Diff-Quik staining is a rapid staining method improved on the basis of Wright staining, and is one of the commonly used staining methods in cytological examination. The staining solution is prepared according to the rapid staining method recommended by the World Health Organization (WHO). Similar to Wright Stain, it is also modified based on the principle of Romanowsky Stain. The staining results are very similar to those of Wright stain, but Diff-Quik rapid staining requires an extremely short time, and can generally be completed within 90 seconds. Diff-Quik staining solution can be used for the evaluation of sperm morphology. Rapid Sperm Morphology Staining Solution (Diff-Quik method) is a sperm morphology staining method recommended by WHO. Its staining principle is that proteins with different isoelectric points in sperm and cells carry different charges at the same acidity and can selectively bind to corresponding dyes for coloration. Amino groups dissociated from eosinophilic proteins are positively charged and can bind to negatively charged acidic dyes (such as eosinophilic xanthene, eosin) and be stained red. Carboxyl groups dissociated from basophilic proteins are negatively charged and can bind to positively charged basic dyes (such as basophilic thiazine, methylene blue) and be stained blue. Neutrophilic proteins have equal numbers of positively charged amino groups and negatively charged carboxyl groups after dissociation; they bind equal amounts of acidic and basic dyes simultaneously and appear purplish red, but the staining intensity is weak because the net charge is zero. This staining solution is mainly used for the evaluation of sperm (cell) morphology. It contains fixative, is very suitable for batch staining, and features a clear background without sediment. This product is for research use only and is not suitable for clinical diagnosis or other purposes.
Product Components and Storage Conditions:
S1511533
| Component
| 3×20mL
| 3×100mL
| Storage
|
S1511533A
| Diff-Quik Fixative
| 20mL
| 100mL
| RT
|
S1511533B
| Diff-QuikⅠ
| 20mL
| 100mL
| RT. Store in the dark.
|
S1511533C
| Diff-QuikⅡ
| 20mL
| 100mL
| RT. Store in the dark. |
Materials Required (User-supplied):
Glass slides, distilled water, microscope
Protocol (For Reference Only):
1. Smear preparation: Clean two glass slides thoroughly, wash with 70% ethanol and air-dry. Place 5–20 μl of sample on the slide. Drag the droplet of semen across the clean slide surface using the edge of a second slide to prepare a smear. If sperm density is excessively high, dilute appropriately with physiological saline.
2. Fix in Diff‑Quik Fixative or by air drying for 15–20 seconds.
3. Stand the slide vertically on absorbent paper to remove excess fluid.
4. Stain the slide in Diff‑Quik Stain I for 10–20 seconds, then stand vertically on absorbent paper to remove excess stain.
5. Stain the slide in Diff‑Quik Stain II for 5–10 seconds, then stand vertically on absorbent paper to remove excess stain.
6. Dip and rinse 10–15 times in running water to remove excess dye.
7. Stand the slide vertically on absorbent paper to remove excess moisture and allow to dry completely. Examine under a microscope.
Staining Results:
Acrosomal region of sperm head: Pale blue
Post-acrosomal region of sperm head: Dark blue
Midpiece of sperm: Pale red (may be)
Sperm tail: Blue or pale red (variable)
Precautions:
1. If sperm density > 20×10⁶/mL, use 5 μL; if sperm density < 20×10⁶/mL, use 5–20 μL.
2. Diff‑Quik Fixative is the dedicated fixative for sperm Diff‑Quik staining. Methanol may be used as an alternative if a large volume is required.
3. Sperm samples should be fresh, and smears should be of uniform thickness to avoid affecting staining results. If sperm density is excessively high, dilute appropriately with physiological saline.
4. Do not remove the staining solution first or rinse the smear forcefully during staining. Do not pour off the stain first, to prevent dye precipitation on the smear.
5. The staining solution can be reused, but not repeatedly. If sediment is present, filter before use.
6. If overstained, appropriate decolorization may be performed with methanol or ethanol; restaining is not recommended.
7. If staining is too intense or too faint, adjust the staining time or working solution concentration.
8. pH affects staining. Slides must be clean and free of acid or alkali contamination.
9. For your safety and health, wear lab coat and disposable gloves during operation.
10. This product is for research use only. Strictly prohibited for other purposes.
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