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Staining technique: new methylene blue stain

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Synonym(s): NMB

Introduction

  • New methylene blue (NMB) is a supra vital stain that is now rarely used as a cytological stain.
  • It is an aqueous stain that allows instant examination after application to air-dried cells.
  • Mixed with whole blood, RNA and DNA stain deep blue.
  • Reticulocyte count: RNA in cytosol of immature RBC stains deep blue when mixed with NMB.
  • Heinz bodies: denatured, precipitated hemoglobin is seen as spherical pale blue structures found at the periphery of erythrocytes.

Uses

  • New methylene blue stain is used for reticulocyte staining and counting procedures.
  • It can also be used to examine cytology specimens.

Advantages

  • Provides excellent nuclear and nucleolar detail.
  • NMB stains cytoplasm weakly so nuclear detail can be visualized in clumped, thick, hemodiluted smears.
  • Prepared stain, if stored in a dark evaporation free bottle will keep indefinitely.

Disadvantages

  • Precipitate often forms requiring regular filtering of stains.
  • Stained reticulocyte films often fade when stored.
  • NMB does not stain RBCs, therefore hemodilution is not a serious problem.
  • NMB is toxic, therefore skin exposure should be avoided.

Alternative techniques

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

Time required

Procedure

  • 15-20 min to stain, plus 5 min to examine slide.

Requirements

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Preparation

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Technique

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Aftercare

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Outcomes

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Further Reading

Publications

Refereed papers

Other sources of information

  • Campbell T W (2015) Stains and Solutions Used in Hematology and Cytology. In: Exotic Animal Hematology and Cytology. 4th edn. Wiley-Blackwell, USA. pp 378.
  • Dunn J K & Gerber K (2005) Diagnostic Cytology. In: BSAVA Clinical & Feline Clinical Pathology. 2nd edn. Eds: Villiers E & Blackwood L. BSAVA, UK. pp 305-339.