Top Agar Medium (Ames Test) - BioReagent

Cat. No.: T1528384

QUALITÄT & REINHEIT BioReagent, Suitable for microbiology

Synonyms

Top Medium

Storage

Protected from light,Room temperature

Shipped In

Normal

Application

1126

Übersicht

Suitable for the Salmonella‑mammalian microsome enzyme system mutagenicity assay (Ames Test) for genotoxicity detection of test articles.

Medium Formula：

Components	Content
Sodium Chloride	5.0 g/L
L‑histidine	7.8 mg/L
Biotin	12.2 mg/L
Agar	6.0 g/L

Instructions for Use:

Weigh 11.0 g of this product and add it into 1 L of distilled or deionized water. Boil for 1 minute to dissolve completely, replenish water to 1000 mL, dispense at 2‑2.5 mL per aliquot, autoclave at 121 ℃ for 15 minutes. Mix well immediately after autoclaving to prevent agar sedimentation and solidification at the bottom. Keep warm at 45 ℃ and set aside for use.

Precautions:

1. pH 7.2±0.2 (25 ℃).

2. Avoid direct contact with skin and eyes during handling. In case of accidental contact, immediately flush with plenty of clean water.

3. This product is for laboratory research purposes only and shall not be used for clinical diagnosis.

Spezifikationen

Synonyme

Top Medium

Spezifikationen & Reinheit

BioReagent, Suitable for microbiology

Stabilität und Lagerung

Store at room temperature long term (36 months).Store in the dark.

Storage

Protected from light,Room temperature

Verschickt in

Normal

Note

BioReagent, Suitable for microbiology

Product Line

Biologische Produkte

Chemische und physikalische Eigenschaften

• Empfindlichkeit: Light-sensitive

Application Protocols

The following is general literature guidance for using top agar in Ames tests. Item-specific instructions are not specified; follow your SOP/regulatory guideline (e.g., OECD 471).

Plate incorporation method (literature):

• Pre-warm minimal agar plates to room temperature.
• Hold molten top agar at ~45 °C.
• In a sterile tube, combine: 0.1 mL overnight bacterial culture, 0.5 mL S9 mix or buffer (as required), and test article at desired dose in compatible vehicle.
• Add ~2.0 mL molten top agar; mix gently but quickly to avoid bubbles.
• Immediately pour onto a minimal agar plate and rotate to spread evenly.
• Allow to solidify on a level surface; invert and incubate ~37 °C for ~48 h.
• Count revertant colonies; compare against vehicle and positive controls.

Preincubation method (literature):

• In a sterile tube, preincubate 0.1 mL bacterial culture with test article ± S9 for ~20–30 min at 37 °C with gentle shaking.
• Add ~2.0 mL molten top agar, mix, and pour as above.

Best practices:

• Use strain-specific positive controls each run.
• Limit solvent to guideline-acceptable levels to avoid toxicity (e.g., DMSO ≤ recommended percent; consult SOP).
• Verify even lawn formation and absence of contamination; record lot numbers, temperatures, and times for traceability.

Biological Roles

• Functional role: Serves as a semi-solid growth matrix enabling uniform distribution and limited diffusion of bacteria and test chemicals in the Ames assay, facilitating precise scoring of revertant colonies (literature).
• Nutritional context (literature):
  • Top agar overlays for Salmonella typically include sodium chloride and may include trace amounts of L-histidine and biotin to support limited, transient growth until reversion; E. coli WP2 variants may utilize tryptophan supplementation. The minimal nature of the overlay ensures that only revertant mutants form prototrophic colonies.
• Microbiological behavior:
  • Provides a soft gel consistency that allows colony expansion without overgrowth into confluent lawns.
  • Maintains local microenvironments for interaction between cells, mutagens, and metabolic activation systems (e.g., S9).
• Relevance to assay biology:
  • The restricted nutrient environment and physical immobilization maximize signal-to-background ratio for detecting reverse mutations.
  • Consistency of gel strength and supplement concentration is crucial to maintain expected baseline revertant counts for negative controls.
• Note for this item: Specific supplement content and concentrations are not specified; users should consult the item’s CoA/Spec Sheet and align preparation with their validated method and the tester strains employed.

