Redox Indicator · Orthogonal Colorimetric Reference · v3.4

Anaerobic Indicator & Buffer Reference Chart

Display Mode Showing: Indicator in aqueous solution

Indicator
Only

+ Broth
Mix

Broth baseline
1% Peptone + 0.25% YE

Compensation Parameters

🌡️

Temperature ref. 25 °C (Nernst)

0 °C 100 °C

25 °C

At reference — no compensation applied

⬡

Concentration ref. 1 mg L⁻¹ (Beer-Lambert)

1 5 mg L⁻¹

1.0 mg L⁻¹

At reference — no compensation applied

Compensated E°′ (pH 7): MB +0.011 V RZ₁ −0.051 V / RZ₂ −0.105 V IC −0.125 V NR −0.325 V · pKₐ 6.80

Apparent colour of 1 mg L⁻¹ aqueous indicator solutions as a function of pH and dissolved-oxygen tension. pH rows ordered by each indicator's principal acid–base transition interval. Toggle Display Mode to compare pure-indicator colours against those observed in peptone/yeast-extract broth (1% w/v + 0.25% w/v).

GMExpression

O₂ Level

Aerobic

Microaerophilic

Anaerobic (Colourless / Broth in mix mode)

Strict Anaerobic ≤0.001 mg L⁻¹

EOS <0.5 ppm gas-phase

Indicator

	Methylene Blue MB · C₁₆H₁₈ClN₃S E°′ = +0.011 V (pH 7)					Resazurin RZ · C₁₂H₇NO₄ E°′ = −0.051 / −0.105 V					Indigo Carmine IC · C₁₆H₈N₂Na₂O₈S₂ E°′ = −0.125 V (pH 7)					Neutral Red NR · C₁₅H₁₇ClN₄ · pKₐ 6.8 E°′ = −0.325 V (pH 7)
	☀ Aerobic [O₂]≥0.1 mg L⁻¹	◑ Microaerophilic 0.01–0.1 mg L⁻¹	☽ Anaerobic ≤0.01 mg L⁻¹	☾☾ Strict Ana. ≤0.001 mg L⁻¹	⬤ EOS <0.5 ppm gas	☀ Aerobic	◑ Microaerophilic	☽ Anaerobic	☾☾ Strict Ana.	⬤ EOS	☀ Aerobic	◑ Microaerophilic	☽ Anaerobic	☾☾ Strict Ana.	⬤ EOS	☀ Aerobic	◑ Microaerophilic	☽ Anaerobic	☾☾ Strict Ana.	⬤ EOS
Strongly AcidicpH < 4.0	Deep Blue	Mid Blue	Colourless	Colourless	Colourless	Amber (pH-shift)	Pale Peach	Peach-Pink	Faint Peach	Colourless	Deep Indigo	Olive Green	Yellow-Green	Pale Yellow	Very Pale Yellow	Bright Red	Bright Red	Bright Red	Faded Red	Colourless
Moderately AcidicpH 4.0 – 6.0	Deep Blue	Sky Blue	Colourless	Colourless	Colourless	Blue-Violet	Salmon Pink	Salmon Pink	Faint Pink	Colourless	Indigo Blue	Forest Green	Golden Yellow	Pale Yellow	Very Pale Yellow	Red	Red	Red	Pale Rose	Colourless
Weakly AcidicpH 6.0 – 6.8	Rich Blue	Pale Blue	Colourless	Colourless	Colourless	Indigo	Rose Pink	Rose Pink	Faint Rose	Colourless	Indigo	Bright Green	Bright Yellow	Pale Yellow	Very Pale Yellow	Orange-Red	Orange-Red	Orange-Red	Pale Orange	Colourless
Near-NeutralPhysiologicalpH 6.8 – 8.0	Vivid Blue	Steel Blue	Colourless	Colourless	Colourless	Deep Indigo	Hot Pink / Magenta	Hot Pink	Faint Pink	Colourless	Blue-Indigo	Mid Green	Yellow	Pale Yellow	Very Pale Yellow	Orange (pKₐ zone)	Orange (pKₐ zone)	Orange	Pale Amber	Colourless
AlkalinepH > 8.0	Bright Blue	Light Blue	Colourless	Colourless	Colourless	Cobalt Blue	Vivid Magenta	Vivid Magenta	Faint Magenta	Colourless	Indigo-Blue	Light Green	Bright Yellow	Pale Yellow	Very Pale Yellow	Amber Yellow	Amber Yellow	Amber Yellow	Pale Yellow	Colourless

