Giemsa, Wright, Romanowsky for Smears

Reagents for this procedure are sold as individual stain solutions and are available for purchase under separate part numbers with storage requirements and expiration date designated per bottle.

Product Options:

Part # Non-Kit

SOLUTIONS: 500 ml 6 X 500 ml 1 Liter 1 Gallon
Giemsa Stock Stain, Romanowsky Part 11215A Part 11215A    

 

Additionally Needed:             

Alcohol, Methanol Anhydrous, ACS Part 12236
Wright Stain Buffer, pH 6.8 Part 1430

                                                                                            

For storage requirements and expiration date refer to individual bottle labels.

 

APPLICATION:

Newcomer Supply Wright-Giemsa, Romanowsky Stain for Smears is deemed the classic Wright-Giemsa stain for hematology. It is designed to demonstrate differential staining of cell types in peripheral blood smears and bone marrow smears/films as well as a method for detecting parasites, bacteria, and inclusion bodies.

A Romanowsky-type stain refers to a stain made from water-soluble eosin, methylene blue and methanol.  Wright-Giemsa stains, comprised of polychrome methylene blue, azure B and eosin Y dyes, are classified as Romanowsky stains.   

 

METHOD:

Solutions: All solutions are manufactured by Newcomer Supply, Inc.

All Newcomer Supply stain procedures are designed to be used with Coplin jars filled to 40 ml following the staining procedure provided below. 

 

STAINING PROCEDURE:

  1. Prepare within an accepted time frame, a well-made blood smear or bone marrow smear/film per your laboratories protocol, with a focus on uniform cell distribution.
  2. Allow smear to thoroughly air-dry prior to staining.
  3. Fix smear in Alcohol, Methanol Anhydrous (Part 12236) for 3-5 minutes.
  4. Air-dry slides in a vertical position.
  5. Prepare Wright-Giemsa, Romanowsky Working Stain Solution; combine, mix well and filter if particulates are present.
    1. For thin smears:

Giemsa Stock Stain, Romanowsky                                20 ml

Wright Stain Buffer, pH 6.8                             20 ml

  1. For thick smears:

Giemsa Stock Stain, Romanowsky                                  4 ml

Wright Stain Buffer, pH 6.8                             36 ml

  1. Stain in Wright-Giemsa, Romanowsky Working Stain Solution for 30-45 minutes.
    1. See Procedure Notes #1 and #2.
  2. Wash in distilled water.
  3. Air-dry slides in a vertical position; examine microscopically.
  4. If coverslip is preferred, air-dry slides and coverslip with compatible mounting medium.

 

RESULTS:                  

Erythrocytes Orange-pink to rose
Platelets Red to purple granules with light blue halo

 

Granulocytes

Neutrophils Nucleus - Dark blue to violet
  Cytoplasm - Pink
  Granules - Purple to lilac
Eosinophils Nucleus - Blue
  Granules - Orange to pink
Basophils Nucleus - Deep blue to violet
  Granules - Deep blue to violet

 

Mononuclear Cells

Lymphocytes Nuclei - Deep blue to violet
  Cytoplasm - Light blue
Monocytes Nuclei - Light blue/purple
  Cytoplasm - Pale gray/blue
Mast cells Nuclei - Deep blue to violet
  Granules - Deep blue-violet
Malarial parasites Nucleus - Red chromatin dot
  Cytoplasm - Blue
Bacteria Blue

 

PROCEDURE NOTES:

  1. The timings provided in this procedure are suggested ranges.  Optimal staining times will depend upon staining intensity preference.
  2. Smears containing primarily normal cell populations require minimum staining time; immature cells may require a longer staining time. Bone marrow smears/films may also require a longer staining time.
  3. The color range of the stained cells may vary depending upon the pH of the buffer and the pH of the rinse water used.
    1. Alkalinity is indicated by red blood cells being blue-grey and white blood cells only blue.
    2. Acidity is indicated by red blood cells being bright red or pink and lack of proper staining in white blood cells. 
    3. If necessary adjust buffer pH accordingly to 6.8 +/ - 0.2.

 

REFERENCES:

  1. Bauer, John D. Clinical Laboratory Methods. 9th ed. St. Louis: Mosby, 1982. 111-112.
  2. Conn's Biological Stains. Edited by Richard Horobin and John Kiernan. 10th ed. Oxford, UK: BIOS Scientific Publishers, 2002. 303-312.
  3. Lillie, R. D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 744-748.
  4. McPherson, Richard and Matthew Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia:  Elsevier Saunders, 2011. 522-532.
  5. Modifications developed by Newcomer Supply Laboratory.