mutagenicity test

["Collaborating Expert | Jiang Jiahong, M.S.", "Biophysics Dalian Maritime University"], ["Reviewed Expert | Nie Yifeng, Ph.D.", "Nano-biomedicine University of Chinese Academy of Sciences"]

Principle

The Microbial Ames Test, fully known as the Contaminant Mutagenicity Assay, is a simple, rapid and powerful bacterial assay consisting of different strains of Salmonella typhimurium/Escherichia coli and applications for determining mutagenic potential.

The Ames test utilizes histidine-deficient strains of Salmonella typhimurium, which are unable to synthesize histidine, so that only a few spontaneous revertant mutants grow on histidine-deficient media; in the presence of a mutagenic agent, the nutrient-deficient bacteria induce a revertant mutation into a prototrophic form, and thus grow to form colonies, which are then determined to be mutagenic or not.

Appliance

Evaluates the mutagenicity of various environmental carcinogens and toxins and is widely used in carcinogen screening.

Operation method

Mutagenicity testing of contaminants: Ames test

Principle

The microbial Ames test, known as the Contaminant Mutagenicity Assay, is a simple, rapid and powerful bacterial assay consisting of different strains of Salmonella typhimurium/E. coli and applications for determining mutagenic potential.The Ames test utilizes histidine-nutrient-deficient strains of Salmonella typhimurium, which are incapable of synthesizing histidine, and therefore only a few spontaneously revert to mutant bacteria grow in histidine-deficient media; if a mutagen is present, the nutrient-deficient bacteria induce revert mutation to the native form and therefore grow to form colonies. The Ames test utilizes a histidine-deficient strain of Salmonella typhimurium, which is unable to synthesize histidine. Therefore, on a medium lacking histidine, only a few spontaneous revertant mutant bacteria will grow.

Materials and Instruments

0.5 mM histidine/biotin solution, nutrient broth, agar, 2-aminoanthracene, mitomycin, sodium azide, 2-nitrofluoride, sodium phosphate buffer, rat liver S9 mix, ampicillin
Sodium azide, 2-nitrofluoride, sodium phosphate buffer, rat liver S9 mix, ampicillin
Glucose, histidine, tryptophan, biotin, MgSO
4
MgSO 4 -7H
2
O, sodium citrate-2H
2
O
K2HPO4 -3H2O, KH2PO4, (NH4)2SO
4
Nicotinamide adenine dinucleotide phosphate (NADP), glucose-6-phosphatase (G-6-P)
KCl, MgCl
2
etc.

Move

(1) Bacterial freezing and thawing solution was mixed with nutrient broth and placed in a water bath shaker at 37 ℃ and 120 r/min for 10 h. After amplification, the optical density was detected using an enzyme marker, and the concentration of viable bacteria was estimated to reach ^1×109/mL and then used in the experiment;

(2) Prepare fresh mutagen for each experiment (see recipe).

Negative control: pressurized distilled water

For TA 98, TA 100 and TA 102 without S9 metabolic activation Positive control (S9 mixture): sodium azide (1 μg/ml), 2-nitrofluoro (1 μg/ml) and mitomycin (0.125 μg/ml)

For TA 98, TA 100 and TA 102 (S9 mixtures) with S9 metabolic activation: 2-aminoanthracene (2 μg/ml);

(3) Preparation of minimal glucose agar (MGA) plates: agar 7.5 g, distilled water 465 ml, 50x VB salt 10 ml, 40% glucose 25 ml, add agar to 465 ml of distilled water and autoclave for 20 min at 121 °C. After cooling, salt and glucose were gently added. Mix the medium for the minimal glucose agar plate and pour 25 ml into each petri dish;

(4) Label all Minimum Glucose Agar plates and Eppendorf tubes prior to the experiment;

(5) To each of the 2 ml sterile Eppendorf tubes, add the following:

a. 0.1 ml freshly cultured Salmonella strain

b. 0.2 ml His/Bio solution

c. 0.5 ml sodium phosphate buffer (S9 mixture not present) or 0.5 ml S9 (S9 mixture present)

d. 0.1 ml of sample or 0.1 ml of positive or negative control

e. Replenish to 1 ml with autoclaved distilled water.

(6) Mix the contents of the Eppendorf tube, pour into a petri dish, and apply to the surface of the MGA plate using an L-shaped applicator. Cover the Petri dish with sterile aluminum foil to protect the test sample from light-reactive substances.

(7) After 48 hours of incubation at 37 °C, spontaneous revertant colonies appear and are visible to the naked eye. The test is repeated 3 times.

(8) The revertants formed uniform colonies of nutrient-deficient bacteria on the surface of the medium and on the background.

(9) The number of revertant colonies per dish for each dose group of the subject was recorded and the mean and standard deviation were obtained.

After the subject has been assayed by the three test strains described above, as long as one of the test strains, either with or without S9, is positive, the subject is reported to be mutation-positive for Salmonella typhimurium. If the subject is negative for three test strains, both with and without S9, the subject is reported to be mutation-negative.

Caveat

1. As Salmonella typhimurium is a pathogenic bacterium, precautions are taken and standard biosafety guidelines are applied every time;2. handling of chemicals and strains should be done in a biological safety cabinet. Before and after use, cabinets must be sterilized with 75% ethanol and exposed to UV light for 15 minutes;3. all contaminated materials (e.g., test tubes, pipettes and pipette tips, gowns, and gloves) should be properly autoclaved before disposal.

Common Problems

1. The colonies are unevenly distributed and concentrated to one side.

A: The petri dish was not placed horizontally when the medium was poured;

2. How to determine whether the Ames test result is false positive.

A: Inoculate the Ames colonies treated with the test substance and positive control on histidine-free medium after enrichment culture and observe and compare the growth of bacteria. If the test-treated strain fails to grow in the histidine-free medium and the positive control strain grows in the histidine-free medium, the test-treated strain has not mutated and the increase in the number of colonies observed in the test is a false positive.

Ref: Vijay, U., Gupta, S., Mathur, P., Suravajhala, P. and Bhatnagar, P. Microbial Mutagenicity Assay: Ames Test. bio-protocol, 2018,8(6): e2763.

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