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| Product Name | Crystal Violet Solution, 0.5%, for Gram Staining and Colony Staining |
| Catalog No. | ATRSA-0033 |
| Description | Ready-to-use 0.5% w/v crystal violet aqueous solution for Gram staining of bacteria, colony formation assays, and cell migration/invasion (Boyden chamber) assays. In Gram staining, crystal violet serves as the primary stain that complexes with Gram's iodine to form crystal violet-iodine (CV-I) complexes within the bacterial cytoplasm. Gram-positive bacteria retain the CV-I complex through the decolorization step due to their thick, cross-linked peptidoglycan layer, appearing purple. In colony formation assays, crystal violet stains fixed adherent cell colonies, enabling quantitative measurement of clonogenic survival. In Boyden chamber assays, crystal violet stains migrated cells on the membrane for counting. |
| Intended Use | Primary stain for Gram classification of bacteria in clinical and environmental microbiology; quantitative staining of cell colonies for clonogenic survival and proliferation assays; staining of migrated cells in transwell migration and invasion assays; general nuclear and cellular staining in histology. |
| Principle / Technology | Crystal violet is a triarylmethane basic dye that binds to negatively charged components (DNA, peptidoglycan, cell surface); in Gram staining, CV-I complex formation with iodine creates a large molecular complex trapped in Gram-positive peptidoglycan; decolorization (acetone/alcohol) extracts CV-I from Gram-negative bacteria due to thin peptidoglycan and outer membrane lipid extraction. |
| Detection Method | Gram staining: apply crystal violet 1 min → water rinse → Gram's iodine 1 min → water rinse → decolorize with acetone/alcohol 5-10 sec → water rinse → safranin counterstain 30 sec → wash, dry. Colony assay: fix colonies with 4% PFA or methanol 10 min → stain with crystal violet 10-20 min → wash with water and air dry → solubilize with 10% acetic acid or 1% SDS and measure absorbance at 570-595 nm. |
| Sample Type | Heat-fixed bacterial smears; fixed adherent cell colonies in multiwell plates; transwell membranes with migrated cells; formalin-fixed tissue sections. |
| Performance Range / Specifications | Gram staining: Gram-positive bacteria — purple/blue-violet; Gram-negative bacteria — pink/red (safranin counterstain); colony assay: linear absorbance range 0.1-3.0 OD at 595 nm; cell number range 50-50,000 cells per well (96-well format). |
| Sensitivity / LOD | Colony assay: detection of colonies from as few as 10-50 seeded cells; Gram staining: bacteria visible at 1000× magnification (oil immersion); crystal violet detectable at <0.01 OD at 595 nm after solubilization. |
| Specificity | Crystal violet stains most bacteria and eukaryotic cells non-specifically; Gram differentiation depends on CV-I complex retention in Gram-positive peptidoglycan layer; in colony assays, stains fixed cells and extracellular matrix indiscriminately. |
| Reaction Conditions / Protocol | Gram stain: 1 min staining; colony assay: 10-20 min staining at RT; decolorization critical for Gram differentiation; solubilization in 10% acetic acid for 15-30 min at RT for quantification. |
| Components / Formulation | Crystal violet solution (0.5% w/v in 20% ethanol or aqueous), Gram's iodine solution, safranin counterstain (0.5% w/v), decolorizing solution (acetone:ethanol 1:1 or 95% ethanol), protocol. |
| Storage Conditions | Store all components at room temperature (15-25 °C); protect from light; keep tightly capped to prevent evaporation of solvents. |
| Shelf Life | 24 months from date of manufacture. |
| Package Specifications | 250 mL crystal violet solution, 250 mL Gram's iodine, 250 mL safranin, 250 mL decolorizer (complete Gram stain kit); or individual components. |
| Product Form | Crystal violet: deep purple liquid; Gram's iodine: dark brown liquid; safranin: pink-red liquid; decolorizer: clear liquid. |
| Quality Control | Each lot tested on standardized Gram-positive (S. aureus) and Gram-negative (E. coli) bacterial smears for correct differential staining; crystal violet concentration verified spectrophotometrically. |
| Key Features | 0.5% ready-to-use solution; classic Gram stain primary dye; also suitable for colony formation and migration assays; consistent staining intensity; long shelf life; includes complete Gram stain reagents. |
| Purity | Crystal violet ≥90% dye content (certified by Biological Stain Commission); Gram's iodine ACS grade; safranin O certified. |
| Concentration | Crystal violet: 0.5% w/v; Gram's iodine: 1% iodine + 2% KI; safranin: 0.5% w/v; decolorizer: acetone:95% ethanol 1:1 v/v or 95% ethanol. |
| Activity / Unit Definition | Gram stain differential verified on standardized mixed bacterial smear per lot. |
| Molecular Weight | Crystal violet: 407.98 g/mol (C25H30ClN3). |
| Source / Origin | Synthetic triarylmethane dye; iodine from mineral source. |
| pH Range / Optimal pH | Crystal violet solution near neutral pH; Gram's iodine acidic. |
| Shipping Conditions | Ambient temperature. |
| Expiration Date / Stability | 24 months at RT; crystal violet may develop surface film over time — filter before use. |
| Regulatory / Compliance | For in vitro diagnostic use in microbiology; manufactured under ISO 13485 quality system. Decolorizer contains flammable solvents — store and handle accordingly. |
| Compatibility | Compatible with bacterial smears from culture plates or clinical specimens. Fixed cell monolayers should be thoroughly washed before staining to remove medium components. For solubilization: 10% acetic acid (colony assay) or 1% SDS (migration assay). Crystal violet is not compatible with live/dead cell discrimination — it stains all fixed cells. |
| Recommended Buffer System | Not applicable; aqueous or 20% ethanolic solution. |
| Application Notes / Precautions | For Gram staining: the decolorization step is the most critical — over-decolorization will make all bacteria appear Gram-negative. Apply decolorizer dropwise until the runoff is clear (5-10 seconds typically). For colony assays: wash plates gently with water to avoid detaching colonies; air dry completely before scanning or photography. For solubilization: 100 µL 10% acetic acid per well (96-well), shake gently for 15-30 min at RT. Measure absorbance at 570-595 nm (peak absorbance 590 nm). Use fresh overnight bacterial cultures (<24 hours old) for Gram staining — older cultures may give variable results. |
| Batch-to-Batch Consistency | Crystal violet dye content within ±5% of specification; Gram stain differential performance verified per lot. |
For research use only, not for clinical use.
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