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SOP for AMES Test: Overview in line with OECD Guidelines 471

SOP for AMES Test: The AMES test (Bacterial Reverse Mutation test) developed by Bruce Ames in the 1970s, is a widely used method for identifying chemicals that can cause mutations, which in turn can lead to cancer. This test uses different strains of the bacterium Salmonella typhimurium that carry mutations in genes involved in histidine synthesis, allowing them to revert back to being histidine-independent if exposed to a mutagen.

SOP for Ames Test: Overview in line with OECD Guidelines 471
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SOP for AMES Test According to OECD 473

Materials needed:

  • Mutant strains of Salmonella typhimurium (e.g., TA98, TA100, TA102, TA1535 etc.)
  • Minimal glucose agar plates
  • Top agar (with and without histidine/biotin)
  • Test substances
  • Liver microsomal fraction (S9 mix) for metabolic activation, if required
  • Sterile distilled water
  • Positive and negative control substances
  • Incubator (37 degrees Celsius)

Procedure:

  1. Prepare bacterial cultures:
    • Inoculate the chosen strain of S. typhimurium into a tube of nutrient broth.
    • Incubate the culture at 37 degrees Celsius for 12-14 hours.
  2. Prepare agar plates:
    • Pour a thin layer of minimal glucose agar into each sterile Petri dish and allow to set.
  3. Prepare test samples:
    • Dissolve or dilute test substances in sterile distilled water or dimethyl sulfoxide (DMSO).
    • If metabolic activation is required, mix the test substance with the S9 mix.
  4. Perform the test:
    • Pipette 0.1 ml of overnight bacterial culture into a sterile test tube.
    • Add 0.1 ml of the test substance or control substance (with or without S9 mix as required).
    • Add 2.0 ml of top agar (containing a trace amount of histidine and biotin) to the tube and vortex to mix thoroughly.
    • Immediately pour the mixture onto a minimal glucose agar plate and gently rotate the plate to ensure even distribution of the mixture.
    • Allow the top agar to set, and then invert the plates.
  5. Incubation:
    • Incubate the plates at 37 degrees Celsius for 48 hours.
  6. Colony counting:
    • After incubation, count the number of colonies on each plate.
    • The presence of colonies on the plate suggests that the bacteria have mutated back to being histidine-independent, indicating the test substance is a potential mutagen.
  7. Interpretation:
    • Compare the number of revertant colonies on the test plates with those on the control plates. An increase in the number of revertant colonies on the test plates suggests that the test substance is mutagenic.
    • According to OECD Guidelines for the Testing of Chemicals, No. 471 (Bacterial Reverse Mutation Test), interpretation of the Ames test is as follows:
    • Negative Result: If there is no increase in the number of revertant colonies in test substance-treated cultures compared to the negative control (either with or without metabolic activation), the test substance can be considered non-mutagenic under the test conditions.
    • Positive Result: If there is a reproducible increase in the number of revertant colonies at one or more dose levels in at least one strain (with or without metabolic activation), the test substance is considered mutagenic.
    • Inconclusive Result: If the results are ambiguous or conflicting, and do not allow a clear positive or negative decision, the test is declared inconclusive and needs to be repeated.
    • The OECD guideline also provides certain criteria to aid in interpretation:
    • A result is generally considered positive if there is a dose-dependent increase in the number of revertant.
    • A test substance can be considered positive if at least a doubling of revertants is observed in any strain at any dose level.
    • The test must also satisfy the validity criteria; e.g., the negative controls are within the normal range, and the positive controls show an expected increase in the number of revertants.

Remember to always follow good laboratory practices and biosafety measures while handling bacterial cultures and potentially mutagenic substances. Always dispose of biohazardous waste according to your institution’s guidelines. This protocol is a standard approach to the Ames test; however, slight modifications may be made depending on specific laboratory protocols.

Strains Information

  • TA98: This strain is defective in the SOS response, which is a DNA repair pathway. This makes TA98 cells more sensitive to DNA-damaging agents, such as mutagens and carcinogens.
  • TA100: This strain is defective in the ability to repair single-strand breaks in DNA. This makes TA100 cells more sensitive to agents that cause single-strand breaks, such as UV radiation.
  • TA102: This strain is defective in the ability to repair double-strand breaks in DNA. This makes TA102 cells more sensitive to agents that cause double-strand breaks, such as ionizing radiation.
  • TA1535: This strain is defective in the ability to replicate its DNA. This makes TA1535 cells more sensitive to agents that interfere with DNA replication, such as antibiotics.

Summary

The Ames test is a widely utilized assay that identifies potential mutagenic substances. This article outlines a detailed standard operating procedure for conducting the Ames test, employing Salmonella typhimurium mutant strains. The procedure involves preparing bacterial cultures, agar plates, test samples, and then incubating the plates following exposure to the test substance.

Colony counting is performed post-incubation to assess the mutagenic potential of the test substance. The interpretation of results is guided by OECD Guidelines for the Testing of Chemicals, No. 471, which categorizes outcomes as positive, negative, or inconclusive, based on the increase in revertant colonies. The Ames test is a crucial tool in assessing the safety of new chemicals and aiding the fight against carcinogenesis.

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