Methylene Blue Staining Solution (0.5%) - BioReagent

CAS: 7220-79-3 Cat. No.: M1528794 分子式: C₁₆H₂₄ClN₃O₃S 分子量: 373.9 Beilstein Registry Number: 3599847 EC番号: 615-731-6

グレード＆純度 BioReagent, Biological Stain, 0.5% in water

Storage

Protected from light,Room temperature

Shipped In

Normal

Application

Tissue Staining

概要

Methylene blue, also known as methylthioninium chloride, basic blue 9, etc., has the molecular formula C₁₆H₂₄ClN₃O₃S (hydrated form), a molecular weight of 373.9, and a CAS number of 7220-79-3.

Methylene blue staining solution is commonly used for staining silk, paper, bacteria, cells, etc. Because it is easily oxidized, staining results are not suitable for long-term storage.

Procedure (for reference only):

1. Sample Processing

① Paraffin sections

Deparaffinize in xylene for 5–10 min.

Replace with fresh xylene and deparaffinize again for 5–10 min.

Immerse in anhydrous ethanol for 5 min.

Rinse in distilled water for 2 min.

② Frozen sections

Rinse in distilled water for 2 min.

③ 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 again for 2 min.

2. Methylene Blue Staining

Stain with Methylene Blue Staining Solution (0.5%) for 3–10 min (the time can be adjusted according to the staining results and requirements).

Wash thoroughly with distilled water or tap water, then observe and photograph.

Staining Results:

Tissues or cells: blue

Precautions:

1.When using this reagent for the first time, it is recommended to perform a preliminary experiment with 1–2 samples.

2.For your safety and health, please wear a lab coat and disposable gloves during operation.

3.This product is for research use only and must not be used for any other purposes.

仕様

仕様と純度

BioReagent, Biological Stain, 0.5% in water

安定性と保管

Store at RT long term (12 months). Store in the dark.

保管条件

Protected from light,Room temperature

入荷

Normal

等級

Biological Stain, BioReagent

Product Line

生物製品

名前と識別子

異性体SMILES

CN(C)C1=CC2=C(C=C1)N=C3C=CC(=[N+](C)C)C=C3S2.O.O.O.[Cl-]

WGKドイツ

2

分子量

373.9

Beilstein

3599847

Reaxy-Rn

16692608

Reaxys-RN_link_address

https://www.reaxys.com/reaxys/secured/hopinto.do?context=S&query=IDE.XRN=16692608&ln=

化学的性質と物理的性質

• 感度: Light-sensitive
• 融点（°C）: 235°C

Application Protocols

General guidance for using a 0.5% methylene blue staining solution (research use only). Adjust to your sample type and institutional SOPs. These are literature-style protocols, not item specifications.

1. Simple bacterial smear stain

• Prepare smear, air dry, and fix (heat or 100% methanol, 1–2 min; air dry).
• Flood slide with 0.5% methylene blue for 30–90 s. Thicker smears may require up to 3 min.
• Rinse gently with distilled water until runoff is nearly colorless.
• Blot dry and examine with oil immersion as needed.

2. Rapid eukaryotic cell/tissue touch prep stain

• Fix cells (e.g., 70–100% ethanol for 1–3 min) or use air-dried touch preps.
• Apply 0.5% methylene blue for 15–60 s.
• Rinse with buffered saline or water; briefly differentiate with 70% ethanol if background is high (optional; monitor microscopically).
• Air dry and mount with aqueous medium.

3. Nucleic-acid–biased contrast (optional differentiation)

• After staining 1–2 min, perform a brief dip in 0.5–1% acetic acid or 70% ethanol to reduce cytoplasmic background; quickly re-rinse with water.

Practical tips

• Filter stain if particulates are observed (0.22–0.45 μm).
• Protect from strong light during longer incubations to minimize photofading.
• Optimize time and rinsing for your specimen and fixation; overexposure increases background.

