Nitrate Iron Agar - BioReagent

Cat. No.: N1528514

GRADE & PURITY BioReagent

Storage

Room temperature

Shipped In

Normal

Übersicht

For selective isolation of Legionella

Specifications

Spezifikationen & Reinheit

BioReagent

Storage

Room temperature

Verschickt in

Normal

Note

BioReagent

Product Line

Biologische Produkte

Application Protocols

General workflow for nitrate reduction and H2S detection on Nitrate Iron Agar (literature/general). Adjust steps to match the item label, CoA, and your SOPs.

1. Medium preparation

• Weigh the manufacturer‑specified grams per liter. Suspend in purified water with heating until agar is fully dissolved.
• Dispense into culture tubes (e.g., 8–10 mL per tube for deep butts). Cap/loosen caps and autoclave at 121 C for ~15 min. Cool upright to solidify.

2. Inoculation

• Using a sterile straight needle, stab inoculate a single, central track to near the tube bottom. Optionally streak the surface.
• Include appropriate positive and negative control organisms with known nitrate/H2S phenotypes.

3. Incubation

• Incubate at organism‑appropriate temperature (commonly 30–37 C) for 18–48 h. Longer may be required for slow growers.

4. Nitrate reduction test

• Add 2–3 drops each of Nitrate Reagent A (sulfanilic acid in acetic acid) and Reagent B (α‑naphthylamine in acetic acid) to the surface.
• Interpret within 5 minutes: Red color = nitrite present (positive for nitrate reduction to NO2−).
• If no color, add a pinch/small amount of zinc dust. Red after zinc = unreduced nitrate (true negative). No color after zinc = nitrate reduced beyond nitrite (positive for denitrification).

5. H2S detection

• Examine the stab line and surrounding medium: Blackening indicates FeS formation from H2S production.

6. Documentation and QC

• Record lot number, incubation conditions, controls, and results. Dispose of inoculated media as biohazard after decontamination.

Note: Volumes, reagent concentrations, and timings should follow validated SOPs.

Biological Roles

This medium interrogates central microbial respiratory pathways and sulfur metabolism (literature/general):

• Nitrate respiration/denitrification:
  • Enzymology: Nitrate reductases (Nar/Nap) reduce NO3− to NO2−. Subsequent nitrite reductases (Nir), nitric oxide reductases (Nor), and nitrous oxide reductases (Nos) can convert NO2− to gaseous N species (NO, N2O, N2).
  • Ecological role: Denitrification supports anaerobic energy conservation in diverse bacteria (e.g., Pseudomonas, Paracoccus) and influences the nitrogen cycle. In clinical/environmental isolates, nitrate reduction phenotypes assist in species differentiation.
  • Detection chemistry: Nitrite forms a diazonium intermediate with sulfanilic acid; coupling with α‑naphthylamine yields a red azo dye—basis of the classic nitrate test.
• Sulfur metabolism and H2S:
  • H2S may arise from reduction of thiosulfate/sulfate or from desulfurylation of cysteine by cysteine desulfhydrase. Generated sulfide reacts with Fe2+ in the medium to form insoluble FeS (black precipitate) along the stab line.
• Semi‑solid matrix effects:
  • The agar concentration creates an oxygen gradient: surface aerobic, deeper anaerobic. This supports detection of anaerobic nitrate respiration and may reveal motility patterns (diffuse growth from the stab path).

Interpretation caveats (literature): Organisms that reduce nitrate fully to gaseous end‑products will test negative with reagents A/B but remain negative after zinc addition (true positive for complete denitrification). False negatives may occur if medium composition, pH, or incubation conditions are suboptimal.

Buffer Applications

This product is not designed or used as a buffer system. While some culture media include phosphate or other salts that incidentally provide buffering capacity, Nitrate Iron Agar is intended for microbiological differentiation rather than pH control. For dedicated buffer preparations (e.g., phosphate, Tris, HEPES), refer to appropriate buffer products and protocols.

Green Alternatives

Nitrate Iron Agar is an aqueous, solvent‑free preparation based on benign components (peptones, inorganic salts, agar). Environmental considerations and greener practice tips (literature/general):

• Solvent footprint: Water is the sole solvent—no VOC emissions associated with use.
• Energy use: Autoclaving is the primary energy input. Batch media preparation, load optimization, and validated 121 C/15 min cycles minimize energy while ensuring sterility.
• Reagent choices: The colorimetric nitrate test uses sulfanilic acid and α‑naphthylamine. Handle and dispose of these aromatic amines responsibly; consider premade, validated, low‑volume dropper bottles to reduce waste. Avoid chromate‑based nitrate tests (where still in use) in favor of the standard diazotization assay.
• Waste minimization: Prepare only the volume needed; solidify and autoclave spent media prior to disposal. Use re‑usable glass tubes when feasible to reduce plastic waste.
• Alternatives and tradeoffs (literature):
  • Nitrate Broth (no agar) reduces agar use but sacrifices the oxygen‑gradient benefits and H2S readout.
  • Combined multi‑test media (e.g., SIM) can reduce the number of tubes used but may not support nitrate testing, requiring an additional assay.

