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3.4: Ames Test

  • Page ID
    63340
  • Learning Objectives

    • Explain the procedure of the Ames Test including use of histidine auxotrophs and replica plating. 

    Ames Test for Mutagenicity

    The Ames test, developed by Bruce Ames (1928–) in the 1970s, is a method that uses bacteria for rapid, inexpensive screening of the carcinogenic potential of new chemical compounds. The test measures the mutation rate associated with exposure to the compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames test uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are plated onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that arise in the absence of the potential mutagen.

    The Ames Test is possible because Ester Lederberg developed replica plating.

    In replica plating, a piece of fabric is used to create an exact copy of bacterial colonies on two plates that differ in their media or environment. In the Ames test, the bacterial colonies can be grown in the presence and absence of histidine

    Hear or read about how Ester Lederberg developed this critical technique in Genetics Unzipped (https://geneticsunzipped.com/news/2019/3/28/powder-puffs-and-plasmids-esther-lederberg).

     

    Exercise \(\PageIndex{1}\)

    Histidine auxotrophs can synthesize their own histidine, which is required for cell viability. Histidine mutants can be identified by replica plating and identifying colonies that can grow on plates with histidine but not on plates without histidine in the media. Which colonies in the figure below could have mutations in a histidine synthesis gene (his¯)?

    petri dish with histidine has colonies A B C D E and F. petri dish without histidine has colonies A C E and F.

    Answer

    The his¯ colonies are B and D. 

     

    Chemicals that are more mutagenic will bring about more mutants with restored histidine synthesis in the Ames test. Because many chemicals are not directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested for their potential carcinogenic properties by using other models, including animal models like mice and rats.

     

    Diagram of the process of identifying auxotrophic mutants. First a medium containing histidine grows colonies of bacteria. Then, a sterile velvet surface is pressed onto the plate to pick up cells from bacterial colonies. Next the cells on the velvet are transferred to new plates, one with histidine and one without. A mark on the plate ensures that the orientation of the colonies is the same for all plates. After the plates are incubated, compare the growth on the plates to identify auxotrophic mutants that grow on medium containing histidine but do not grow on medium lacking histidine. A colony that is missing on the medium lacking histamine but present on the medium with histamine is the auxotrophic mutant.
    Figure \(\PageIndex{2}\): Identification of auxotrophic mutants, like histidine auxotrophs, is done using replica plating. After mutagenesis, colonies that grow on nutritionally complete medium, but not on medium lacking histidine, are identified as histidine auxotrophs.
    Diagram of the Ames test. 1 – Add rat liver extract and Salmonella to control tube. Add rat liver extract, possible mutagen, and Salmonella to experimental tube. The Salmonella strain in this test requires histidine. 2 – Plant and incubate both samples using medium lacking histidine. 3 – Compare growth on plates to identify revertants, which suggest mutagen causes mutations. In the image the plate without the possible mutagen has a few colonies (control with natural revertants). The plate from the sample with the possible mutagen has many colonies (high number of revertants his- to his+).

    Figure \(\PageIndex{3}\): The Ames test is used to identify mutagenic, potentially carcinogenic chemicals. A Salmonella histidine auxotroph is used as the test strain, exposed to a potential mutagen or carcinogen. The number of reversion mutants capable of growing in the absence of supplied histidine is counted and compared with the number of natural reversion mutants that arise in the absence of the potential mutagen.

    Questions \(\PageIndex{1}\)

    1. What mutation is used as an indicator of mutation rate in the Ames test?
    2. Why can the Ames test work as a test for carcinogenicity?
    3. Why do you think the Ames test is preferable to the use of animal models to screen chemical compounds for mutagenicity?

    Exercise \(\PageIndex{2}\)

    Which of the following regarding the Ames test is true?

    A. It is used to identify newly formed auxotrophic mutants.
    B. It is used to identify mutants with restored biosynthetic activity.
    C. It is used to identify spontaneous mutants.
    D. It is used to identify mutants lacking photoreactivation activity.

    Answer

    B

     

    Contributors and Attributions

    • Nina Parker, (Shenandoah University), Mark Schneegurt (Wichita State University), Anh-Hue Thi Tu (Georgia Southwestern State University), Philip Lister (Central New Mexico Community College), and Brian M. Forster (Saint Joseph’s University) with many contributing authors. Original content via Openstax (CC BY 4.0; Access for free at https://openstax.org/books/microbiology/pages/1-introduction)

     

    Exercise \(\PageIndex{3}\)

    Zinc oxide nanoparticles have been used in food processing and packaging as well as animal farming. Mittag and colleagues tested two different sizes of zinc oxide nanoparticles for mutagenicity using the Ames Test (2021). A small portion of their results is shown in the table.

    1. What is the genotype of the revertant colonies?
    2. Why do revertants occur in all conditions (even the negative control)?
    3. Does the data suggest that the ZnO nanoparticles are mutagenic?
    4. Does this data that ZnO nanoparticles are not harmful to living things?
    Treatment Average number of revertants/plate
    Negative control (no nanoparticles) 148
    10 μg/plate ZnO NP (<50nm) 148
    10 μg/plate ZnO NP (<100nm) 143
    Positive Control (a known mutagen) 1136

    Source: CC BY Mittag et al (2021) via https://www.mdpi.com/2305-6304/9/5/96/htm 

    Answer

    1. In an Ames Test, his¯ mutants are treated with the potential mutagen. Any DNA changes that restore the ability of the cell to synthesize histidine will produce his+ cells that have the ability to grow on media lacking histidine. 

    2. Random errors that occur during DNA replication are a constant source of new mutations; therefore the Ames Test is looking to detect a rate of mutations that occurs above this baseline level. 

    3. No. The number of revertants does not differ substantially from the negative control. (The complete paper reports that the p values from statistical tests of the data are not significant.)

    4. Not necessarily. Other tests suggest that the nanoparticles could increase cell death. DNA damage is only one aspect to consider when determining whether a chemical is harmful to living things. 

     

    References

    • Mittag A, Hoera C, Kämpfe A, Westermann M, Kuckelkorn J, Schneider T, Glei M. Cellular Uptake and Toxicological Effects of Differently Sized Zinc Oxide Nanoparticles in Intestinal Cells. Toxics. 2021 Apr 27;9(5):96. doi: 10.3390/toxics9050096. PMID: 33925422; PMCID: PMC8146923.
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