Controls and QC

• Include a known positive smear/slide to benchmark intensity.
• Optional: Record absorbance of the working solution at ~664 nm (literature) for batch-to-batch tracking (no specification implied).

Biological Roles

Scope

• This section addresses the dye’s interactions in biological systems as relevant to research staining; it does not imply any clinical/therapeutic use.

General biological interactions (literature)

• Charge-driven binding: As a cationic phenothiazinium dye, methylene blue associates with negatively charged biomolecules, notably nucleic acids (DNA/RNA) and acidic polysaccharides/proteoglycans, enhancing contrast in light microscopy.
• Subcellular localization: Staining tends to highlight nuclei and RNA-rich cytoplasmic regions; intensity reflects fixation method, permeability, and ionic conditions.
• Redox behavior: The dye undergoes reversible reduction/oxidation, which can modulate color intensity; hypoxic/reducing microenvironments may transiently lighten stain (leuco form), reoxidizing upon air exposure.
• Aggregation: Dye dimerization and higher-order aggregates occur at elevated concentrations/ionic strengths (literature), affecting spectral properties and potentially staining hue.

Experimental implications

• Fixation dependency: Methanol or heat fixation enhances cell adherence and permeability, improving consistent uptake.
• Ionic strength and pH: Increased ionic strength can weaken electrostatic binding; mild buffers may produce cleaner backgrounds than pure water by controlling pH/ionic competition—optimize empirically.
• Compatibility: Generally compatible with standard aqueous mounting media; certain protein-rich mountants can modestly alter hue due to microenvironment effects.

Note

• The above are literature-based behaviors of methylene blue and not specifications for this particular product. For protocol-critical parameters (e.g., pH of this solution), consult the CoA/Spec Sheet.

Buffer Applications

Not typically applicable.

• This catalog item is a ready-to-use 0.5% methylene blue staining solution rather than a buffering agent or buffering system. It can be used alongside common buffers (e.g., PBS, saline) during staining and rinsing steps, but it is not intended for pH control or buffer preparation.
• For buffer recipes and pH-range selections, consult dedicated buffer products (e.g., phosphate, Tris, HEPES).

Green Alternatives

Context

• This product is an aqueous staining solution, aligning with green chemistry principles by avoiding volatile organic solvents during the staining step. Environmental considerations mainly involve dye persistence and wastewater handling.

Greener practice within methylene blue use (general)

• Minimize waste: Prepare small working volumes; reuse short-term working baths when permissible by your QA practices.
• Capture and treat: Collect spent dye solutions for appropriate disposal; consider activated carbon treatment for adsorptive removal prior to disposal if institutionally supported.
• Energy: Store at room temperature (Product Data), reducing cold-chain energy.

Alternatives and tradeoffs (literature/general)

• Toluidine Blue O (aqueous): Similar cationic dye with metachromatic properties; may allow shorter contact times. Tradeoff: Different specificity and color balance.
• Azure dyes (A/B): Offer nucleic acid preference in some protocols; tradeoff: May require adjusted formulation and differentiation steps.
• Neutral stains (e.g., neutral red) or biological stains like methylene green: Different binding profiles; selection depends on target structures rather than being categorically “greener.”

Comparison snapshot (general)

• Aqueous methylene blue vs. alcohol-based stains
  • Solvent footprint: Water-based is generally greener (lower VOCs).
  • Performance: Alcohol accelerates drying/differentiation but can extract lipids and alter morphology.

Bottom line

• For many routine stains, this aqueous 0.5% methylene blue is already a relatively low-impact choice; focus on waste minimization and proper disposal to enhance sustainability.

Pharmaceutical Uses

Not applicable for this product’s intended use.

• This is a BioReagent-grade laboratory staining solution for research use only (Product Data). No claims are made regarding GMP status, pharmacopeial monographs, or suitability as an excipient.
• Any discussion of therapeutic/clinical applications is outside scope and not implied. For manufacturing or quality requirements beyond research staining (e.g., sterility, endotoxin), consult the CoA/Spec Sheet or select products specifically qualified for those uses.