Always follow institutional environmental health and safety policies for disinfectant selection, autoclave verification, and waste segregation.

Pharmaceutical Uses

No therapeutic or clinical diagnostic use is implied. For research use only (per Product Data).

Industry context (literature/general):

• Microbiology QC labs in pharmaceutical, biotech, food, and environmental sectors may employ nitrate reduction and H2S detection tests during isolate characterization or method development. Choice of medium and acceptance criteria must be defined in site‑specific SOPs.
• Pharmacopeial status: Not specified for this item; refer to CoA/Spec Sheet for any compendial references or suitability testing claims.
• Role in workflows: Supports phenotypic differentiation complementary to modern identification methods (e.g., MALDI‑TOF MS, biochemical panels). May be used to verify metabolic capabilities of production or environmental isolates as part of hygiene monitoring or contamination investigations.
• Documentation: Maintain traceability to lot numbers and retain growth‑promotion/performance test records when used in regulated environments.

Note: Any use in GMP settings requires formal qualification (sterility, growth promotion with control strains, pH verification) under your quality system. This product is not cleared for clinical diagnostic procedures.

Physical Properties

For a complex dehydrated culture medium, item‑specific physicochemical specifications are not provided unless on the CoA.

• Appearance (dehydrated powder): Not specified for this item; refer to CoA/Spec Sheet. (Literature: typically a tan to beige free‑flowing powder.)
• Prepared medium appearance (literature): semi‑solid, clear to slightly opalescent; darkening/blackening may occur upon H2S production after inoculation due to FeS.
• Solubility: Intended to disperse/dissolve in purified water with heating (literature). Do not use organic solvents.
• pH (at 25 C, prepared medium): Not specified for this item; refer to CoA/Spec Sheet. (Literature for nitrate media is often near neutral, e.g., ~7.0–7.4 before sterilization.)
• Melting/softening: Governed by agar gelation; no discrete melting point (literature: agar gels set ~35–40 C and melt ~85–90 C).
• Density, refractive index, logP, pKa, UV cutoff: Not applicable or not defined for complex mixtures; Not specified for this item; refer to CoA/Spec Sheet.
• Autoclave stability (literature): Nutrient/ion components are generally heat‑stable at 121 C for 15 minutes; avoid overheating/extended sterilization to minimize Maillard reactions and nutrient degradation.
• Typical preparation strength: Not specified for this item; refer to CoA/Spec Sheet. (Literature guidance: weigh per label instructions to achieve manufacturer’s recommended grams per liter.)

Note: Always verify lot‑specific pH range, fill volumes, and g/L on the product label or CoA before use.

Quality and Grades

• Supplied grade: BioReagent (per Product Data). This denotes suitability for laboratory research workflows, including preparation of culture media and biochemical assays, but is not a certification of clinical/diagnostic use.
• Item‑specific specifications (e.g., microbial performance, growth promotion, moisture content, pH range): Not specified for this item; refer to CoA/Spec Sheet.
• Performance considerations (general, literature):
  • Growth/biochemical performance in nitrate/H2S assays depends on precise composition, pH, and semi‑solid consistency. Iron(II) availability and nitrate concentration must be within validated ranges to avoid false negatives/positives.
  • Reduced agar content is important to produce a clear stab track and to limit oxygen diffusion for anaerobic nitrate respiration assessment.
• Stabilizers/additives: Not specified for this item; refer to CoA/Spec Sheet. (Literature: some media incorporate phosphate buffers or reducing agents; exact presence should be confirmed per lot.)
• Documentation: For regulated laboratories, retain the manufacturer’s CoA and any growth‑promotion or performance testing results. Lot‑to‑lot verification can include control organisms with known nitrate/H2S phenotypes (e.g., Enterobacteriaceae strains) under laboratory SOPs.
• UV absorbance/trace metals/contaminants: Not specified for this item; refer to CoA/Spec Sheet.

Note: If compendial compliance (e.g., pharmacopeial suitability tests) is required, confirm against the CoA and perform in‑house qualification with appropriate reference strains.

