Eosin Methylene blue according to Wright is a composite dye consisting of the acidic dye eosin and the basic dye methylene blue. It appears as a pale green powder and acts as a neutral stain, capable of differentially staining components of the protoplasm. It exerts specific staining effects on neutrophilic, basophilic, and eosinophilic cellular parts, and is primarily used as a microscopic staining agent for smears, such as staining of blood, bone marrow, and blood-borne parasites (including protozoa, Leishmania, filarial worms, etc.).
Cell staining involves both physical adsorption and chemical affinity. Different cellular components possess distinct chemical properties, resulting in varying affinities for different dyes. Thus, after staining with this dye solution, a variety of colors can be observed on the same blood smear. For instance, hemoglobin and eosinophilic granules are alkaline proteins; they react with the acidic dye eosin to stain pink and are therefore referred to as eosinophilic substances. Nuclear proteins and the cytoplasm of lymphocytes are acidic; they bind to the basic dyes methylene blue or azure to stain purple-blue and are thus called basophilic substances. Neutrophilic granules are in an isoelectric state, enabling them to bind to both eosin and methylene blue, resulting in a pale purple stain, and they are known as neutrophilic substances.
The pH of the staining environment has a significant impact on cell staining. All cellular components are proteins, which are amphoteric electrolytes. The net charge carried by proteins is determined by the pH of the solution. In an acidic environment, proteins carry more positive charges, making them prone to binding with eosin and leading to a reddish stain. In an alkaline environment, proteins carry more negative charges, facilitating binding with methylene blue or azure and resulting in a bluish stain. Therefore, cell staining is highly sensitive to hydrogen ion concentration. Slides used for staining must be clean and free from acid-base contamination. High-quality methanol must be used for preparing Wright's stain solution. Buffered solutions should be employed for diluting the stain, and the washing water should be nearly neutral. Failure to adhere to these requirements can cause abnormal staining reactions of various cells, leading to difficulties in identification and even diagnostic errors.
Wright Stain Solution (ready-to-use) is formulated by grinding high-quality imported Wright's stain as the primary raw material, delivering clear and distinct cell staining results. This stain solution is ready for direct use without any prior preparation. A trace amount of neutral glycerol is added to the solution to prevent methanol from volatilizing or oxidizing, while also enhancing the clarity of blood cell staining. This product is intended for research use only and not for any other purposes.
Materials to Be Prepared by User:
1. Glass slides
2. Distilled water
3. Staining racks
4. Microscopes
Operating Procedures (For Reference Only):
1. Prepare blood smears, bone marrow smears or bacterial smears using standard protocols, and allow the smears to air dry naturally.
2. Place the prepared smears onto the staining racks.
3. Add drops of Wright Stain to completely cover the smear surface. Stain at room temperature, gently agitate the slide or mix using other appropriate methods, then let it stand undisturbed at room temperature.
4. Rinse one end of the slide gently with tap water or distilled water.
5. Allow the slide to dry.
6. Microscopic Examination: First examine the smear under the low-power objective lens, then switch to the oil immersion objective lens for detailed observation.
Staining Results:
Bacteria, cell nuclei
| Blue
|
Eosinophilic granules
| Pink or orange-red
|
Cytoplasm of lymphocytes and basophils
| Purple-blue or blue
|
Fully mature red blood cells
| Pink |
Precautions:
1. Blood smears or bone marrow smears should be of uniform thickness to avoid affecting the staining results.
2. During smear staining, do not remove the stain first or rinse the smear vigorously directly. Never pour off the stain prematurely, as this may cause dye precipitation on the smear.
3. The staining solution can be reused but not for excessive times. If sediment appears, filter the solution before use.
4. For over-stained smears, appropriate destaining can be performed with methanol or ethanol, and restaining is not recommended if possible.
5. If the staining is too dark or too light, adjust the staining duration or the concentration of the working solution accordingly.
6. pH has a certain impact on staining. Glass slides must be clean and free from acid-base contamination to prevent adverse effects on the staining results.
7. For your safety and health, wear a lab coat and disposable gloves during the operation.
ZJ26F0332394