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Microbial Mutagenicity Assay: Ames Test
微生物诱变分析:Ames试验   

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Inventi Impact - Pharm Biotech & Microbio
Oct 2014

Abstract

The Microbial mutagenicity Ames test is a bacterial bioassay accomplished in vitro to evaluate the mutagenicity of various environmental carcinogens and toxins. While Ames test is used to identify the revert mutations which are present in strains, it can also be used to detect the mutagenicity of environmental samples such as drugs, dyes, reagents, cosmetics, waste water, pesticides and other substances which are easily solubilized in a liquid suspension. We present the protocol for conducting Ames test in the laboratory.

Keywords: Mutagenicity (诱变作用), Carcinogenicity (致癌作用), Salmonella strains (沙门氏菌菌株), Gene mutation (基因突变), Revertants (回复突变体)

Background

The Microbial Ames test is a simple, rapid and robust bacterial assay consisting of different strains and applications of Salmonella typhimurium/E. coli, used for ascertaining the mutagenic potential (Levin et al., 1982; Gupta et al., 2009). In 1975, Ames and his followers standardized the traditional Ames assay protocol and reappraised in 1980’s (Maron and Ames, 1983). Induction of new mutations replacing existing mutations allows restoring of gene function. The newly formed mutant cells are allowed to grow in the absence of histidine and form colonies, hence this test is also called as ‘Reversion assay’ (Ames, 1971). While traditional Ames test is quite laborious and time consuming for initial monitoring of mutagenic compounds, miniaturization of liquid suspension significantly impacted the usability by making it more convenient. The standard doses (2 µl, 5 µl, 10 µl, 50 µl and 100 µl) were set to evaluate the mutagenicity from lower to higher concentration (Hayes, 1982). Mice liver has been used as a tissue for preparing homogenate 9,000 x g (S9 hepatic fraction) whereas in S9 mix, hepatocytes are used to minimize the mammalian metabolic activation formed in the mice liver. In Ames bioassay, the sensitivity of a compound for mutagenicity is based on the knowledge that a substance which is mutagenic in the presence of liver enzymes metabolizing compound might be a carcinogen (Mathur et al., 2005).

Genetic Approach: The Salmonella/E. coli tester strains: Several strains of Salmonella typhimurium have been used in Ames assay which requires histidine synthesis to assess the mutagenicity. In the histidine operon, each tester strain contains a different mutation. In addition to the histidine mutation, the standard tester strain of Salmonella typhimurium contains other mutations that greatly enhance their ability to detect the mutations (Figure 1). One of the mutations (rfa) causes partial loss of the lipopolysaccharides barrier that coats the surface of the bacteria and increases permeability to large molecules such as benzo[a]pyrene allowing not to penetrate in the normal cell wall (Mortelman and Zeiger, 2000). The mutagens present in the tested samples give rise to induced revertants on a minimal medium (absence of histidine). They are further used to observe revertants in previously mutated strains (that are not able to grow in a medium without histidine). The other mutation (uvrB) is a deletion mutation in which deletion of a gene, coding for the DNA excision repair system, causing gradually increased sensitivity in detecting many mutagens (Ames et al., 1973a). The reason behind this mutation is the deletion excising the uvrB gene emulsifying these bacteria requiring biotin for growth. The standard strains such as TA 97, TA 98, TA 100 and TA 102 contain the R-factor plasmid, pKM101. These R-factor strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains (Ames et al., 1975a).


Figure 1. Genetic approach for assessing the mutagenicity in Salmonella strains (modified from https://en.wikipedia.org/wiki/Ames_test)

Many studies (Ames et al., 1975b; Levin et al., 1982) revealed that development of plasmid pKM101 in TA 1535 and TA 1538 strains leads to complement other isogenic strains such as TA 98, TA 100, TA 104 and TA 102. The his G46 mutation in TA 100 and TA 1535 codes for the first enzyme of histidine biosynthesis (hisG) (Ames et al., 1975b). This mutation, determined by DNA sequence analysis, substitutes proline (-GGG-) for leucine (-GAG-) in the wild type organism (Barnes et al., 1982). The tester strains TA 1535 and its R-factor derivative present in TA 100, detect mutagens which causes base-pair substitutions generally at one of these G-C pairs. The hisD3052 mutation in TA 1538 and TA 98 is in the hisD gene coding for histodinol dehydrogenase. TA 1538 and its R-factor derivative TA 98 detect various frameshift mutagens in repetitive sequences as ‘hot spots’ resulting in a frame shift mutation (Walker and Dobson, 1979; Shanabruch and Walker, 1980) (Table 1).

