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Staining techniques: Wright's stain

ISSN 2398-2950


Synonym(s): Romanowskys stain

Introduction

  • Romanowsky stains Staining techniques: Romanowsky-type stains are neutral stains composed of a mixture of oxidized methylene blue (azure) dyes and Eosin Y.
  • The azures are basic dyes that bind acid nuclei and result in a blue to purple color.
  • The acid dye, eosin, is attracted to the alkaline cytoplasm, producing red coloration.

Uses

  • Hematology and cytology.

Advantages

  • If 'hemofast' is used, it contains neutral methyl alcohol, ie does not contain acetone, therefore there is no need for prior fixing of the smeared slides.

Disadvantages

  • Must use distilled water for 'hemofast'.
  • Cannot use tap water since the latter contains interfering substances, particularly calcium which can cause precipitation of eosin to an insoluble calcium salt.

Alternative techniques

Procedure

  • 5 to 15 minutes depending on whether the stain has been prepared or is ready mixed.

Requirements

Materials required

Ideal equipment

  • Coplin jars, light microscope.

Minimum consumables

  • Distilled water, clean microscope slides, Wright's stain and buffer or Hemofast.

Preparation

Technique

Approach

Step 1 - Wright's stain

  • Prepare Wright's stain and Wright's buffer or use commercially prepared solutions, eg Hemofast by Mascia Brunelli.

Step 2 -

  • Prepare thin blood smears or cytology preparations, air dry quickly.
  • Cover slide with Wright's stain.

Step 3 -

  • Leave immersed for 3 minutes.
  • Add an equal volume of Wright's buffer.

Step 4 -

  • Gently rock the slide to mix the solutions until a green metallic sheen forms on the surface of the mixture.
  • Leave stain - buffer solution on for 6 minutes.

Step 5 -

  • Rinse under running tap water.
  • Air dry.
  • Examine with light microscope as recommended for blood film Blood smear.

Core procedure

Step 1 - Manual staining with Hemofast

  • Prepare Wright's stain and Wright's buffer or use commercially prepared solutions, eg Hemofast by Mascia Brunelli.
  • Prepare thin blood smears or cytology preparations, air dry quickly.

Step 2 -

  • Fill a coplin jar with hemofast stain.

Step 3 -

  • Immerse the slides for 5 minutes at room temperature.
  • Gently rinse slides under running distilled water.

Step 4 -

  • Place slides in another coplin jar filled with distilled water for 10 minutes.

Step 5 -

  • Remove slides and allow to drain and dry.
  • Examine with light microscope.

Aftercare

Immediate Aftercare

Monitoring

  • Colors of cells/components vary slightly with the variation of pH of the distilled water (hemofast stain).

Potential complications

  • Under or overstained preparations: need to adjust method (staining times) slightly according to the total protein concentration (of fluid) and thin/thickness of smear, eg thick smears and fluids with high protein concentration need increased staining time.
  • Excessive blue staining/tint: preparation too thick, one of the stages are too alkaline or exposure to formalin fumes.
  • Excessive pink/magenta tint: any of the preparation stages are too acidic, heparin used for anticoagulants (color due to presence of mucopolysaccharides)
  • Stain precipitation: dirty slides, inadequate stain filtration or inadequate washing.  Can be misinterpreted as parasites, inclusion bodies, dohle bodies or cytoplasmic granules.
  • Refractile artifact: especially with red blood cells
    in diffquick due to moisture in the fixative. Interpreted as artifacts.

Outcomes

Reasons for treatment failure

  • Must be stored in dark (non-metal) container in a dry area, since light and  humidity alter hemofast.
  • Poisonous and flammable (therefore store away from heat sources).

Further Reading

Publications

Refereed papers

Other sources of information

  • Lewis H B & Rebar A H (1979) Bone Marrow Evaluation in Veterinary Practice. Ralston Purina Company, USA. p 59.

Organisation(s)

  • Mascia Brunelli S.P.A.package insert for Hemofast, Viale Monza 272-20128 Milano, Italy. Tel: 02/25:51.641 - telex 330838 Mabios 1 - Telefax 02/2576428.