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.
Operating Procedures (For Reference Only):
1. Sample Preparation
① For paraffin sections:
Deparaffinize in xylene for 5–10 min.
Replace with fresh xylene and deparaffinize for another 5–10 min.
Treat with absolute ethanol for 5 min.
Treat with 90% ethanol for 2 min.
Treat with 70% ethanol for 2 min.
Rinse with distilled water for 2 min.
② For frozen sections:
Rinse with distilled water for 2 min.
③ For cultured cells:
Fix with 4% paraformaldehyde for at least 10 min.
Wash with distilled water for 2 min.
Replace with fresh distilled water and wash for another 2 min.
2. Crystal Violet Staining
① Stain with crystal violet staining solution for 5–10 min; adjust the time according to staining results and requirements.
② Wash thoroughly with distilled water or tap water, then observe and photograph.
Staining Result:
Tissues or cells – Deep 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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