Wright Stain, Buffered

SOLUTION:

Additionally Needed:

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

APPLICATION:

Newcomer Supply Wright Stain, Buffered for Smears provides a quick staining technique 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.

PRESTAINING PREPARATION:

1. 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 slides to thoroughly air-dry prior to staining.
   3. Filter Wright Stain, Buffered prior to use with quality filter paper.
      1. 1. For flat staining rack method, filter sufficient stain to allow 1 ml of stain per slide.
   4. Prepare 25% Aqueous Methanol Rinse; combine and mix well.
      1. 1. Distilled Water 30 ml  or  3 ml
         2. Methanol (Part 12236) 10 ml  or  1 ml

STAINING PROCEDURE:

1. 5. Coplin Jar Method: See Procedure Notes #1 and #2.
      1. 1. Fix smears in Methanol for 15 seconds.
         2. Stain in filtered Wright Stain, Buffered for 1-2 minutes.
         3. Place directly into Wright Stain Buffer, pH 6.8 (Part 1430), for 1-4 minutes. Do Not Agitate!
         4. Dip quickly in 25% Aqueous Methanol Rinse (Step #4).
         5. Rinse in distilled water.
         6. Air-dry slides in a vertical position; examine microscopically.
         7. If coverslip is preferred, allow slides to air-dry and coverslip with compatible mounting medium.
   6. Flat Staining Rack Method: See Procedure Notes #1 and #2.
      1. 1. Place slides on flat staining rack suspended over sink.
         2. Fix by flooding slide with Methanol for 15 seconds.
         3. Drain off Methanol.
         4. Flood each slide with 1 ml of filtered Wright Stain, Buffered for 1 minute.
         5. Retain Wright Stain, Buffered on slides.
         6. Directly add 2 ml of Wright Stain Buffer, pH 6.8 to each slide; agitate gently to mix with retained Wright Stain.
         7. Stain for an additional 3 minutes.
         8. Flood smears with 25% Aqueous Methanol Rinse (Step #4) for 1 second.
         9. Rinse in distilled water.
         10. Air-dry slides in a vertical position; examine microscopically.
         11. If coverslip is preferred, allow slides to air-dry and coverslip with compatible mounting medium.

RESULTS:

PROCEDURE NOTES:

1. 1. Timings provided are suggested ranges. Optimal times will depend upon staining intensity preference.
   2. Smears containing primarily normal cell populations require minimum staining time; immature cells and bone marrow smears/films may require longer staining time.
   3. The color range of stained cells may vary depending on buffer pH and pH of rinse water.
      1. 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. 1. Lillie, R. D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 747-748.
   2. McPherson, Richard and Matthew Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders, 2011. 522-532.
   3. Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 154-155.
   4. Modifications developed by Newcomer Supply Laboratory.