Wright-Giemsa Stains Overview

Definition

• Wright-Giemsa Stains, also known as Romanowsky Stains, are utilized in hematology and cytological studies.
• Primarily used for differentiating cells in microscopic examinations of peripheral blood and bone marrow specimens.

Applications

• Can detect blood parasites, such as Plasmodium (malaria), for rapid results.
• Different stain types:
  • Giemsa stain
  • Wright and Wright-Giemsa stain
  • May-Grunwald stain
  • Leishman stain

Historical Context

• Named after Dmitri Leonidovich Romanowsky (1891) for his work in diagnosing hematological diseases using blood samples.
• James Homer Wright modified the stain in 1902 for better differentiation of blood cells.
• Gustav Giemsa further modified the stain in 1904, stabilizing it with glycerol, leading to the creation of the Wright-Giemsa stain.

Staining Principles

• Composed of oxidized methylene blue dyes and Eosin Y.
• Basic azure dyes bind to acidic nuclei producing blue to purple colors.
• Acid Eosin binds to alkaline cytoplasmic components producing red-orange or pink coloration.
• This results in the Romanowsky effect (metachromasia), allowing differentiation of cellular components.

Preparation for Staining

Blood Smear Technique

1. Materials Needed:

   • Two clean slides
   • Capillary tube or Diff-Safe blood dispenser
   • Blood sample (preferably from EDTA tube)

2. Slide Preparation:

   • Ensure slides are clean and free from oils.
   • Label slides with a chemical-resistant marker.
   • Place a small drop of blood near the frosted end of one slide.
   • Use a clean spreader slide to create a thin smear (one cell thick).
   • Allow the smear to air dry.

Stain Preparation

• Perform all staining steps in a fume hood or well-ventilated area.

• Prepare three containers for staining:

  • Container 1: Wright-Giemsa stain
  • Container 2: Hematology buffer solution
  • Container 3: Hematology rinse solution

• Maintain reagent freshness and replace solutions when discoloration occurs.

Staining Procedure

1. Fixation:
   • Fix slides using absolute methanol for 30 seconds, air dry (do not use heat).
2. Staining:
   • Dip slide in Container 1 (Wright-Giemsa stain) for 60 seconds (15-30s for Rapid Wright’s One-Step Stain).
3. Draining:
   • Drain excess stain without touching the smear.
4. Buffering:
   • Dip in Container 2 (buffer) for 60 seconds (15-45s for Rapid stain).
5. Rinsing:
   • Dip in Container 3 (rinse solution) for 2-10 seconds (25 seconds for Rapid stain).
6. Drying:
   • Dry the slide vertically, avoiding contact with the smear.
7. Microscopy:
   • Examine under high dry lens (40x), switch to oil immersion lens for 1000x magnification.

Final Notes

• Staining Quality:
  • The overall color of red blood cells is an indicator of stain quality.
• Supplies Available:
  • Wright-Giemsa stain kits, methanol, Coplin jars, premium slides, and other lab supplies can be obtained from Hardy Diagnostics.
• Educational Resources:
  • YouTube channel with tutorials and product videos.

Company Information

• Hardy Diagnostics is employee-owned and has been serving microbiologists for over 40 years.