The Salmonella (Ames) Test for Mutagenicity

Errol Zeiger1, Kristien Mortelmans2

1 Environmental Toxicology Program/National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, 2 SRI International, Menlo Park, California
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 3.1
DOI:  10.1002/0471140856.tx0301s00
Online Posting Date:  May, 2001
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The Ames test is a widely accepted short‐term assay for detecting chemicals that induce mutations in the DNA of organisms. The assay is a reverse mutation assay that detects the mutational reversion of his‐dependent Salmonella to his‐independent colonies. Mutation‐causing chemicals increase the frequency of occurrence of his‐independent colonies, usually in a dose‐dependent fashion. This unit includes protocols for the basic plate test with and without preincubation as well as procedures for highly volatile chemicals and gases, chemicals that require reductive metabolism, and chemicals that are available in small quantities. In addition there are protocols for dose selection, strain maintenance and preservation, freezing working and permanent cultures, and preparing the S‐9 metabolic activation system.

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Table of Contents

  • Aseptic Technique
  • Safety Considerations
  • Basic Protocol 1: Standard Plate Incorporation Test
  • Alternate Protocol 1: Plate Assay with Preincubation Procedure
  • Alternate Protocol 2: Desiccator Assay for Volatile Liquids
  • Alternate Protocol 3: Desiccator Assay for Gases
  • Alternate Protocol 4: Reductive Metabolism Assay
  • Alternate Protocol 5: Modified (Kado) Microsuspension Assay
  • Support Protocol 1: Toxicity Test for Dose Selection
  • Support Protocol 2: Strain Maintenance and Preservation
  • Support Protocol 3: Freezing of Permanent and Working Cultures
  • Support Protocol 4: Genetic Analysis of Strains
  • Support Protocol 5: Preparation of Metabolic Activation System (S‐9)
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
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Basic Protocol 1: Standard Plate Incorporation Test

  • Cultures of Salmonella tester strains (see protocol 8 and protocol 9)
  • Nutrient broth (see recipe), sterile
  • Glucose minimal agar plates (GM plates; 100 × 15 mm; see recipe)
  • Metabolic activation system (rat liver S‐9 homogenate; see protocol 11)
  • Cofactors for the S‐9 mix (see recipe)
  • Test compound
  • Positive control chemicals (Table 3.1.2)
  • Negative control (solvent)
  • Top agar supplemented with biotin and limited histidine (see recipe), maintained at 43°C
    Table 3.1.2   Materials   Recommended Positive Control Chemicals and Test Concentrations a   Recommended Positive Control Chemicals and Test Concentrations

    Strain Control Chemical b
    Without metabolic activation
    TA97 9‐Aminoacridine 50 µg/plate
    TA98 4‐Nitro‐o‐phenylenediamine 2.5 µg/plate
    TA100 Sodium azide 5 µg/plate
    Methyl methane sulfonate 250 µg/plate
    TA102 Mitomycin C 0.5 µg/plate
    TA104 Methyl methane sulfonate 250 µg/plate
    TA1535 Sodium azide 5 µg/plate
    Methyl methane sulfonate 250 µg/plate
    TA1537 9‐Aminoacridine 50 µg/plate
    TA1538 4‐Nitro‐o‐phenylenediamine 2.5 µg/plate
    With metabolic activation
    TA97 2‐Aminoanthracene 1‐5 µg/plate c
    TA98 2‐Aminoanthracene 1‐5 µg/plate c
    TA100 2‐Aminoanthracene 1‐5 µg/plate c
    TA102 2‐Aminoanthracene 5‐10 µg/plate c
    TA104 2‐Aminoanthracene 5‐10 µg/plate c
    TA1535 2‐Aminoanthracene 2‐10 µg/plate c
    TA1537 2‐Aminoanthracene 2‐10 µg/plate c
    TA1538 2‐Aminoanthracene 2‐10 µg/plate c

     aOther chemicals have been recommended for use as positive controls (Ames et al., ; Maron and Ames, ; Kier et al., ). For other strains, refer to the publications in which the strains are described.
     bConcentrations based on 100 × 15–mm GM plates containing 20 to 25 ml agar (see protocol 1 and Reagents and Solutions).
     cThe optimum positive control concentration will depend on the source of S‐9 and its concentration and must be determined empirically.
  • 37°C shaking incubator set at 100 to 120 rpm for cultivation of overnight cultures
  • Sterile glass test tubes (16 × 150–mm and 13 × 100–mm)
  • Boiling water or microwave oven to melt top agar
  • Oven, heating block, or water bath set at 43° to 48°C to maintain temperature of top agar
  • Bottle‐top dispensers: 2‐ml for delivering top agar and 0.5‐ml for delivering buffer and S‐9 mix to test tubes
  • Colony counter (optional)
  • Additional reagents and equipment for maintaining and growing bacterial stocks ( appendix 3A), maintaining and growing Salmonella indicator strains (see protocol 8), dose selection for Salmonella test (see protocol 7), preparation of metabolic activation system for Salmonella test (see protocol 11), and performing strain check for genetic integrity (see protocol 10)

