Wrights staining

Tuesday, March 16, 2004

Description
Wright's stain is a Romanowsky type metachromatic stain made by mixing old or specially treated methylene blue dye with eosin in a methanol diluent. Basic components of the cell, such as hemoglobin or certain inclusions or granules, will unite with the acidic portion of the stain, eosin, and are said to be eosinophilic. These components are stained varying shades of pink or red. Acidic cell components, such as nucleic acids, reactive cytoplasm, etc. take up the basic dye components, methylene azure, and stain blue or purple. pH must be carefully controlled through the use of a buffer of 6.4-6.7. If the pH is too acidic the stain will take on a pinkish tint, and nuclear structures will be poorly stained. A basic pH will cause all intracellular structures, nuclei, etc. to be blue-black in color, with poorly defined structure.

Procedure
1)Prepare a solution of 20 ml Giemsa stain + 240 ml deionized H2O. This must be made fresh daily.

2)Using a Beral pipette, overlay a properly prepared blood smear slide with enough Wright's stain to completely cover the slide with a layer of stain approximately 1/8" thick. The stain will be held to the slide by surface tension, and should not run off. Assure that the slide is level and does not touch the side of the staining rack.

3)After 2 minutes, cover the slide with an equal amount of the Giemsa solution prepared in step 1 above. Blow gently to mix and watch for metallic sheen to appear. Allow to stand for 4 minutes.

4)After 4 minutes, wash the slide for 30 seconds with distilled water.

5)Allow slide to dry at room temperature before examination.

Recipes

Supplies

Tips
Too Acid Stain:

1) insufficient staining time
2) prolonged buffering or washing
3) old stain

Correction: 1) lengthen staining time
2) check stain and buffer pH
3) shorten buffering or wash time

Too Alkaline Stain:

1) thick blood smear
2) prolonged staining
3) insufficient washing
4) alkaline pH of stain components

Correction:
1) check pH
2) shorten stain time
3) prolong buffering time

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Wrights staining