Physicochemical Properties & Reduction Mechanisms

Methylene Blue (MB)

FormulaC₁₆H₁₈ClN₃S

Mᵣ319.85 g mol⁻¹

E°′ (pH 7, 25 °C)+0.011 V

Electrons / step2e⁻ + 2H⁺

Oxidised λmax664 nm (Blue)

Reduced formColourless (leuco-MB)

pH sensitivityLow

ReversibilityFully reversible

Working conc.0.5–2 mg L⁻¹

Reduction Mechanism — Single-Step Reversible

MBBlue · Oxidised

⇌+ 2H⁺ + 2e⁻
Reversible

Leuco-MBColourless · Reduced

pH effect: Phenothiazine chromophore is pH-insensitive (pH 3–11). Re-oxidation to blue occurs spontaneously upon O₂ re-exposure.

Resazurin (RZ)

FormulaC₁₂H₇NO₄

Mᵣ229.19 g mol⁻¹

E°′ RZ→Resorufin−0.051 V

E°′ Resorufin→DH−0.105 V

Oxidised λmax600 nm (Blue)

Intermediate λmax573 nm (Pink)

Reduced formColourless (dihydroresorufin)

pH sensitivityHigh — bathochromic < pH 3.8

Isobestic point~602 nm

Reduction Mechanism — Sequential Two-Step

ResazurinBlue · Oxidised

→+ 2H⁺ + 2e⁻
Irreversible

ResorufinPink · Intermediate

⇌+ 2H⁺ + 2e⁻
Reversible

DihydroresorufinColourless · Reduced

pH < 3.8: Protonation of the oxazine nitrogen causes a bathochromic shift: Resazurin → amber/orange; Resorufin → pale peach. pH-driven, not redox-driven.

Indigo Carmine (IC)

FormulaC₁₆H₈N₂Na₂O₈S₂

Mᵣ466.36 g mol⁻¹

E°′ (pH 7, 25 °C)−0.125 V

Electrons / step2e⁻ + 2H⁺

Oxidised λmax610 nm (Indigo Blue)

IntermediateGreen (semi-leucoindigo)

Reduced formYellow (leucoindigo)

pH sensitivityLow — colours pH-stable

Unique feature3 visually distinct states

Reduction Mechanism — Two-Step (3 Colour States)

Indigo
CarmineIndigo · Oxidised

⇌+ 2H⁺ + 2e⁻
Reversible

Semi-leuco-
indigoGreen · Intermediate

⇌+ 2H⁺ + 2e⁻
Reversible

LeucoindigoYellow · Fully Reduced

Diagnostic advantage: Three chromatically distinct states (blue → green → yellow) observable under ambient illumination. Both steps are reversible upon re-oxidation.

Neutral Red (NR)

FormulaC₁₅H₁₇ClN₄

Mᵣ288.78 g mol⁻¹

E°′ (pH 7, 25 °C)−0.325 V

pKₐ6.8

Acid form λmax540 nm (Red, pH < 6.8)

Base form λmax460 nm (Amber-Yellow, pH > 8)

Reduced formColourless (leuco-NR)

Dual indicatorpH and redox

pH sensitivityVery high

Dual Mechanism — Acid–Base + Redox

① Acid–Base Equilibrium (pH indicator)

NRH⁺Red · pH < 6.8

⇌− H⁺ (pKₐ 6.8)
Reversible

NRAmber-Yellow
pH > 8.0

② Redox Reduction

NR (ox.)Red/Yellow
(pH-dependent)

⇌+ 2H⁺ + 2e⁻
Reversible

NRH₂ (red.)Colourless
Fully Reduced

Caution: NR responds independently to both pH and Eh. Red → yellow may indicate alkalinisation rather than reduction. E°′ = −0.325 V renders NR the most reductive-condition-sensitive indicator in this panel.