Table 1. Genotype of the Salmonella strain used for mutagenesis testing


Levin et al. (1982) described a standard strain Salmonella typhimurium bacterium called TA 102 which was used to evaluate the effect of some compounds reacting with nucleotides AT. Tester strain TA102 containing nucleotides AT, present in hisG gene carrying plasmid pAQ1. There are certain mutagenic agents which are detected by TA 102 but not by TA 1535, TA 1537, TA 1538, TA 98 and TA 100 (Wilcox et al., 1990). Before performing experiment, a new set of fresh strains are prepared; and the genotypes are assessed (R-factor, His, rfa and uvrB mutations). For these, we refer readers to many excellent reviews (Walker, 1979; Czyz et al., 2002; Fluckiger-Isler et al., 2004).

Certain carcinogens present in active forms in biological reaction are easily catalyzed by cytochrome-P450. Metabolic activation system is absent in Salmonella, and in order to improve the potentiality of bacterial test systems, liver extracts of Swiss albino mice are used. This serves as a rich source in converting carcinogens to electrophilic chemicals that are incorporated to detect in vivo mutagens and carcinogens (Garner et al., 1972; Ames et al., 1973a). The crude liver homogenate as 9,000 x g S9 fraction contains free endoplasmic reticulum, microsomes, soluble enzymes and some cofactors set with S9 concentration to 10% (Franz and Malling, 1975). The oxygenase requires the reduced form of Nicotinamide Adenine Dinucleotide Phosphate (NADP) which is generally in situ by the action of glucose-6-phosphate dehydrogenase and reducing NADP both work as cofactors in assay (Prival et al., 1984; Henderson et al., 2000). While water is considered as a negative control, sodium azide, 2-nitrofluorine and mitomycin for TA 98, TA 100 and TA 102 without S9 metabolic activation and 2-anthramine with S9 hepatic fraction are used as positive controls for conducting the test (Table 2). Before performing the experiment, fresh solutions must be prepared.

Table 2. Positive controls with and without S9 metabolic activation (DeFlora et al., 1984)


Spontaneous Reversion Control: Each strain of Salmonella contains a specific mutant range. Selection of solvents shows the effect on the frequency range of spontaneous mutant (Maron and Ames, 1983) (Table 3). The range of revertants varies in research laboratories. The spontaneous revertants are visible through unaided eyes (Figure 2).

Table 3. Spontaneous revertants control values for various strain types and number of revertants (Mortelmans and Stocker, 1979)



Figure 2. Spontaneous revertants colonies obtained after addition of waste water from health center in Salmonella mutagenicity assay at different concentrations, viz. 2 µl, 10 µl, 50 µl, 100 µl (Vijay, 2014)

Materials and Reagents

  1. Materials
    1. Tips (1,000 µl, 200 µl, 10 µl) (Tarsons)
    2. Sterile Petri plates (HiMedia Laboratories, catalog number: PW001 )
    3. Erlenmeyer flask and beaker (SchottDuran,10 ml, 250 ml, 500 ml)
    4. Eppendorf tubes (Tarsons,1.5 ml, 2.0 ml)
    5. Metal loop holder (metal loop Ch-2, HiMedia Laboratories, catalog number: LA012 )
    6. L shaped spreader(HiMedia Laboratories, catalog number: PW1085 )