Alternate Protocol 1: Plate Assay with Preincubation Procedure

  • Volatile test chemical
  • Desiccator (9‐liter) with perforated ceramic shelf
  • Glass petri plate (100 × 15–mm) or watch glass, sterile
  • Magnetic stir bar
  • Magnetic stirrer

Alternate Protocol 2: Desiccator Assay for Volatile Liquids

  • Gaseous test chemical
  • Desiccator (9‐liter) with stopcock inlet (e.g., for use as vacuum desiccator) and perforated ceramic shelf
  • Glass petri plate (100 × 15–mm) or watch glass, sterile
  • Magnetic stir bar
  • Polyvinyl tubing
  • Pressure gauge
  • Vacuum pump
  • Magnetic stirrer

Alternate Protocol 3: Desiccator Assay for Gases

  • Cofactors for reductive metabolism (see recipe)
  • Metabolic activation system (uninduced hamster liver S‐9 homogenate; see protocol 11, but use hamsters and omit injection of Arochlor)
  • Flavin mononucleotide (FMN) solution (see recipe)

Alternate Protocol 4: Reductive Metabolism Assay

  • 0.1 M sodium phospate buffer, pH 7.4 (see recipe)
  • Centrifuge and rotor
  • Sterile test tubes (10 × 70–mm), with sterile caps

Alternate Protocol 5: Modified (Kado) Microsuspension Assay

  • Salmonella strain (Table 3.1.1): available from Dr. Bruce Ames, Biochemistry Department, University of California, Berkeley, Calif. 94720
  • Nutrient agar plates (see recipe)
  • Nutrient broth (see recipe)
  • Glucose minimal agar plates (GM plates; see recipe)
  • Enriched GM agar plates (see recipe)
  • Cryogenic vials
  • Additional reagents and equipment for growth and manipulation of bacteria ( appendix 3A)

Support Protocol 1: Toxicity Test for Dose Selection

  • Permanent or working culture (see protocol 8, step or ), with OD 540 between 0.1 and 0.2)
  • Glycerol or DMSO, sterile
  • 2‐ml cryogenic vials, sterile

Support Protocol 2: Strain Maintenance and Preservation

  • Overnight culture of Salmonella (see protocol 8, step or )
  • Enriched GM agar plates (see recipe) with the following additives:
  •  An excess of biotin (B plates)
  •  An excess of histidine (H plates)
  •  An excess of biotin and histidine (BH plates)
  •  An excess of biotin and histidine, and 24 µg/ml ampicillin (BHA plates)
  •  An excess of biotin and histidine, and 2 µg/ml tetracycline (BHT plates)
  • 0.1% (w/v) crystal violet solution (see recipe)
  • Sterile filter paper disk (6‐mm)

Support Protocol 3: Freezing of Permanent and Working Cultures

  • Chemical inducer: e.g., polychlorinated biphenyl (Aroclor 1254) or phenobarbital
  • Corn oil
  • Male Sprague‐Dawley rats weighing ∼200 g each
  • 0.15 M KCl
  • Syringes and 27‐G needles
  • Sterile forceps and scissors
  • Potter‐Elvehjem tissue homogenizer with loose Teflon pestle
  • 1‐ or 5‐ml cryogenic vials
  • Centrifuge with speeds up to 9000 × g
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Literature Cited

Literature Cited
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Key References
   Ashby, J. and Tennant, R.W. 1991. Definitive relationships among chemical structure, carcinogenicity, and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res. 257:229‐306.
   The key references will provide additional insight into how data collected from large studies are analyzed and interpreted and how such data are used to predict carcinogenicity.
   Claxton, L.D. 1998. The development, validation, and analysis of Salmonella mutagenicity test methods in environmental situations. In Microscale Testing in Aquatic Toxicology: Advances, Techniques, and Practice (P.G. Wells, K. Lee, and C. Blaise, eds.) pp. 591‐605, CRC Press, Boca Raton, Fla.
   Zeiger, E. 1997. Genotoxicity database. In Handbook of Carcinogenic Potency and Genotoxicity Databases (L.S. Gold and E. Zeiger, eds.) pp. 687‐729. CRC Press, Boca Raton, Fla.
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