Reaction and Applications

Chemical reaction applications are not the primary use of this product. Instead, Nitrate Iron Agar is used for microbiological biochemical assays. Below are key laboratory applications and interpretation principles (literature/general):

• Nitrate reduction test:
  • Principle: Many bacteria respire anaerobically by reducing nitrate (NO3−) to nitrite (NO2−) via nitrate reductase; some carry out further reduction to N2O/N2 (denitrification).
  • Workflow: After incubation, add Nitrate Reagent A (sulfanilic acid in acetic acid) and Reagent B (α‑naphthylamine in acetic acid). A red azo dye indicates nitrite presence (positive for nitrate reduction to nitrite). If no color develops, add zinc dust: a red color after zinc indicates unreduced nitrate (true negative), while no color after zinc suggests complete reduction beyond nitrite (positive for denitrification).
• H2S detection:
  • Principle: H2S produced from sulfur‑containing substrates reacts with iron(II) to form insoluble FeS, giving blackening along the stab line.
• Semi‑solid format advantages:
  • Creates an oxygen gradient to favor anaerobic respiration in deeper regions; supports assessment of motility by diffuse growth from the stab.
• Typical use cases:
  • Characterization of Enterobacteriaceae and other Gram‑negative rods; differentiation of environmental isolates; educational microbiology labs.

Note: Exact reagent recipes, volumes, and incubation parameters should follow the product label/SOP. This medium is for research use only.

Reaction Conditions

Classical “reaction conditions” do not apply to this formulated medium; however, the performance of the nitrate/H2S microbiological assays depends on preparation and incubation parameters (literature/general):

• Medium preparation:
  • Weigh the manufacturer‑specified amount of dehydrated medium per liter (per label/CoA). Suspend in purified water with gentle heating to fully dissolve/disperse agar.
  • Dispense into tubes (deep butt recommended for oxygen gradient), cap loosely, and autoclave at 121 C for ~15 minutes. Allow to cool and solidify in an upright position.
• Inoculation and incubation:
  • Stab inoculate with a straight needle to near the tube bottom; optionally streak the slant/surface if present.
  • Incubate aerobically at the organism‑appropriate temperature (often 30–37 C) for 18–48 h; extended incubation may be needed for slow reducers.
• Nitrate test reagents (post‑incubation):
  • Add a few drops of Nitrate Reagent A (sulfanilic acid) and B (α‑naphthylamine). Observe for red color within minutes.
  • If negative, add a small amount of zinc dust to reveal unreduced nitrate (red = true negative; no color = complete reduction beyond nitrite).
• H2S readout:
  • Observe for black FeS precipitate along the stab line or spreading from it.

Always follow your SOPs and the product label for exact quantities, sterilization times, and safety practices.

Safety and Handling

• GHS/CLP status for this specific item: Not specified for this item; refer to SDS for authoritative hazard classification, pictograms, signal word, and H‑statements.
• General hazards (literature): Dehydrated culture media may cause eye, skin, and respiratory irritation as nuisance dust. Prepared/inoculated media must be handled as potential biohazard according to organism biosafety level (BSL) requirements.
• PPE: Laboratory coat, gloves (nitrile), and safety goggles. Use a dust mask/respirator if dust generation is likely. For inoculated cultures, follow BSL‑appropriate PPE and containment practices.
• Engineering controls: Prepare and dispense media near a fume hood or dust‑controlled area to limit aerosolized powder. Inoculation and manipulations should be performed in a certified biological safety cabinet when working with BSL‑2+ organisms.
• Sterilization: Autoclave at 121 C for 15 min (literature) unless label indicates otherwise. Do not autoclave zinc dust or nitrate reagents in the same vessel; add test reagents aseptically after incubation.
• Incompatibilities: Strong oxidizers may degrade organic components; moisture uptake causes clumping and affects performance. Store tightly closed and desiccated. Avoid light/air exposure that may promote oxidation of iron(II) salts in prepared media.
• First aid (overview): In case of eye/skin contact with powder, rinse with water for several minutes. If inhaled, move to fresh air. If ingested, rinse mouth. Seek medical attention if irritation persists. Defer to SDS for detailed measures.
• Waste disposal: Autoclave or chemically disinfect spent/inoculated media before disposal according to institutional biosafety and local regulations.

Solvent Selection

This product is a culture medium and is intended to be prepared in purified water; organic solvent selection is not applicable.

