1 g 100 ml distilled water).

Method

  • Fix smeared slides in a Coplin jar filled with methyl alcohol for a minimum of 10 min (overnight preferable).
  • Then place in fresh 1% acidified potassium ferrocyanide solution for 10 min.
  • Gently wash in running tap water for 10 min.
  • Shake off excess water.
  • Dip smears into 1% Safranin counter stain solution for 10 s.
  • Rinse smears in tap water.
  • Dry.
  • Examine under 100 X oil immersion objective.

Result data

  • Pathological: indicates previous hemorrhage if iron is identified in macrophages from body fluids or bronchoalveolar lavage.

Storage

  • 2% potassium ferrocyanide must be kept in a brown bottle, stored in a dark cupboard to keep it stable.

Specificity

  • Good.

Further Reading

Publications

Refereed papers

Other sources of information

  • Harvey J W (2001) Blood and Bone Marrow of Domestic Animals. In: Atlas of Veterinary Hematology. W B Saunders. pp 4-15.
  • Lewis H B & Rebar A H (1979) Bone Marrow Evaluation. In: Veterinary Practice. Ralston Purina Co, USA. pp 64-65. ISBN 78-6855 6.