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AVIACOLOR®
a stain for avian leukocytes
| PRINCIPLE: | |
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After a brief exposure in a methanolic solution of an acid anionic dye, smears of avian blood are stained in an aqueous mixture containing two basic cationic dyes and one acid anionic dye. Following a vigorous rinse in an acidic phosphate buffer, slides are air dried and viewed under the light microscope. The method provides differential coloration of avian leukocytes, making their identification possible. |
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| A. Reagents: | |
| 1. Acid dye A: a solution of an acid anionic dye in absolute methanol. | |
| 2. Staining mixture B: an aqueous solution of an acid anionic dye and two basic cationic dyes. | |
| 3. Rinse buffer C: a phosphate buffer at pH 5.6, prepared by adding the test tube containing buffer salts to one liter deionized water. Agitate until salts are completely dissolved, add the small vial of liquid phenol to the liter of buffer, and store in a clean plastic bottle at room temperature. Prepared and stored this way, the buffer is stable for one year. | |
| B. Materials: | |
| 1. Two Coplin staining jars with screw tops. Use small Coplin jars if coverslips are used. | |
| 2. One 250 mL glass or plastic beaker. | |
| 3. One tube cyanoacrylate adhesive contained in the kit. | |
| 4. One tube liquid phenol contained in the kit. | |
| C. Procedure: | |
| 1. Make coverslip or slide preparations of avian blood from a tube of peripheral venous blood anticoagulated with heparin, and air dry. | |
| 2. Put slide or coverslip into a covered Coplin jar containing Solution A for 1 minute. | |
| 3. Without rinsing, transfer slide or coverslip using forceps from the Coplin jar containing Solution A into a separate Coplin jar containing Solution B. | |
| 4. Without any repetitive dipping, leave slide or coverslip in the Coplin jar containing Solution B for 8-10 minutes. | |
| 5. Using a forceps, remove slide or coverslip from the Coplin jar containing Solution B, and agitate very vigorously for 5-10 seconds in a beaker containing Solution C. | |
| 6. Remove slide or coverslip from buffer with forceps, and blot on a piece of filter paper. Air dry, and mount with a drop of cyanoacrylate adhesive contained in the kit. Avoid contact of skin with the adhesive. | |
| 7. Discard and replace Solution C after rinsing 30-40 slides or coverslips in it. | |
| EXPECTED RESULTS: | |
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Heterophils contain granules ranging in color from yellow to orange, brown, and black. In some species, granules are angular or crescent shaped, or may be round. Nuclei stain red to purple.. Eosinophils contain turquoise granules. In some species granules are globular or round. In others, granules are angular and appear tightly packed together in the cytoplasm. Nuclei are bilobular and stain brown. Basophils contain bright red to red orange stained granules, and have red brown nuclei. Monocytes display violet cytoplasm that may contain vacuoles and a few red granules. Nucleated erythrocytes stain purple and contain brown nuclei. In immature erythrocytes, the cytoplasm is pale purple. Avian thrombocytes are dark blue to dark purple They are smaller than lymphocytes, and may contain several red granules and vacuoles. |
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