Physical Properties

Item-specific properties

• Appearance: Not specified for this item; refer to CoA/Spec Sheet. (Typically a blue aqueous solution.)
• Concentration: 0.5% w/v (item-specific, from Product Name).
• Density, pH, refractive index, and conductivity: Not specified for this item; refer to CoA/Spec Sheet.
• Storage conditions: Room temperature (Product Data).

Literature/general properties of methylene blue (dye component; not item-specific)

• Absorption maximum (λmax): ~660–668 nm in water; secondary band ~610–620 nm (literature), with solvatochromic dependence and concentration/dimerization effects.
• Redox behavior: Reversible two-electron reduction to leucomethylene blue; E° values depend on medium and pH (literature).
• Aqueous solubility: High (tens of g/L) for the salt form; solubility increases with ionic strength (literature); prone to aggregation/dimerization at higher concentrations.
• Partitioning: Strongly hydrophilic as the cationic salt; binds to negatively charged substrates (e.g., nucleic acids, acidic polysaccharides) (literature).

Practical implications (general)

• The 0.5% concentration provides strong contrast in typical microbiology and cytology stains with short contact times.
• Spectrophotometric QC can leverage λmax ~664 nm to verify dye presence and monitor photofading; however, quantitative specs for this item are not provided.

Note

• Do not treat these literature values as specifications. For exact pH, conductivity, stabilizers (if any), and UV/Vis profile of this catalog item, consult the CoA/Spec Sheet.

Quality and Grades

Item-specific grade

• Grade: BioReagent (Product Data).

What BioReagent typically signifies (general)

• Intended for laboratory and life-science workflows, such as histology, cytology, and microbiology staining.
• Emphasis on consistency, low levels of common biological interferents, and suitability for routine imaging or educational/industrial QC staining workflows.
• May include internal QC on color strength/absorbance and appearance; however, specific acceptance criteria for this item are Not specified for this item; refer to CoA/Spec Sheet.

Formulation-related quality considerations (general guidance)

• Concentration accuracy: A 0.5% w/v solution should deliver robust staining within short exposures; users may verify with a quick UV–Vis scan at ~664 nm (literature) if method validation is needed.
• Particulate control: Staining solutions should be free of visible particulates to avoid tissue/slide artifacts. If particulates are observed, a 0.22–0.45 μm filtration may improve uniformity (validate for your application).
• Photostability: Phenothiazinium dyes can photobleach; amber containers and light-minimized storage support lot-to-lot utility.
• Bioburden: For microscopy use, low bioburden helps prevent microbial growth and precipitate formation over time. If sterility is required (e.g., certain cell-associated workflows), users should sterile-filter on receipt as appropriate.

What is not claimed for this item

• No claims are made here for clinical/diagnostic grade, pharmacopeial conformity, or trace-metal/purity specifications. For any required attributes (e.g., sterility, pH range, stabilizers, UV cutoff), consult the item’s CoA/Spec Sheet.

Reaction and Applications

Primary use (this item)

• Ready-to-use 0.5% methylene blue for routine biological staining in microscopy and simple visualization workflows (Product Name; research use only).

Typical laboratory applications (literature/general)

• Microbiology: Simple staining of bacteria to assess morphology, cell density, and smear quality. Can serve as a counterstain in some differential methods.
• Cytology/histology: Staining of nuclei and acidic tissue components (RNA-rich regions, cartilage matrix) due to the dye’s cationic nature; useful for rapid intra-procedural checks.
• Yeast and protozoa: Contrast enhancement for counting and viability assessment (in conjunction with other markers as required by protocol).
• Redox indicator: Acts as a qualitative redox reporter in teaching labs (color change to leucomethylene blue under reducing/anaerobic conditions), though this item is formulated for staining rather than analytical standardization.

