Staining principle:
Wright's pigment is a composite dye composed of the acid dye Eosin and the basic dye Methylene Blue, which has a good distinguishing effect on the dyeing of protoplasm. The chemical properties of various cell components are different, and the affinity for various dyes is also different. For example, hemoglobin and eosinophilic particles are basic proteins that combine with the acid dye eosin and dye pink, called eosinophils; nucleoprotein, lymph The cytoplasm of cells and basophils is acidic. After being combined with basic dyes, dyed purple or blue, called basophils; neutral particles in an isoelectric state can be combined with eosin and methylene blue. Stained with lavender red, called neutrophils, primitive red blood cells, cytoplasm and nucleolus of early young red blood cells contain more acidic materials, dyed into a thicker blue; young red blood cells contain both acidic and alkaline substances , Dyed red-blue or gray-red; fully mature red blood cells, after the acidic substances have completely disappeared, dyed pink. Wright's stain is a blue and clear liquid with a specific fragrance of methanol. It is mainly composed of methylene blue, eosin Y, methanol, and glycerin. The role of methanol is to dissolve methylene blue and eosin and fix cell morphology.
Kimsa dye is composed of azure and eosin (quality). The dyeing principle and result are basically the same as the Wright staining method. However, this method can color the nucleus (blue) and parasites better, and the structure is more unclear, while the cytoplasm and neutral particles are poorly colored. In order to take into account the advantages of both, a compound dyeing method can be used. That is, the diluted Kimsa solution was used instead of the buffer solution and stained for 10 minutes according to the Wright staining method. Or first stain with Wright's staining method, and then counterstain with diluted Giemsa.
Kit composition:
Swiss staining solution; Giemsa staining solution; phosphate buffer pH6.8
Tissue fixation
According to the conventional smear, air drying, 4% paraformaldehyde fixation.
Steps
1. Prepare blood smears or bone marrow smears or bacterial smears by conventional methods, and wait for the smears to dry naturally.
2. Place the blood smear or bone marrow smear on the staining rack.
3. Add Wright stain dropwise to cover the smear, stain at room temperature for 1 to 2 minutes, gently shake the slide or mix in other ways, after 1-2 minutes, drop in phosphate buffer (pH 6.4-6.8) (dye and buffer) The ratio of liquid is about 1:1.5, 1:1 in winter and 1:2 in summer). Gently pump air on the glass slide with an air bag to mix the dye and buffer evenly. Generally speaking, it is not suitable to blow up by mouth, so as to prevent the exhaled carbon dioxide from changing the pH of the buffer and affecting the staining.
4. Rinse gently from one end of the slide with tap water or distilled water
5. Dilute the staining solution (take 9 parts of phosphate buffer solution and 1 part of Giemsa staining solution and mix thoroughly), drop the staining solution on the tissue or cells, and stain for 10-15 minutes.
6. Rinse with distilled water, and add a cover glass for microscopy when it is wet;
7. Dry.
8. Microscopic examination: first observe the blood smear with a low-power lens, and then use an oil lens.
result:
The dried blood smear was observed under a microscope. Observe the blood cells at the junction of the blood smear body and tail with a low-power microscope. Under the microscope, mature red blood cells are stained pink; platelets are stained purple; neutrophil cytoplasm is stained pink with purple-red granules; eosinophils contain large orange granules; basophils cytoplasm Contains a large number of dark purple-blue particles; the cytoplasm of monocytes is stained gray-blue; the cytoplasm of lymphocytes is stained light blue.
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