Under normal circumstances, bacteria are easily stained with basic dyes. The chromogenic ions of basic dyes carry a positive charge after ionization and readily bind to negatively charged stained substances. Since the isoelectric point of bacteria ranges from pH 2 to 5, bacteria carry a negative charge in alkaline, neutral and weakly acidic environments, and are thus prone to combining with positively charged dyes for coloration. Commonly used dyes include basic fuchsin, crystal violet and methylene blue.
Crystal violet, also known as methyl violet, has a molecular formula of C₂₅H₃₀N₃Cl·9H₂O, a molecular weight of 407.98 and a CAS number of 548-62-9. Together with methyl green, neutral red, methylene blue, safranin, hematoxylin, malachite green, methyl violet and basic fuchsin, it is a commonly used synthetic basic dye at present. It is soluble in water, ethanol and chloroform, but insoluble in diethyl ether. Its aqueous and ethanol solutions appear deep purple, and turn orange when exposed to concentrated sulfuric acid. It is used as an acid-base indicator with a pH color change range of 0.1 (green) to 2.0 (blue), a non-aqueous titration indicator in analytical chemistry, a biological stain and a chromogenic agent in colorimetric determination. Crystal violet is also an important dye for Gram staining of bacteria.
Crystalline violet PBS solution (0.1%) is mainly composed of 0.1% crystal violet, PBS Buffer and other components, and is mostly used for cell staining. This reagent is for research use only and not intended for clinical diagnosis or any other applications.
Operating Procedures (For Reference Only):
Cell Staining (Taking 6-well Plate as an Example)
Step 1: Cell Fixation
1. Aspirate and discard the cell culture medium in the culture plate.
2. Gently rinse the cells with pre-cooled 1× PBS for 1–2 times to completely remove serum and dead cell debris.
3. Add a fixative (e.g., 4% paraformaldehyde or methanol) to ensure the bottom of each well is fully covered.
Paraformaldehyde: Fix at room temperature for 15–20 minutes.
Methanol: Fix at room temperature for 10–15 minutes (methanol serves as both a fixative and a permeabilizer).
4. Aspirate and discard the fixative.
Step 2: PBS Washing
1. Add an adequate volume of 1× PBS and wash with gentle shaking for 1–2 minutes.
2. Aspirate and discard the PBS, and repeat the washing step once to completely remove residual fixative.
3. (Optional) Invert the plate and gently pat dry on absorbent paper to ensure no liquid remains in the wells.
Step 3: Crystal Violet Staining
1. Add an appropriate volume of 0.1% crystal violet solution to each well (ensure the cell layer is fully covered; approximately 1 mL per well for a 6-well plate).
2. Incubate at room temperature in the dark.
Recommended incubation time: 15–30 minutes. The specific time can be adjusted according to cell density and staining intensity. Longer staining time results in a deeper color but may also cause a darker background.
3. After staining, carefully aspirate and discard the staining solution (Note: Crystal violet is highly prone to splashing and may contaminate the environment and skin).
Step 4: Thorough Washing (Critical Step)
1. Gently rinse the plate several times with a slow, fine stream of deionized water or 1× PBS until the rinse solution becomes colorless and transparent.
2. Immerse the plate in a large container filled with deionized water and shake gently on a shaker for 10–15 minutes to remove non-specific background staining. The water can be replaced 1–2 times until it remains clear.
3. Pour off the water, invert the plate on clean absorbent paper, and air-dry completely at room temperature.
Step 5: Result Observation
Observe the cell morphology and staining under a regular light microscope. The cells will stain blue-purple.
Precautions:
1. Crystal violet is harmful to the human body; proper protective measures are recommended.
2. It is advised to conduct a pre-experiment with 1–2 samples when using this reagent for the first time.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
4. This product is for research use only and shall not be used for any other purposes.
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