Buffer Applications

This product is a culture medium rather than a classical buffering agent. Nevertheless, pH control is important for Ames overlays (literature):

• pH environment (literature): Ames top agar is commonly used near neutral pH (approximately 7.2–7.6) to support tester strain viability and S9 enzyme activity. The bottom minimal agar and any diluents/vehicle contribute to final pH.
• Preparation approaches (literature):
  • Some laboratories prepare top agar using a buffered saline base (e.g., phosphate-buffered saline) to stabilize pH during holding and pouring; others rely on the buffering capacity of the complete assay mixture (bacteria + vehicle ± S9).
• For this item: Specific buffer system, if any, and target pH are not specified and must be confirmed from the CoA/Spec Sheet or internal SOPs.

If a dedicated buffer solution is required for your validated method, prepare it separately according to guideline documents (e.g., OECD 471) and confirm compatibility with your tester strains and S9 mix.

Green Alternatives

Top agar media are water-based and agar is a renewable, seaweed-derived polysaccharide, which aligns with several green chemistry/green lab principles. While “alternatives” are less about replacing the base matrix and more about optimizing the workflow, the following sustainability considerations apply (literature/general):

• Matrix choice:
  • Agar (renewable) vs. synthetic gelling agents (e.g., polyacrylamide for other applications): Agar offers biodegradability and avoids toxic monomers.
• Energy and resource use:
  • Optimize batch size to reduce repeated autoclave cycles.
  • Use insulated water baths and tight-fitting lids to minimize heat loss when holding molten agar at ~45 °C.
• Waste management:
  • Segregate uncontaminated packaging for recycling; autoclave biological waste prior to disposal to prevent contamination.
• Reusability/reduction:
  • Prepare only the volume needed per assay day to limit discard of solidified excess.
• Comparison snapshot (literature):
  • Conventional practice: Frequent small autoclave runs, excess overlay prepared “just in case,” increased energy/waste.
  • Optimized practice: Planned batching, validated hold times, and prewarmed dispensers to reduce waste and energy.

Tradeoffs: Longer hold times at 45–50 °C can lead to increased water loss and potential changes in gel texture; balance energy savings with performance consistency. Always verify that any sustainability change does not impact revertant counts or guideline compliance.

Pharmaceutical Uses

• Intended use domain: Research use only (per Product Data). Not for diagnostic or therapeutic applications.
• Role in pharmaceutical R&D/QC (literature):
  • Widely employed in nonclinical genotoxicity screening workflows (Ames test) that support decision-making in drug discovery and development.
  • Used by quality and safety laboratories to assess mutagenic potential of active ingredients, intermediates, impurities, and degradants in alignment with ICH, OECD, and regional regulatory expectations.
• Methodological fit:
  • Suitable for plate incorporation and preincubation variants of the Ames test, with or without S9 metabolic activation, as defined in SOPs.
• Compliance note: While the Ames test is part of standardized genotoxicity batteries, this product itself is not certified for GMP or compendial use unless explicitly stated. Lot qualification with positive/negative controls is recommended for regulated environments.
• Documentation: Record lot numbers and preparation details in notebooks or electronic systems to maintain data integrity and traceability for regulatory submissions.

Item-specific pharmacopeial status or certifications: Not specified for this item; refer to CoA/Spec Sheet.

Physical Properties

• Physical state/appearance: Not specified for this item; refer to CoA/Spec Sheet. (Ames top agar media are commonly supplied as a dry, free-flowing powder; literature.)
• Odor: Not specified for this item.
• pH (prepared): Not specified for this item; laboratories commonly target near-neutral pH for Ames overlays (e.g., ~7.2–7.6, literature), adjusted by preparation buffer.
• Solubility: Dispersible in water upon heating; agar dissolves above its melting/solubilization temperature (literature).
• Gelation/melting (literature values for agar matrices):
  • Gelation (setting) temperature typically ~32–40 °C.
  • Melting temperature typically ~80–95 °C, depending on agar grade and concentration.
• Density, refractive index, logP, pKa: Not applicable for a complex medium; not specified for this item.
• Typical working concentrations (literature): "Soft"/top agar overlays are often prepared at ~0.6–0.75% agar (w/v) with salts; exact values should follow the validated SOP for the assay.
• Clarifying note: The above are general literature characteristics of agar-based overlays. This item’s definitive specifications (e.g., pH of a defined preparation, recommended agar percentage, any supplements present) are not provided here and should be verified on the CoA/Spec Sheet.

