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The level of difficulty for this activity would be considered moderate. It is helpful for the student to have prior knowledge of mammalian blood smear and staining techniques.
Materials & Equipment
Note: the stains may be purchased already prepared from the manufacturer through suppliers, such as Fisher Scientific Company, Pittsburgh, PA and American Scientific Products, McGraw Park, IL.
Procedure for Slide Preparation
When used with avian blood, the traditional method of wedge slide preparation used for mammalian smears often yields non-diagnostic slides that contain many smudge cells. This method creates a feathered edge where there is a consolidation of the larger leukocytes. This results in a less than random distribution of cells throughout the slide. A preferred method is the coverglass slide preparation. This method produces fewer smudge cells with a more even distribution of cells. The utilization of a 22 x 50mm cover glass with a 1" x 3" slide generates a superior slide.
1. Use a microhematocrit tube to place a small drop of blood near the end of the slide.
2. Drop the cover glass directly on the top of the blood droplet, perpendicular to the slide.
3. Before the blood spreads completely to the edges of the slide, pull the cover glass horizontally to the side in one swift motion, being careful not to lift the coverglass from the surface of the slide. You may need to practice a few times to become proficient at this technique.
4. Allow to air dry before staining.
5. Discard the used coverglass in the sharps container.
6. Disinfect the work surface.
7. Wash hands with the hand disinfectant.
Numerous staining techniques have been used for avian slides. One of the preferred methods is the May-Grűnwald technique, which will be used for this wet lab.
1. Fix the slide using pure methanol in a coplin jar for 2 minutes and air dry.
2. Mix 1 part May-Grűnwald stain, 1 part Wright-Giemsa stain, and 1 part 6.8pH buffered water (distilled or deionized).
3. Place the slide on the rack over the sink, and flood it with the above mixture.
4. Add additional buffered water until a green sheen appears. Blow on the slide to mix. Do not allow the stain to dry on the slide.
5. Leave the stain on the slide for 6 minutes, being careful not to let the stain dry on the slide by adding more stain mixture if needed, and then rinse the slide with buffer solution briefly. Air-dry or blot with bibulous paper.
Note on buffers: The lower the pH the more eosinophilic the cells stain. The higher the pH, the more basophilic the cells stain. Pre-measured buffer powders can be purchased with a wide range of pH choices. Buffer solutions can be hand-made with distilled or deionized water and buffer salts.