Resazurin Sodium Salt l ASD-622
Resazurin Sodium Salt
Redox indicator for anaerobic culture media, microbial viability assays, AlamarBlue-type cell metabolic activity, and antibiotic susceptibility testing. Blue → Pink → Colourless on reduction.
Resazurin Sodium Salt is a phenoxazine redox indicator dye that undergoes a well-defined, visually striking colour change in response to cellular metabolic activity and reducing conditions. It is one of the most versatile indicators in microbiology and cell biology — used as an anaerobic media indicator, a viability dye, and a metabolic activity reporter.
Non-fluorescent
Fluorescent λᴇm ~590 nm
Non-fluorescent
Colour change is irreversible in the first step (resazurin → resorufin) and reversible in the second (resorufin ⇌ dihydroresorufin).
Resazurin (blue, oxidised form) is reduced to resorufin (pink/red, fluorescent) by cellular reductants — primarily NADH, NADPH, and the flavoenzymes of the electron transport chain in metabolically active cells. This reduction reflects cellular metabolic activity and is proportional to the number of viable, metabolically active cells. The resorufin product can be further reduced to the colourless, non-fluorescent dihydroresorufin by highly reduced environments or dense cell populations.
In anaerobic media, resazurin serves as an anaerobiosis indicator — the medium turns from blue/purple (oxidised) to pink/colourless (reduced) as oxygen is consumed, confirming that anaerobic conditions have been achieved. Re-oxygenation causes the medium to return to blue (the reaction is partially reversible).
1. Anaerobic Media Indicator
| 1 | Prepare stock solution: Dissolve 0.1 g Resazurin Sodium Salt (ASD-622) in 100 mL sterile distilled water (1 mg/mL stock). Protect from light. Store at 4°C. |
| 2 | Add to medium: Add 1 mL of stock per litre of anaerobic broth or agar after autoclaving (≤50°C). Final concentration: 1 mg/L (1 ppm). Mix gently. |
| 3 | Interpret: Blue/purple = oxidised (aerobic — do not inoculate). Pink or colourless = reduced (anaerobic conditions achieved — safe to inoculate). Discard or re-reduce if medium re-oxidises before use. |
2. AlamarBlue-Type Microbial Viability Assay
| 1 | Prepare working solution: Dilute stock to 0.01% w/v (100 µg/mL) in sterile PBS or culture medium. Filter-sterilise (0.22 µm). |
| 2 | Add to cell/microbial suspension: Add resazurin working solution to wells containing organisms at 10% v/v final concentration. Incubate at 37°C (bacteria) or 30°C (yeasts). |
| 3 | Read: Measure fluorescence (Ex 560 nm / Em 590 nm) or absorbance (570 nm vs 600 nm reference) at 1–4 h intervals. Viable, metabolically active cells reduce resazurin → resorufin → fluorescence increase proportional to cell number. |
| 4 | Antibiotic susceptibility (REMA/MABA): Inoculate test organism into antibiotic dilution series in 96-well plate. Add resazurin at 0.002% final concentration. Incubate 5–7 days (Mtb) or 24–48h (rapid growers). Pink = growth (resistant). Blue = inhibited (susceptible). MIC = lowest concentration maintaining blue colour. |
| Indicator | Redox Change | Fluorescent? | Primary Use |
|---|---|---|---|
| Resazurin (ASD-622) | Blue → Pink → Colourless | Yes (resorufin, 590 nm) | Anaerobic media; viability; REMA/MABA; milk testing |
| Methylene Blue (ASD-601) | Blue → Colourless (leucoform) | No | Redox indicator; bacterial staining; RNA gels |
| Neutral Red | Red → Yellow (pH dependent) | No | pH indicator in media; cell staining |
| MTT | Yellow → Purple (formazan) | No | Cell viability (requires solubilisation step) |
| XTT | Orange → Red | No | Cell viability (soluble product, no wash step) |
| Manufacturer | Product Name | Cat. No. | Notes |
|---|---|---|---|
| Sigma-Aldrich (Merck) | Resazurin sodium salt | R7017 | Equivalent grade; CAS 62758-13-8 |
| Merck Millipore | Resazurin sodium salt | 1.01605 | Equivalent grade |
| Thermo Fisher Scientific | Resazurin, Sodium Salt | R12204 | Equivalent grade |
| BioLegend (ThermoFisher) | AlamarBlue® Cell Viability Reagent | DAL1025 | Proprietary formulation; active ingredient = resazurin |
AuSaMicS ASA-2059 is supplied locally in Melbourne at significantly lower cost than proprietary AlamarBlue formulations — same compound, same performance.
