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:
100 µL cultured strain (step 1)
200 µL 0.5 mM histidine/biotin solution
500 µL S9 mixture OR 500 µL sodium phosphate buffer
100 µL of the sample, positive control, or negative control
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.
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:
5 g magnesium sulfate heptahydrate
50 g citric acid monohydrate
250 g potassium phosphate anhydrous, dibasic
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
12.5 mL sodium phosphate buffer (recipe 3)
1 mL NADP solution (recipe 6)
0.125 mL 1 M G6P solution (recipe 7)
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
Sodium azide
2-nitrofluorine
2-antramine
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