• Solvent for preparation: Use high‑purity water (e.g., distilled, deionized, or ASTM/ISO Type II or better) to avoid inhibitory ions (chlorine, heavy metals) that can affect microbial growth and the nitrate/H2S readouts.
• Water quality guidance (literature):
  • Conductivity/resistivity: Prefer ≥1 MΩ·cm resistivity or better to minimize ionic variability.
  • TOC and endotoxin control are generally not critical for basic biochemical differentiation tests but good laboratory‑grade water reduces background variability.
• When to choose this medium vs alternatives:
  • Choose Nitrate Iron Agar for simultaneous assessment of nitrate reduction (via post‑incubation colorimetric reagents) and H2S production (black FeS along stab).
  • Use Nitrate Broth (no agar) when only nitrate reduction is evaluated and turbid growth assessment is preferred.
  • Use SIM medium or TSI/KIA agars when broader sulfur reduction, indole, motility, and carbohydrate fermentation profiles are required.

Comparison (literature, qualitative):

• Nitrate Iron Agar: semi‑solid; nitrate test + H2S capture; stab inoculation.
• Nitrate Broth: liquid; nitrate test only; avoids oxygen gradient effects.
• SIM medium: semi‑solid with thiosulfate + iron; H2S + indole + motility; no nitrate test.

Storage and Reconstitution

• Storage conditions (per Product Data): Room temperature. Keep container tightly closed in a cool, dry place. Protect from moisture uptake; use a desiccant if available. Avoid prolonged exposure to light and air to limit oxidation of iron(II) components after preparation.
• Shipped in: Normal (ambient) conditions (per Product Data).
• Shelf life and retest: Not specified for this item; refer to CoA/Spec Sheet.
• Reconstitution (literature/general):
  • Weigh the manufacturer‑specified grams per liter of dehydrated medium.
  • Suspend in purified water with heating until fully dissolved. Do not boil excessively.
  • Dispense into tubes or bottles, cap loosely, and sterilize by autoclaving at 121 C for ~15 min, unless label indicates otherwise.
  • Allow to cool and solidify upright if dispensing as deep butts; avoid condensation and excessive drying.
• Prepared medium storage (literature): Store sealed tubes/bottles at 2–8 C and use within a few weeks for optimal performance. Warm to room temperature before inoculation. Discard if drying, cracking, contamination, or precipitate unrelated to inoculation is observed.
• Freeze–thaw: Do not freeze dehydrated powder or prepared agar; freezing disrupts gel integrity.
• Research use note: For research use only (per Product Data). Always consult the product CoA/SDS and follow institutional SOPs for handling and disposal.

Structure and Identity

Nitrate Iron Agar is a complex, multi‑component microbiological culture medium formulated to assess nitrate reduction and detect H2S production in facultative and anaerobic bacteria. As a formulated medium, it is not a single defined chemical entity and therefore lacks a unique CAS number, SMILES, or InChIKey.

• Item identity: Nitrate Iron Agar (SKU: N1528514), BioReagent grade
• Composition nature (general, literature): typically contains a peptone/protein digest base and inorganic salts; nitrate (e.g., potassium nitrate) as terminal electron acceptor; reduced agar concentration for stab cultures; and an iron(II) salt (e.g., ferrous sulfate or ferrous ammonium sulfate) as an H2S indicator forming black FeS precipitate. Exact formulation for this item: Not specified for this item; refer to CoA/Spec Sheet.
• Functional components (general description):
  • Carbon/nitrogen sources to support growth
  • Phosphate or similar salts to moderate pH (if present)
  • Nitrate source for respiratory reduction testing
  • Iron(II) salt to capture H2S as FeS (blackening)
  • Agar (semi‑solid) to enable stab‑line assessment and limit oxygen diffusion
• Structural features: As a heterogeneous mixture, the “structure” is an ensemble of amino acids/peptides (from peptones), carbohydrates (if present), and inorganic ions; no single 2D molecular structure applies.
• Molecular formula: Not applicable to complex mixture.
• Molecular weight: Not applicable to complex mixture.

Use context: Designed for differential biochemical testing—nitrate respiration (nitrate → nitrite → N2/N2O) and H2S production (sulfide + Fe2+ → FeS).

Synthetic Utility

Not applicable. Nitrate Iron Agar is a formulated microbiological medium, not a discrete synthetic reagent or building block. It is not typically used in chemical synthesis, catalysis, or materials fabrication. For chemical transformations involving nitrate, nitrite, or iron salts, use defined reagents with known purity and specifications. Refer instead to the Reaction & Applications and Application Protocols sections for microbiological use guidance.

Target Specificity

Not applicable. This product is a general microbiological medium and does not possess molecular target specificity (no antigen/epitope, clone, isotype, or species‑restricted reactivity). It supports growth and differential biochemical testing across a range of bacteria capable of nitrate reduction and/or H2S production.