Ames Test Protocol

Background

The Ames test is a widely used assay for evaluating the mutagenicity of a chemical substance. It uses several strains of auxotrophic Salmonella typhimurium, known to carry a mutation that prevents histidine biosynthesis, an essential amino acid for their growth. As a result, histidine must be supplemented in the culture media. Otherwise, his^- S. typhimurium cannot grow. In the Ames test, mutagenicity is determined by exposing his^- S. typhimurium to varying concentrations of the suspected substance and then selecting for reversion events. To facilitate selection, bacteria must be inoculated on HIS-selective media. This allows only bacteria that have undergone reverse mutations and reverted to their prototrophic state to survive and grow. A positive test, illustrated by the formation of visible colonies, indicates the chemical is mutagenic.

Fig. 1

Illustration of Ames Test protocol (created in BioRender).

This protocol includes three S. typhimurium strains - TA 98, TA 100, and TA 102 - (table 1) that are offshoots of the original strains used by Ames. In addition to lacking proper histidine biosynthesis, they contain the R-factor, which has mutations that impair their ability to repair DNA and to properly form the lipopolysaccharide cell wall. The impaired DNA repair enhances the mutation rate, and the abnormal cell wall formation allows the entry of compounds that would normally be unable to enter the intracellular space. As a result, these strains have a higher basal mutation rate and are more accessible to possible mutagens. The three strains also include plasmids to detect what type of mutation occurred.

Further adaptions to the Ames test include rodent liver extract (S9). Bacteria lack the metabolic capabilities of higher-ordered organisms, and the inclusion of liver extract enables additional biochemical reactions to occur. In organisms with hepatic systems, enzymes like P450 catalyze many xenobiotics. The metabolized product may become mutagenic, so including liver extract makes it possible to discover mutagenic metabolites that wouldn't be detected with just the bacteria.

Experimental Conditions

Each experiment requires the following conditions for each of the three strains. The appropriate positive control depends on whether the S9 mix is present. Strains TA 98, TA 100, and TA 102 that have the S9 mix present require 2-anthramine as a positive control (2 µg/mL). If there is no S9 mix present, then use the following as a positive control: sodium azide (1 µg/mL), 2-nitrofluorine (1 µg/mL), and mitomycin C (125 ng/mL). The negative control is distilled water (dH₂O) for samples with or without the S9 mix.

Materials

Petri plates, sterile. 90 mm diameter, 15 mm depth
Sodium azide
2-aminofluorene
Mitomycin C
200 proof/100% ethanol, laboratory-grade
Magnesium sulphate heptahydrate
Ammonium sodium phosphate dibasic tetrahydrate
Potassium chloride
Oxoid nutrient broth #2
Citric acid monohydrate
Potassium phosphate anhydrous, dibasic
L-histidine hydrochloride
D-biotin
NADP sodium salt
Magnesium chloride hexahydrate
Sodium hydroxide
Hydrochloric acid (for adjusting the pH of sodium phosphate buffer)
Disodium hydrogen phosphate
Glucose-6-phosphate (G6P) monosodium
Agar

Protocol

Set up new colonies for each strain. Each S. typhimurium strain should be incubated overnight in Oxoid nutrient broth #2 at 37°C on a shaker set to 120 rpm.
On the day of the experiment, prepare stock solutions of the positive control mutagens for tubes containing S9 and those without.
Label 10 mL Falcon tubes appropriately for each strain. See Table 2 for the necessary conditions.
Prepare minimal glucose plates.
- Aliquot 20 mL into each 90mm x 15 mm Petri plate.
Label all minimal glucose agar plates.
Prepare heated agar solution. Keep around 45°C in a water bath.
Aliquot 5 mL of the heated agar into each 10 mL Falcon tube.
Add the following to each tube containing heated agar:
1. 100 µL cultured strain (step 1)
2. 200 µL 0.5 mM histidine/biotin solution
3. 500 µL S9 mixture OR 500 µL sodium phosphate buffer
4. 100 µL of the sample, positive control, or negative control
5. 100 µL dH₂O
Vortex each 10 mL tube quickly and distribute evenly onto minimal glucose plates.
Let the mixture cool for 2-3 mins.
Cover plates with aluminum foil to protect them from light.
Incubate for 48 hours at 37°C. Examine for colony formation.
The colonies should be distributed evenly.

