Wright Stain, Buffered
SOLUTION: | 500 ml | 1 Liter | 1 Gallon |
Wright Stain Solution, Buffered | Part 1422A | Part 1422B | Part 1422C |
Additionally Needed:
Alcohol, Methanol Anhydrous, ACS | Part 12236 |
Wright Stain Buffer Solution, pH 6.8 |
For storage requirements and expiration date refer to individual bottle labels.
APPLICATION:
Newcomer Supply Wright Stain, Buffered for Smears provides a quick staining technique, used for differential staining of cell types in peripheral blood smears as well as bone marrow smears/films.
METHOD:
Technique: Coplin jar or flat staining rack method
Solutions: All solutions are manufactured by Newcomer Supply, Inc.
STAINING PROCEDURE:
- 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.
- Allow slides to thoroughly air-dry prior to staining.
- Filter Wright Stain Solution, Buffered prior to use.
- Prepare 25% Aqueous Methanol Rinse; combine and mix well.
- Distilled water 30 ml or 3 ml
- Methanol 10 ml or 1 ml
- Coplin Jar Method: See Procedure Notes #1 and #2.
- Fix smears in methanol for 15 seconds.
- Stain in Wright Stain Solution, Buffered for 1-2 minutes.
- Place smear directly into Wright Stain Buffer Solution, pH 6.8, 1 to 4 minutes. Do Not Agitate!
- Dip smears in 25% Aqueous Methanol Rinse for 1 second.
- Rinse in distilled water.
- Air-dry slides in a vertical position, then examine microscopically.
- If coverslip is preferred, allow slides to air-dry and coverslip with compatible mounting medium.
- Flat Staining Rack Method: See Procedure Notes #1 and #2.
- Place slides on a flat staining rack suspended over a sink.
- Fix smear by flooding slide with methanol for 15 seconds.
- Shake off excess methanol; apply 1 ml Wright Stain Solution, Buffered to each slide for 1 minute.
- Retain Wright Stain Solution, Buffered on slides, directly add 2 ml of Wright Stain Buffer Solution, pH 6.8 to each slide; gently agitate to mix. Stain for an additional 3 minutes.
- Flood smears with 25% Aqueous Methanol Rinse for 1 second.
- Rinse in distilled water.
- Air-dry slides in a vertical position, then examine microscopically.
- If coverslip is preferred, allow slides to air-dry and coverslip with compatible mounting medium.
RESULTS:
Erythrocytes | Pink |
Neutrophils | Granules - Purple |
Eosinophils | Granules - Pink |
White blood cells | Chromatin - Purple |
Lymphocytes | Cytoplasm - Blue |
Monocytes | Cytoplasm - Blue |
Bacteria | Deep Blue |
PROCEDURE NOTES:
- The timings provided in this procedure are suggested ranges. Optimal staining times will depend upon staining intensity preference.
- 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.
- The color range of the stained cells may vary depending upon the pH of the buffer and the pH of the rinse water used.
- Alkalinity is indicated by red blood cells being blue-grey and white blood cells only blue.
- Acidity is indicated by red blood cell being bright red or pink and lack of proper staining in white blood cells.
- If necessary adjust buffer pH accordingly to 6.8 +/ - 0.2.
REFERENCES:
- Lillie, R. D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 747-748.
- McPherson, Richard and Matthew Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders, 2011. 522-532.
- Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 154-155.
- Modifications developed by Newcomer Supply Laboratory.