Quality and Grades

• Item grade: BioReagent (from Product Data). This denotes suitability for general laboratory biological applications, prepared and packaged to minimize bioburden and meet consistency expectations for research workflows.
• What BioReagent typically implies (general guidance):
  • Raw materials and processing selected for biological assays (e.g., microbiology, cell-free systems).
  • Controlled lot-to-lot variability, suitable for qualitative/quantitative research use.
  • Not intended for clinical/diagnostic use or GMP manufacturing unless otherwise specified.
• Item-specific specs such as pH of prepared medium, gel strength, supplementation (histidine/biotin/tryptophan), metal content, or UV impurities: Not specified for this item; refer to CoA/Spec Sheet.
• Stabilizers/preservatives: Not specified for this item.
• Practical notes for Ames testing (literature):
  • Consistency of soft-agar viscosity is critical for even lawn formation and reproducible revertant counts.
  • If supplemented top agar is used, confirm the supplement concentration aligns with your validated method (e.g., OECD 471, EPA 870.5100, ICH S2). Record lot IDs in your assay records.
• Suitability checks: Run positive/negative controls at receipt of a new lot to confirm expected revertant ranges for each Salmonella/E. coli tester strain.

Reaction and Applications

Chemical reaction use is not applicable. Instead, this medium is used in the Ames bacterial reverse mutation assay.

• Core application (literature): Formation of a thin, low-strength agar overlay containing the tester strain (e.g., Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA102; or E. coli WP2 uvrA) and the test article, poured onto minimal agar plates. Mutagenicity is assessed by counting revertant colonies.
• Modes of use:
  • Plate incorporation method: Mix bacterial culture, top agar, and test article (± S9) and pour immediately over minimal glucose agar plates.
  • Preincubation method: Incubate bacteria with test article (± S9) briefly before adding molten top agar and plating; can increase sensitivity for certain chemicals.
• Metabolic activation: Incorporation of an S9 liver fraction (typically Aroclor-1254- or phenobarbital/β-naphthoflavone-induced rat liver; literature) within the top agar mix or added during preincubation, to reveal promutagens requiring metabolic activation.
• Practical tips (literature):
  • Maintain molten top agar at ~45 °C to avoid thermal killing while preventing premature solidification.
  • Work rapidly and uniformly to ensure even overlays and consistent lawn density.
  • Validate with strain-specific positive controls each run.
  • Minimize air bubbles; allow plates to set on a level surface before incubation.
• Readout: After incubation (often 48 h at 37 °C; literature), count revertant colonies; compare with solvent controls and historical ranges per guideline.

Reaction Conditions

Chemical reaction parameters do not apply. The relevant “conditions” pertain to microbiological preparation and assay setup (literature/general guidance):

• Sterilization: Autoclave the medium as directed by your SOP (commonly 121 °C for ~15 min) if supplied non-sterile. Avoid overheating to prevent caramelization or texture changes.
• Holding temperature: Maintain molten top agar at ~45 °C (range ~43–48 °C) prior to mixing with bacteria/test article to prevent thermal damage and premature solidification.
• Overlay thickness: Typical pour volumes are ~2 mL per 90-mm plate; adjust per guideline to ensure even coverage without excessive thickness.
• Incubation: Plates are commonly incubated inverted at ~37 °C for ~48 h (strain- and method-dependent) before counting revertants.
• Additives: Metabolic activation (S9 mix) is included as required by the test design. Avoid solvents or concentrations that are cytotoxic to tester strains or inhibit S9 enzymes.
• Controls: Include vehicle control and strain-specific positive controls each run to verify assay sensitivity and historical control ranges.

For this item, any manufacturer-recommended preparation parameters are not specified; follow your validated SOP and consult the CoA/Spec Sheet.

Safety and Handling

• GHS classification, signal word, pictograms, H-statements: Not specified for this item; refer to SDS.
• General hazards (literature/practice):
  • Dry media dust may cause mechanical irritation to eyes/respiratory tract—avoid inhalation and contact.
  • Heated molten agar presents burn risk; handle with heat-resistant gloves and caution around steam/autoclaves.
  • After inoculation with bacteria or S9 mix, materials are potentially biohazardous; handle as per BSL-1/2 as appropriate and autoclave waste before disposal.
• PPE: Lab coat, safety glasses/goggles, disposable gloves; dust mask/respirator where dust generation cannot be controlled (per risk assessment/SDS).
• Incompatibilities (general): Strong oxidizers/strong acids and bases are not typically used with prepared agar. Avoid contamination with solvents or reagents that may inhibit bacterial growth in the assay.
• First aid (overview; defer to SDS):
  • Inhalation: Move to fresh air; seek medical attention if irritation persists.
  • Skin/eye contact: Rinse with water for several minutes; remove contaminated clothing; seek medical advice if irritation continues.
  • Ingestion: Rinse mouth; obtain medical advice.
• Spill/cleanup: Avoid dust formation; sweep/vacuum with HEPA filtration; dispose per institutional biosafety procedures (autoclave if contaminated).
• Fire: Agar-based media are combustible when dry; use water spray, CO2, foam, or dry chemical; firefighters wear SCBA.