For proper statistical analysis and comparison, refer to the: Statistical analysis of the Ames Salmonella/microsome test - PubMed.

Recipes

0.5 mM histidine+biotin stock solution

Heat 125 mL dH₂O to 45°C
Dissolve 15.45 mg D-biotin and 12 mg L-histidine hydrochloride in the prewarmed dH₂O
Autoclave the solution around 120°C for 20 mins and store at 4°C

Vogel-Bonner Medium E (VBME) 50X stock solution

Heat 335 mL of dH₂O to 45°C
Dissolve the following into the prewarmed dH₂O:
1. 5 g magnesium sulfate heptahydrate
2. 50 g citric acid monohydrate
3. 250 g potassium phosphate anhydrous, dibasic
4. 87.5 g sodium ammonium phosphate
Keep the solution on a hot plate
Once everything has dissolved, transfer the solution into a new bottle. Autoclave the solution around 120°C for 20 mins
Allow time for the solution to cool off
Seal the bottle and store it at 4°C

0.2 M sodium phosphate buffer

Dissolve 6.9 g sodium dihydrogen phosphate monohydrate into 250 mL dH₂O
In a separate container, dissolve 7.1 g disodium hydrogen phosphate into 250 mL dH₂O
In a sterile third container, aliquot 30 mL of the solution in step 4a and then add 220 mL of the solution from step 4b to a total of 250 mL
Measure pH and adjust to 7.4
Autoclave the combined solution around 120°C for 20 mins

Minimum glucose mixture

Dissolve 7.5 g fresh agar in 465 mL dH₂O
Autoclave the solution around 120°C for 20 mins. Allow time for the solution to cool
Aliquot 25 mL 40% glucose
Aliquot 10 mL 50X VBME stock solution from recipe 2

1 M NADP solution

Dissolve 191.5 mg NADP (sodium salt) into 2.5 mL dH₂O
Vortex the mixture, and once mixed, keep it in an ice bath
Otherwise, store at 4°C for up to 6 months

1 M G6P solution

Dissolve 1.41 g G6P in 5 mL dH₂O
Vortex the mixture, and once mixed, keep it in an ice bath
Otherwise, store at 4°C for up to 6 months

Potassium magnesium solution

Dissolve 30.75 g potassium chloride and 20.35 g magnesium chloride (hexahydrate) into 250 mL dH₂O
Autoclave the solution around 120°C for 20 mins and store at 4°C

S9 mix

Measure out 9.86 mL dH₂O and add the following solutions in order
1. 12.5 mL sodium phosphate buffer (recipe 3)
2. 1 mL NADP solution (recipe 6)
3. 0.125 mL 1 M G6P solution (recipe 7)
4. 0.5 mL magnesium chloride/potassium chloride solution (recipe 8)
Do not freeze

Positive control mutagen stock solutions

Dissolve 10 µg of the compound in 990 µL dH₂O
1. Sodium azide
2. 2-nitrofluorine
3. 2-antramine
4. Mitomycin

References

Langer, P. J., Shanabruch, W. G., & Walker, G. C. (1981). Functional organization of plasmid pKM101. Journal of bacteriology, 145(3), 1310–1316. https://doi.org/10.1128/jb.145.3.1310-1316.1981
Margolin, B. H., Kaplan, N., & Zeiger, E. (1981). Statistical analysis of the Ames Salmonella/microsome test. Proceedings of the National Academy of Sciences of the United States of America, 78(6), 3779–3783. https://doi.org/10.1073/pnas.78.6.3779
Rodríguez, E., Piccini, C., Sosa, V., & Zunino, P. (2012). The use of the ames test as a tool for addressing problem-based learning in the microbiology lab. Journal of microbiology & biology education, 13(2), 175–177. https://doi.org/10.1128/jmbe.v13i2.421
Vijay, U., Gupta, S., Mathur, P., Suravajhala, P., & Bhatnagar, P. (2018). Microbial Mutagenicity Assay: Ames Test. Bio-protocol, 8(6), e2763. https://doi.org/10.21769/BioProtoc.2763

Document: 02.0187.221116r1

Last updated Tue Sep 09 2025