ISSN 2398-2977      

Staining techniques: New Methylene Blue stain

pequis

Introduction

Rationale
  • New Methylene Blue (NMB) is a supravital stain which when mixed with whole blood, stains RNA and DNA deep blue.
  • Reticulocyte count: RNA in cytosol of immature RBC is precipitated into strands and clumps, staining deep blue when mixed with NMB.
  • Heinz bodies: denatured, precipitated, hemoglobin which when stained with NMB are seen as spherical pale blue structures found at the periphery of erythrocytes.

Uses

  • Wet mount: identify Aggregate reticulocytes for count (seen as erythrocyte ghosts containing blue to purple granular material); but punctate reticulocytes not demonstrated.
  • Dry: identify reticulocytes; Heinz bodies and platelets.

Pros

  • Prepared stain stored in a dark evaporation free bottle will keep indefinitely.
  • Good adjunct to Romanowsky stains, since it stains cytoplasm weakly, but gives excellent nuclear and nucleolar detail.
  • Can often see nuclear detail in clumped cells, because NMB does not stain cytoplasm well.

Cons

  • Reticulocyte-stained films fade when stored.
  • Hemodilution not a marked problem, since RBC do not stain with NMB or they only develop a pale blue tint.

Alternatives

  • Reticulocyte stains (0.5 g of NMB mixed with 1.6 g of potassium oxalate in 100 ml of distilled water).

Work time

  • 23 min.

Preparation

  • 18 min staining.

Procedure

  • +/- 5 min depending on cell type and number that need to be identified.

Requirements

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Method

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Result data

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Storage

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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 13-14.
  • Pratt P W (1997) Laboratory Procedures for Veterinary Technicians. 3rd edn. Mosby. pp 574-578.
  • Lewis H B & Rebar A H (1979) Bone Marrow Evaluation in Veterinary Practice. Ralston Purina Co, USA. pp 60-61.

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