Practical tips (general)

• Smear prep: Air-dry fixed smears before staining to minimize uneven uptake. Heat or methanol fixation improves adherence.
• Contact time: 10 s to 3 min is common; optimize empirically for your sample and downstream imaging. Overstaining can increase background; controlled rinsing differentiates.
• Rinsing: Use water or isotonic buffer; avoid vigorous streams that can detach delicate monolayers.
• Photoprotection: Limit bright light exposure during prolonged incubations to reduce photofading.
• Surface artifacts: Filter solution if particulates are observed to prevent speckling.

Notes

• While methylene blue participates in photoredox chemistry, this product is intended as a staining solution; it is not positioned as a synthetic reagent. Exact protocol parameters are user- and sample-dependent.

Reaction Conditions

Not directly applicable to chemical synthesis.

• As a staining solution, this product is used under ambient laboratory conditions for biological sample visualization, not for chemical reactions requiring defined stoichiometry, catalysts, or reaction kinetics.
• If employing methylene blue as a redox indicator or in educational redox demonstrations, typical conditions involve aqueous media at room temperature and visible-light exposure; however, such uses are general literature practices and not specifications for this item.

Safety and Handling

GHS information (item-specific)

• Signal word, hazard statements, pictograms, and classification: Not specified for this item; refer to the SDS for authoritative details.

General safety considerations for methylene blue aqueous staining solutions (literature/good practice)

• Hazards: May cause eye/skin irritation and staining; may stain porous materials and laboratory surfaces. Ingestion and inhalation of aerosols should be avoided. Dye solutions may be harmful to aquatic life in concentrated form.
• PPE: Wear lab coat, safety glasses or splash goggles, and appropriate chemical-resistant gloves (e.g., nitrile). Use in a well-ventilated area; avoid aerosol generation.
• Handling: Prevent contact with strong oxidizers and reducing agents that could degrade the dye. Protect from intense light to minimize photobleaching. Avoid contamination of stock with microbial flora; use clean dispensing techniques.
• First aid (overview; follow institutional procedures):
  • Eyes: Rinse cautiously with water for several minutes; remove contact lenses if present and easy to do; seek medical attention if irritation persists.
  • Skin: Wash with soap and water; remove contaminated clothing; persistent irritation warrants medical advice.
  • Inhalation: Move to fresh air; obtain medical attention if symptoms occur.
  • Ingestion: Rinse mouth; do not induce vomiting; seek medical advice.
• Spills: For small spills, absorb with towels or inert material; collect in suitable container. Rinse area with water/detergent; methylene blue can strongly stain surfaces—clean promptly.

Waste disposal

• Collect dye-containing waste according to institutional chemical waste procedures; avoid direct release to drains where restricted. Follow local regulations.

Always consult the product’s SDS for definitive hazard and handling guidance.

Solvent Selection

Applicability

• This product is a ready-to-use aqueous staining solution; independent solvent selection is generally not required for routine use.

Polarity and miscibility (general behavior of the dye solution)

• Matrix: Water-based; fully miscible with water and common biological buffers (e.g., PBS, saline). Dilutable to lower concentrations as needed.
• Compatibility: Can be mixed with alcohols (e.g., ethanol, isopropanol) for differentiated or rapid-stain protocols; note that alcohol can alter dye–substrate interactions and decolorize to a degree.

When to choose this 0.5% solution vs alternatives

• Choose this item when you need a convenient, robust basic (cationic) stain for rapid visualization of cells, bacteria, yeast, or acidic tissue components without preparing stock solutions.
• Consider alternative media or concentrations when:
  • High-resolution nucleic acid specificity is required (e.g., switch to azure B, toluidine blue O, or thionine depending on protocol; literature guidance).
  • Non-aqueous workflows (e.g., resin-embedded samples) demand alcohol-rich or xylene-compatible stains.

