Jenner's Stain Solution

EMS26033-05

Specimen Collection

Fresh blood film or fresh EDTA anticoagulated blood film and bone marrow films. Specimens should be air dried after smear has been prepared and fixed in absolute methanol for 15 seconds.

Mode of Action

The Jenner Stain Solution is a mixture of several thiazin-dyes in a methanol solvent. Ionic and nonionic forces are involved in the binding of these dyes. The staining solution has anionic and cationic properties. The negatively charged phosphoric acid groups of DNA attract the purple polychromatic cationic dyes to the nuclei. The blue basophilic granules are stained by the polychromatic cationic dyes. Cationic cellular components, such as erythrocytes and eosinophilic granules, are stained by the red and pink anionic dyes. The buffers used in the staining procedure liberate and activate dye ions allowing them to chemically bond with specific cellular components. When staining blood and bone marrow smears, the pH of the staining solution and/or buffer is a critical factor.

Technical Procedures

Immersion Staining Protocol

1. Thoroughly dry blood or bone marrow smears.
2. Fix smears in absolute methanol for 15 seconds to 5 minutes.
3. Stain smears in Jenner Solution for 2 minutes.
4. Stain in mixture of 50ml of Jenner Stain Solution, 75ml of pH 6.6 Phosphate Buffer solution and 175ml deionised water for 5 minutes.
5. Rinse in standing deionised water for 1.5 minutes, or rinse briefly in running deionised water.
6. Air dry smears.
7. Examine smears under a microscope.

Horizontal Staining Protocol

1. Place slide with thoroughly dried film on a horizontal staining rack.
2. Flood smear with absolute methanol for 15-30 seconds and then drain.
3. Flood smear with 1ml Jenner Stain Solution and let stand for 3 minutes.
4. Add 1ml of pH 6.6 Phosphate Buffer solution and 1ml deionised water to smear and let stand for 45 seconds.
5. Rinse briefly with running deionised water.
6. Air dry and examine under a microscope.

Results

Erythrocytes - Pale Pink
Eosinophilic Granules - Reddish Orange
Leukocyte Nuclei - Purple
Cytoplasm - Bluish Purple
Neutrophilic Granules - Light Purple

Discussion

Jenner Stain Solution should be stored at room temperature. This staining reagent is for "In Vitro" use only. Refer to the Safety Data Sheet for Health and Safety Information. All reagents are stable and should not form precipitants under ordinary storage parameters. It is recommended that the Jenner Stain Solution be discarded after each use. All dyes used in the formulation have been certified by the Biological Stain Commission.

Technical Comments

Thicker films and bone marrow preparations will require longer staining times. Because there is variation in the pH tap water use of tap water in procedure may hinder staining results. Distilled or deionised water should be used during the staining procedure. The "ripening" of the polychromed dye is a continuous chemical reaction. Therefore, the stock solution should not be used or diluted after the expiration date. The staining procedure may require modification to suit personal preference. Blood films which have not been thoroughly air dried before staining may show sloughing of cells from slide.

References

1. Raphael, S.S. Lynch's Medical Laboratory Technology, Fourth Edition. Saunders Company, Philadelphia, PA, 1983.
2. Lillie, R.D. H. J. Conn's Biological Stains, Ninth Edition. Williams and Watkins Company, Baltimore, MD, 1977.
3. Brown, B.A. Hematology: Principles and Procedures, Fourth Edition. Lea and Febiger Company, Philadelphia, PA, 1984.