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Short answer: The Ames assay (bacterial reverse mutation test) exposes specially engineered bacterial strains that cannot make an essential amino acid to a test substance, with and without a liver-derived metabolic activation mix (S9), and counts how many colonies regain the ability to grow. A reproducible, concentration-related rise in revertant colonies indicates the substance is mutagenic in that system. It is a screen for gene mutation, not a verdict on cancer risk in people.
Definition and scope
The Ames assay is the common name for the bacterial reverse mutation test described in OECD Test Guideline 471 and in the U.S. FDA’s Redbook 2000 chapter IV.C.1.a. The test strains carry a mutation that makes them dependent on an added amino acid. A mutagen can reverse that defect, so only revertant bacteria form colonies on minimal medium. The assay is one of the standard in vitro genotoxicity tests; for how genotoxicity testing fits into pharmaceutical development see the ICH M3(R2) guide, for medical-device genotoxicity see ISO 10993-3, and for the wider discipline see what is toxicology.
How the assay works in practice
Strains
FDA’s Redbook describes a set of five strains: four S. typhimurium strains (TA1535; TA1537, TA97 or TA97a; TA98; TA100) plus either E. coli WP2 uvrA (or uvrA pKM101) or S. typhimurium TA102. The Redbook notes that TA102 and the E. coli strains help detect oxidizing mutagens, cross-linking agents and hydrazines that guanine-cytosine (GC) base-pair strains may miss. A 2018 paper in Genes and Environment reporting positive-control ranges lists the OECD 471 recommended strains as TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
Laboratories verify each stock before use: the amino-acid requirement and marker phenotypes are checked, and spontaneous revertant counts are compared with historical data. The markers named in the 2018 paper are amino-acid dependence, rfa (crystal violet sensitivity), pKM101 (ampicillin resistance) and the uvr deletion.
Metabolic activation (S9)
Many compounds become mutagenic only after metabolism, which bacteria lack. The assay is therefore run with and without S9, a liver fraction usually from rats treated with an enzyme inducer (Aroclor 1254, or phenobarbitone plus beta-naphthoflavone). The Redbook specifies 10 to 30 percent v/v S9 in the S9 mix and a sterility test for each batch.
Concentrations, replicates and controls
- Concentrations: at least five analyzable concentrations, spaced at about half-log intervals; for soluble, non-cytotoxic substances the maximum is 5 mg/plate or 5 µl/plate.
- Replicates: triplicate plating at each dose level, with duplicate only if justified.
- Controls: a concurrent solvent (vehicle) negative control and strain-specific positive controls, each with and without S9; an untreated control as well unless historical data show the solvent is harmless. FDA states that 2-aminoanthracene should not be the sole indicator of S9 efficacy.
- Conditions: incubation at 37 °C for 2 to 3 days; a preincubation step, where used, is usually at least 20 minutes at 30 to 37 °C.
The 2018 paper names AF-2, sodium azide, 9-aminoacridine and 2-aminoanthracene among positive controls, and gives an example protocol of 20 minutes’ preincubation at 37 °C, top agar on minimal glucose agar, and 48 hours’ incubation before revertants are counted.
Reading Ames assay results
Under the Redbook, a result is positive when there is a concentration-related increase, and/or a reproducible increase at one or more concentrations, in at least one strain, with or without S9. FDA adds that statistical significance should not be the only determining factor. Weak or equivocal results should be verified, possibly under modified conditions. The Redbook (IV.C.1) pairs bacterial gene-mutation testing with other genotoxicity tests, so an Ames result is read alongside them rather than alone.
What to check in an Ames assay report
This checklist restates the Redbook and OECD 471 elements above as questions a lab or study sponsor can put to a report. It is a reading aid, not a regulatory acceptance test.
| Report element | What the sources say it should show |
|---|---|
| Strains | The strain set, with amino-acid requirement and marker phenotypes verified |
| Spontaneous revertants | Counts compared with historical data |
| Metabolic activation | Results with and without S9; S9 source and inducer; sterility check |
| Concentrations | At least five analyzable concentrations, with the top dose within the 5 mg or 5 µl per plate limit for soluble non-cytotoxic substances |
| Replicates | Triplicate plating, or a justification for duplicate |
| Controls | Solvent and strain-specific positive controls with and without S9; untreated control unless solvent history supports omission |
| Interpretation | Concentration-related and/or reproducible increase, not statistics alone; follow-up of equivocal results |
Common misunderstandings
- “Ames positive means it causes cancer in people.” The assay detects mutagenicity in bacteria. It is one input to genotoxicity assessment, not a carcinogenicity finding.
- “One strain is enough.” The guidance specifies a set that includes TA102 or E. coli WP2 so that mutagen classes GC-pair strains may miss are covered.
- “S9 is optional.” Testing is specified with and without S9, because some substances only act after metabolic activation.
- “Only a statistically significant increase counts.” FDA says significance should not be the only determining factor.
FAQ
What is the Ames assay test?
It is a bacterial reverse mutation test in which engineered, amino-acid-dependent strains are exposed to a substance and the colonies that revert to growing without the amino acid are counted.
What is the principle of the Ames assay?
A mutagen can reverse the mutation that makes the strain dependent on an amino acid, so revertant colonies appear on minimal medium, and more colonies at higher concentrations indicate mutagenic activity.
What counts as a positive Ames result?
Per FDA’s Redbook, a concentration-related increase and/or a reproducible increase at one or more concentrations in at least one strain, with or without S9.
Why is S9 used?
Bacteria lack mammalian metabolism, so a rodent liver fraction is added to reveal substances that become mutagenic only after metabolic conversion.
Sources
- U.S. FDA, Redbook 2000, IV.C.1.a Bacterial Reverse Mutation Test (July 2000), read 2026-11-07.
- Genes and Environment (2018), positive-control ranges for the bacterial reverse mutation test, PMC5883876, read 2026-11-07.
