Q&A of Ames Test

Possible reasons:

• Related to the toxicity of a new batch of agar medium.
• Wrong strain of test bacteria was used.
• Too little histidine was added to the top agar.

Solution:

• Each new batch of agar medium should be routinely checked for toxicity before use.
• Repeat the determination for the strain and, if necessary, re-isolate the strain and select clones with a normal spontaneous reversion mutation rate.
• Use top agar containing the correct concentration of histidine.

Possible reasons:

• Most histidine was added to the top agar.
• A high number of His+ bacteria were inoculated from frozen culture species (jack-pot effect).
• Petri dishes may have been sterilised by ethylene oxide, and ethylene oxide residues can induce mutations in TA1535 and TA100.
• Contamination may be present.

Solution:

• Prepare a new top agar and ensure that the histidine concentration is appropriate. It is also possible that the test sample species contains histidine, which usually occurs when testing biological fluids such as urine, or food-related items such as proteins and meat extracts.
• Check the number of spontaneous revertant mutations in several frozen cultures. If the problem persists, reisolate the strain and select a clone with a normal spontaneous reversion mutation rate.
• Use irradiated and sterilised plates or leave in a biological fume hood for one day before using.
• Re-isolate the strain.

Possible reasons:

• The wrong positive control chemical may have been used.
• The chemical may have deteriorated in quality during storage.
• The metabolic activation system has been ignored.
• The metabolic activation system has lost its function.
• The wrong test strain was used.

Solution:

• Double check the positive control chemical to ensure that the correct test strain is being used.
• Prepare a new batch of positive control chemical.
• Retest the positive control chemical to ensure that the metabolic activation system is being used. If the problem persists, it may be that the S9 component or cofactor may be inactive.
• Prepare a new batch of cofactors for testing. If the problem persists, use a new batch of S9-grade components.
• Confirm that the correct strain is being used.

Possible reasons:

• The chemicals are too toxic.
• The temperature of the top agar was too high.
• Solvent is toxic.

Solution:

• When the chemical is very toxic, most bacteria will be killed, resulting in not enough bacteria surviving to mutate to non-histidine dependent. Lower doses of chemicals should be used.
• The temperature of the top agar should not exceed 48°C and the top agar should be kept at 43°C.
• If the solvent is toxic and a pre-culture method is being used, use the plate doping method for testing. If the problem persists, try another solvent, or dilute the solvent to make it less toxic.

Possible reason: The bottom and/or top agar layers were placed at an angle while solidifying.

Solution: Plates should be placed horizontally when inverted.