Practical notes

• Rinsing/differentiation: Water or mild buffer rinses remove excess unbound dye; brief ethanol rinses can accelerate differentiation.
• Mounting: Aqueous mounting media preserve blue coloration; if using organic mountants, ensure compatibility to prevent dye leaching.

Comparison snapshot (general)

• Aqueous methylene blue vs. toluidine blue O: MB provides strong general cationic staining; TBO shows metachromasia in some tissues (e.g., mast cell granules). Selection depends on required contrast and specificity.

Storage and Reconstitution

Item-specific storage

• Storage conditions: Room temperature (Product Data).
• Shipped: Normal (Product Data).

Reconstitution

• Not applicable: this is a ready-to-use 0.5% aqueous staining solution; no dilution or reconstitution is required unless your protocol specifies a working-strength adjustment.

General handling to maintain performance (good practice)

• Light protection: Store in amber or otherwise protect from strong light to reduce photofading of phenothiazinium dyes.
• Container hygiene: Minimize headspace opening time and avoid tip/sample backflow to reduce contamination; if long-term storage is expected, consider aliquoting for routine use.
• Temperature: Ambient storage is appropriate per Product Data; avoid freezing to prevent possible precipitation or container stress. Avoid prolonged exposure to elevated temperatures.
• Filtration: If visible particulates appear, filter through 0.22–0.45 μm membrane into a clean container; verify staining performance after filtration.

Shelf life and specifications

• Expiry dating, pH, and preservative/stabilizer content (if any) are Not specified for this item; refer to CoA/Spec Sheet.

Research use note

• For research use only (Product Data). Not for human or animal therapeutic or diagnostic use.

Structure and Identity

Item-specific identifiers: Not specified for this item; refer to CoA/Spec Sheet.

About this product

• This listing is a ready-to-use aqueous dye formulation: Methylene Blue Staining Solution (0.5%).
• Grade: BioReagent (Product Data).
• Concentration: 0.5% w/v (Product Name; item-specific).

Methylene blue (literature/general description)

• Common name: methylene blue, basic blue 9; a cationic phenothiazinium dye.
• Structural class: phenothiazine core bearing dimethylamino substituents; exists as a chloride salt in typical lab use; dye is cationic (thiazinium).
• Functional features: extended conjugation, redox-active chromophore capable of reversible reduction to leucomethylene blue.
• 2D structural description (words): tricyclic phenothiazine ring system with a central thiazine (S and N in ring), para-dimethylamino anilinium-like substituent patterns; positively charged heteroaromatic nitrogen, typically paired with Cl−.

Canonical identifiers (literature; for the dye component, not this specific solution)

• Molecular formula (dye, chloride form): commonly reported as C16H18ClN3S (literature; may vary by hydration/impurities).
• Molecular weight (dye, chloride form): ~319.85 g/mol (literature).
• SMILES/InChIKey: Not specified for this item; refer to literature databases if needed for the dye entity.

Important note

• The above identifiers are provided as general background for methylene blue dye. Exact salt form, hydration, and formulation details for this specific 0.5% solution are not specified; consult the item’s CoA/Spec Sheet for definitive identity information.

Synthetic Utility

Limited relevance.

• The product is formulated as a 0.5% aqueous methylene blue staining solution for microscopy and related workflows, not as a reagent for organic synthesis.
• General literature note: Methylene blue can act as a photoredox catalyst or redox mediator in certain synthetic and analytical contexts, but those applications utilize controlled concentrations/solvents distinct from this ready-to-use stain.
• Researchers seeking photoredox catalysts or redox mediators should consult reagent-grade solids or appropriately specified solutions rather than staining formulations.

Target Specificity

Not an antibody or affinity reagent.

• No antigen, epitope, clone, or species specificity applies. Staining arises from electrostatic interactions with negatively charged biomolecules (nucleic acids, acidic polysaccharides) rather than sequence- or epitope-specific binding.
• For molecularly specific detection, combine with immunostaining or nucleic acid probes as appropriate to your study design.