Resazurin

Key takeaways

Resazurin belongs to biology; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Resazurin to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Resazurin from memory before moving on to harder problems.

Reference excerpt

Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a phenoxazine dye that is weakly fluorescent, nontoxic, cell-permeable, and redox‐sensitive. Resazurin has a blue to purple color above pH 6.5 and an orange color below pH 3.8. It is used in microbiological, cellular, and enzymatic assays because it can be irreversibly reduced to the pink-colored and highly fluorescent resorufin (7-Hydroxy-3H-phenoxazin-3-one). At circum-neutral pH, resorufin can be detected by visual observation of its pink color or by fluorimetry, with an excitation maximum at 530-570 nm and an emission maximum at 580-590 nm. When a solution containing resorufin is submitted to reducing conditions (Eh < -110 mV), almost all resorufin is reversibly reduced to the translucid non-fluorescent dihydroresorufin (also known as hydroresorufin) and the solution becomes translucid (the redox potential of the resorufin/dihydroresorufin pair is -51 mV vs. standard hydrogen electrode at pH 7.0). When the Eh of this same solution is increased, dihydroresorufin is oxidized back to resorufin, and this reversible reaction can be used to monitor if the redox potential of a culture medium remains at a sufficiently low level for anaerobic organisms.

Resazurin solution has one of the highest values known of Kreft's dichromaticity index. This means that it has a large change in perceived color hue when the thickness or concentration of observed sample increases or decreases. Usually, resazurin is available commercially as the sodium salt.

Cell viability applications Resazurin is reduced to resorufin by aerobic respiration of metabolically active cells, and it can be used as an indicator of cell viability. It was first used to quantify bacterial content in milk by Pesch and Simmert in 1929. It can be used to detect the presence of viable cells in mammalian cell cultures. It was introduced commercially initially under Alamar Blue trademark (Trek Diagnostic Systems, Inc), and now also available under other names such as AB assay, Vybrant (Molecular Probes) and UptiBlue (Interchim). Resazurin based assays show excellent correlation to reference viability assays such as formazan-based assays (MTT/XTT) and tritiated thymidine based techniques. The low toxicity makes it suitable for longer studies, and it has been applied for animal cells, bacteria, and fungi for cell culture assays such as cell counting, cell survival, and cell proliferation. In antimicrobial assays, resazurin is commonly utilized to assess the minimum inhibitory concentration (MIC) or minimum bactericidal concentration (MBC) of antimicrobial agents. To take the place of a standard live/dead assay, resazurin also be multiplexed with chemiluminescent assays, such as cytokine assays, caspase assays to measure apoptosis, or reporter assays to measure a gene or a protein expression. The irreversible reaction of resazurin to resorufin is proportional to aerobic respiration.

Resazurin reduction test Resazurin can be used as one of a series of rapid tests to determine the quality of a milk sample. In this test, resazurin is added as a violet redox dye which turns mauvish-pink due to conversion to resorufin and then to colourless dihydroresorufin. This happens due to lowering of the oxidation-reduction potential in the milk sample caused by presence of bacteria which utilize available oxygen present in the milk for aerobic respiration. The rate of the colour change is used as an index for the number of bacteria present in the milk sample.

Other applications

Resazurin is effectively reduced in mitochondria, making it useful also to assess mitochondrial metabolic activity. Usually, in the presence of NADPH dehydrogenase or NADH dehydrogenase as the enzyme, NADPH or NADH is the reductant that converts resazurin to resorufin. Hence the resazurin/diaphorase/NADPH system can be used to detect NADH, NADPH, or diaphorase level, and any biochemical or enzyme activity that is involved in a biochemical reaction generating NADH or NADPH. Resazurin can be used to assay L-Glutamate, achieving a sensitivity of 2.0 pmol per well in a 96 well plate. Resazurin can also be used to measure the aerobic biodegradation of organic matter found in effluents. Resazurin is used to measure the amount of aerobic respiration in streams. Since most aerobic respiration occurs in the stream bed, the conversion of resazurin to resorufin is also a measure of the amount of exchange between the water column and the stream bed.

Synthesis Resazurin is prepared by acid-catalyzed condensation between resorcinol and 4-nitrosoresorcinol followed by oxidation of the intermediate with manganese(IV) oxide:

Treatment of the crude reaction product with excess sodium carbonate yields the sodium salt of resazurin, which is typically the commercial form of the dye. Running the condensation step in alcohols is possible but results in lower yields of the product; in pure water or acetic acid, the reaction does not proceed satisfactorily.

Related dyes 10-acetyl-3,7-dihydroxyphenoxazine (also known as Amplex Red), structurally related to resazurin, reacts with H2O2 in a 1:1 stoichiometry to produce the same by-product resorufin (used in many assays combining for example horseradish peroxidase (HRP), or NADH, NADPH using enzymes). 7-ethoxyresorufin, a compound used as the substrate in the measurement of cytochrome P450 (CYP1A1) induction using the ethoxyresorufin-O-deethylase (EROD) assay system in cell culture and environmental samples, produced in response to exposure to aryl hydrocarbons. The compound is catalysed by the enzyme to produce the same fluorescent product, resorufin. 1,3-dichloro-7-hydroxy-9,9-dimethylacridin-2(9H)-one (DDAO dye), a fluorescent dye used for oligonucleotide labeling.

References

Illustrations

Worked examples

Example 1 — a first encounter with Resazurin

Start with the simplest possible case. Write down what Resazurin claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In biology, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Resazurin before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Resazurin ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Resazurin

In research: Resazurin appears in biology research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Resazurin in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Resazurin is common in secondary-school and first-year university syllabi. It links to neighbouring topics Amine oxides, Cell culture reagents, Oxazine dyes, so understanding it makes those chapters shorter.
In everyday life: Look for Resazurin outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

How to study Resazurin in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Resazurin means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain Resazurin out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Resazurin in simple terms?

Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a phenoxazine dye that is weakly fluorescent, nontoxic, cell-permeable, and redox‐sensitive. Resazurin has a blue to purple color above pH 6.5 and an orange color below pH 3.8.

Why does Resazurin matter?

Because it connects several biology ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Resazurin?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Resazurin.