| Product Name | Resazurin Sodium Salt |
| Catalogue Number | ASA-2059 |
| IUPAC Name | Sodium 7-hydroxy-3H-phenoxazin-3-one 10-oxide |
| Synonyms | Resazurin sodium; Diazoresorcinol sodium; AlamarBlue active component; CI 51010 |
| CAS Number (sodium salt) | 62758-13-8 |
| CAS Number (free acid) | 550-82-3 (not this product) |
| Molecular Formula | C₁₂H₆NNaO₄ |
| Molecular Weight | 251.17 g/mol |
| Grade | Laboratory Grade |
| Purity | ≥98.0% |
| Appearance | Dark green to brownish-purple crystalline powder with metallic lustre |
| Solution colour (oxidised) | Blue (dilute aqueous solution) |
| Reduction product | Resorufin (pink/red, fluorescent; λex ~560 nm / λem ~590 nm) |
| Solubility (water) | Freely soluble — ~3.5 g/100 mL at 25°C |
| Solubility (ethanol) | Slightly soluble |
| λmax (aqueous, oxidised) | ~600 nm (blue/violet) |
| Fluorescence (resorufin) | λex 560 nm / λem 590 nm |
| Application | Working Concentration | Detection | Notes |
|---|---|---|---|
| Anaerobic media indicator | 0.5–1 mg/L (0.5–1 ppm) | Visual (blue → pink) | Add as aqueous stock post-autoclave |
| Microbial viability assay | 0.002–0.01% w/v | Fluorescence or colorimetric | 10% v/v of 0.02–0.1 mg/mL working solution |
| REMA / MABA (TB MIC) | 0.002% w/v final | Visual (blue = inhibited) | Add 20µL of 0.02% to 96-well plates |
| Milk reductase test | ~0.005–0.01% w/v | Visual (blue → colourless) | Time to decolourisation indicates microbial load |
| Storage Temperature | 2–8°C; protect from light |
| Container | Tightly sealed, amber or light-resistant container |
| Protect From | Direct light (photosensitive), heat above 40°C, moisture |
| Shelf Life (powder) | ≥3 years from manufacture date |
| Aqueous Stock (1 mg/mL) | Stable 1–3 months at 4°C in dark. Discard if colour changes significantly or precipitate forms. |
| # | Reference |
|---|---|
| 1 | O'Brien J, et al. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem. 2000;267(17):5421–5426. [Mechanism of resazurin reduction in cell viability assays] |
| 2 | Palomino JC, et al. Resazurin Microtiter Assay Plate: simple and inexpensive method for detection of drug resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother. 2002;46(8):2720–2722. [Original REMA method paper] |
| 3 | Collins L, Franzblau SG. Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium. Antimicrob Agents Chemother. 1997;41(5):1004–1009. [MABA method development] |
| 4 | Nakayama GR, et al. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J Immunol Methods. 1997;204(2):205–208. [Resazurin viability assay validation] |
| 5 | ISO 11283:1997. Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of β-glucuronidase-positive Escherichia coli. [References resazurin in anaerobic media context] |
| Product Name | Resazurin Sodium Salt |
| Catalogue No. | ASA-2059 |
| Supplier | AuSaMicS Pty Ltd | ABN 56 676 640 467 |
| Address | 31 Longview CT, Thomastown VIC 3074, Australia |
| Emergency | Poisons Information Centre: 13 11 26 (24 hr) |
| Phone | +61 412 520 598 |
| support@ausamics.com.au |
| GHS Classification | Eye Irrit. 2 — H319: Causes serious eye irritation Aquatic Chronic 3 — H412: Harmful to aquatic life with long-lasting effects |
| Signal Word | WARNING |
| GHS Pictograms | ⚠️ Exclamation mark | 🌿 Environmental hazard |
| Note | Relatively low hazard profile at laboratory use concentrations. Primary concern is eye irritation and aquatic toxicity. |
| Inhalation | Remove to fresh air. Seek medical attention if symptoms persist. |
| Skin Contact | Wash with soap and water. Note: may stain skin pink/purple — not a hazard, fades within hours. |
| Eye Contact | Irrigate immediately with water ≥15 min, hold eyelids apart. Seek medical attention if irritation persists. |
| Ingestion | Rinse mouth. Not expected to be acutely toxic at laboratory quantities. Call Poisons Centre 13 11 26 if large amounts ingested. |
| Extinguishing Media | Water spray, foam, dry powder, CO₂ |
| Respiratory Protection | P1 filter when weighing bulk powder |
| Eye Protection | Safety glasses — eye staining risk from solutions |
| Skin Protection | Nitrile gloves, lab coat. Resazurin stains skin and clothing — use disposable gloves. |
| Aquatic Toxicity | Harmful to aquatic life with long-lasting effects (H412). Avoid bulk discharge to waterways. |
| Disposal | Dispose of solutions as chemical waste per local EPA regulations. Small volumes may be diluted and disposed via trade waste drain per institutional policy. |
| Transport | Not classified as dangerous goods — ADG, IMDG, IATA |
| Regulatory Framework | Australian WHS Regulations 2023 | GHS 7th Edition | Safe Work Australia COP 2021 |
| AICIS Status | Listed on Australian Inventory of Industrial Chemicals (AIIC) |
| Poisons Schedule | Not scheduled under SUSMP |
| Parameter | Specification | Method |
|---|---|---|
| Appearance | Dark green to brownish-purple crystalline powder with metallic lustre | Visual |
| Purity | ≥98.0% | HPLC |
| λmax (aqueous, oxidised) | 600 ± 5 nm | UV-Vis spectrophotometry |
| Water Solubility | Complete dissolution in water at 3.5 g/100 mL; blue solution | Visual inspection |
| Redox Response | Colour change blue → pink on addition of ascorbic acid (reducing agent) — confirms active redox indicator function | Functional redox test |
| Loss on Drying | ≤2.0% | Loss on drying (60°C / 2h) |
| Heavy Metals | ≤20 ppm | ICP-OES |
| Batch COA | Available for every production lot | On request or included |
31 Longview CT, Thomastown VIC 3074, Australia | ABN 56 676 640 467
✓ HPLC purity verification and functional redox test per batch
✓ COA, TDS, SDS included with every order
✓ Australian stock — no import delays
✓ Cost-effective alternative to proprietary AlamarBlue formulations
✓ Available in 1 g, 5 g, 10 g, 25 g, 100 g pack sizes