Solvent Selection

This product is a dehydrated culture medium intended to be reconstituted with water; organic solvent selection is generally not applicable.

• Reconstitution vehicle: High-purity water (e.g., ASTM Type 1 or equivalent) is typically used (literature). Sterility is essential; use freshly prepared sterile water or sterilize by autoclaving when appropriate.
• Miscibility profile: Upon heating, agar dissolves in water to form a homogeneous solution; upon cooling, it forms a gel. Insoluble in most organic solvents (literature).
• When choosing water quality (practical guidance):
  • Use low-conductivity, low-TOC water to minimize inhibitory contaminants that could affect bacterial growth or mutagen response.
  • Avoid residual disinfectants (chlorine/chloramine) and chelating agents.
• Comparison (literature):
  • Top agar overlay vs. broth overlays: Agar overlays immobilize bacteria and test chemical for spatially resolved revertant scoring; broths do not provide this feature.
• Bottom line: Focus on water purity, sterility, and proper temperature control rather than solvent polarity considerations.

Storage and Reconstitution

• Storage conditions (as supplied): Room temperature (from Product Data). Protect from moisture; keep container tightly closed. Store away from microbial contamination sources.
• Shipping: Normal (from Product Data).
• Shelf life/expiry: Not specified for this item; refer to CoA/Spec Sheet.

Reconstitution and sterilization (general literature guidance; not item-specific):

• Water: Use sterile, high-purity water. Disperse the medium gradually with stirring to prevent clumps.
• Heating: Heat until fully dissolved; avoid localized overheating. If supplied non-sterile, autoclave (e.g., 121 °C for ~15 min) per SOP.
• Holding: Cool molten medium to ~45 °C before mixing with bacteria/test articles to prevent thermal injury.
• Supplements: If your method requires histidine/biotin or tryptophan supplementation, add sterile-filtered supplements to cooled molten top agar aseptically. The presence and levels of any supplements in this item are not specified.
• Working aliquots: Maintain molten top agar in a 45 °C water bath; minimize hold time to preserve performance and volume (evaporation).
• Freeze–thaw: Not applicable for dry medium. Do not freeze prepared agar; store prepared molten agar only short-term at 45 °C. Discard if contamination or premature gelation occurs.

Always consult the product’s CoA/Spec Sheet and your validated SOP for exact preparation parameters and volumes for your assay.

Structure and Identity

• Item type: Microbiological culture medium (overlay “top agar” for Ames mutagenicity assay).
• Composition details: Not specified for this item; refer to CoA/Spec Sheet.
• Single-compound identifiers (CAS, InChIKey, SMILES, molecular formula, molecular weight): Not applicable to a blended medium and not specified for this item.
• Structural features: As a formulated blend, top agar does not have a single defined molecular structure. It typically contains a gelling polysaccharide (agar) and inorganic salts; some variants are supplemented with trace amounts of L-histidine and biotin for Salmonella assays or tryptophan for E. coli WP2 assays (literature), but the exact composition must be confirmed from the item’s documentation.
• 2D/functional description (literature): The medium functions as a low-strength gel overlay. After melting and cooling to ~45 °C, it remains pourable; once spread over minimal agar plates, it solidifies to immobilize bacteria and test articles uniformly, enabling clear scoring of revertant colonies in the Ames test.
• Identity checks in practice (literature):
  • Gelation behavior consistent with agar-based media.
  • Sterility pre-use (no turbidity after hold), and pH near neutrality when prepared per method SOP.
  • Supports a limited number of background colonies for control strains when supplemented per method requirements.

Synthetic Utility

Not applicable. This product is a formulated microbiological medium and is not used as a reagent in chemical synthesis. For chemists seeking soft-gel matrices for chemical separations or immobilization, agar/agarose systems exist, but those applications are distinct from the Ames top agar use-case. Refer instead to the sections on Reaction & Applications and Application Protocols for assay-relevant guidance.

Target Specificity

Not applicable. This is not an antibody, enzyme inhibitor, or target-directed reagent. It is a culture medium used broadly with Ames tester strains (e.g., Salmonella typhimurium and E. coli WP2 derivatives) as defined by your assay design.