Ames Test Protocol

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

1. 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.
2. On the day of the experiment, prepare stock solutions of the positive control mutagens for tubes containing S9 and those without.
3. Label 10 mL Falcon tubes appropriately for each strain. See Table 2 for the necessary conditions.
4. Prepare minimal glucose plates.
   1. Aliquot 20 mL into each 90mm x 15 mm Petri plate.
5. Label all minimal glucose agar plates.
6. Prepare heated agar solution. Keep around 45°C in a water bath.
7. Aliquot 5 mL of the heated agar into each 10 mL Falcon tube.
8. 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
9. Vortex each 10 mL tube quickly and distribute evenly onto minimal glucose plates.
10. Let the mixture cool for 2-3 mins.
11. Cover plates with aluminum foil to protect them from light.
12. Incubate for 48 hours at 37°C. Examine for colony formation.
13. 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 (return to protocol)

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

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

1. Heat 335 mL of dH₂O to 45°C
2. 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
3. Keep the solution on a hot plate
4. Once everything has dissolved, transfer the solution into a new bottle. Autoclave the solution around 120°C for 20 mins
5. Allow time for the solution to cool off
6. Seal the bottle and store it at 4°C

0.2 M sodium phosphate buffer (return to protocol)

1. Dissolve 6.9 g sodium dihydrogen phosphate monohydrate into 250 mL dH₂O
2. In a separate container, dissolve 7.1 g disodium hydrogen phosphate into 250 mL dH₂O
3. 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
4. Measure pH and adjust to 7.4
5. Autoclave the combined solution around 120°C for 20 mins

Minimum glucose mixture (return to protocol)

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

1 M NADP solution (return to protocol)

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

1 M G6P solution (return to protocol)

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

Potassium magnesium solution (return to protocol)

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

S9 mix (return to protocol)

1. 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)
2. b. Do not freeze

Positive control mutagen stock solutions (return to protocol)

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

References

1. 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
2. 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
3. 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
4. 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