  2. Mutagens
    1. Sodium azide (HiMedia Laboratories, catalog number: GRM1038 )
    2. 4-Nitroquinoline N-oxide (Sigma-Aldrich, catalog number: N8141 )
    3. 2-Aminofluorene (Sigma-Aldrich, catalog number: A55500 )
    4. Benzo(a)pyrene (Sigma-Aldrich, catalog number: B1760 )
    5. Mitomycin C (Roche Diagnostics, catalog number: 10107409001 )
    6. 2,4,7-Trinitro-9-fluorenone (Accustandard, catalog number: R-033S )
    7. 4-Nitro-o-phenylenediamine (Sigma-Aldrich, catalog number: 108898 )

  3. Reagents
    1. Oxoid nutrient broth No. 2 (Sigma-Aldrich, catalog number: 70123 )
      Note: This product has been discontinued.
    2. 70% ethanol
    3. Magnesium sulphate heptahydrate (MgSO4·7H2O) (HiMedia Laboratories, catalog number: RM683 )
    4. Citric acid monohydrate (HiMedia Laboratories, catalog number: GRM1008 )
    5. Potassium phosphate, dibasic (K2HPO4) (anhydrous) (Merck, catalog number: 61788005001730 )
    6. Sodium ammonium phosphate tetrahydrate (NaNH4HPO4·4H2O) (Sigma-Aldrich, catalog number: S9506 )
    7. D-biotin (HiMedia Laboratories, catalog number: TC096 )
    8. L-histidine (HiMedia Laboratories, catalog number: TC076 )
    9. Hydrochloric acid (HCI) (HiMedia Laboratories, catalog number: AS003 )
    10. Potassium chloride (KCl) (Merck, catalog number: 61753305001730 )
    11. Magnesium chloride hexahydrate (MgCl2·6H2O) (HiMedia Laboratories, catalog number: MB040 )
    12. Sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) (Merck, catalog number: 1063700050 )
    13. Disodium hydrogen phosphate (Na2HPO4) (HiMedia Laboratories, catalog number: TC051 )
    14. NADP (sodium salt) (HiMedia Laboratories, catalog number: RM392 )
    15. D-glucose-6-phosphate (monosodium salt) (Sigma-Aldrich, catalog number: G7879 )
    16. Ampicillin trihydrate (Sigma-Aldrich, catalog number: A6140 )
    17. Sodium hydroxide (NaOH) (Merck, catalog number: 106462 )
    18. Crystal violet (Sigma-Aldrich, catalog number: C6158 )
    19. Agar-Agar (Himedia Laboratories, catalog number: RM026 )
    20. Nutrient broth (HiMedia Laboratories, catalog number: M002 )
    21. Tetracycline (Sigma-Aldrich, catalog number: 87128 )
    22. Dimethylsulfoxide (HiMedia Laboratories, catalog number: TC185 )
    23. Vogel-Bonner medium E (50x) (see Recipes)
    24. 0.5 mM histidine/biotin solution (see Recipes)
    25. Salt solution (1.65 M KCl + 0.4 M MgCl2) (see Recipes)
    26. 0.2 M sodium phosphate buffer, pH 7.4 (see Recipes)
    27. 1 M Nicotinamide Adenine Dinucleotide Phosphate (NADP) solution (see Recipes)
    28. 1 M glucose-6-phosphate (see Recipes)
    29. Ampicillin solution (4 mg/ml) (see Recipes)
    30. Crystal violet solution (0.1%) (see Recipes)
    31. Minimal glucose plates (see Recipes)
    32. Histidine/Biotin plates (see Recipes)
    33. Ampicillin and tetracycline* plates (see Recipes)
    34. Nutrient agar plates (see Recipes)
    35. S9 mix (Rat Liver Microsomal Enzymes + Cofactors) (see Recipes)
    36. Sodium azide (see Recipes)
    37. Mitomycin (see Recipes)
    38. 2-Anthramine (see Recipes)

Equipment

  1. Orbital shaking incubator (Remi, model: RIS-24(BL) )
  2. Laminar Flow hood (Bio safety cabinet) (Deepak Meditech Pvt Ltd., Steri clean)
  3. Pipettes (Eppendorf, model: Research® plus, catalog number: 3120000062 , 1,000 μl; catalog number: 3120000046 , 200 μl; catalog number: 3120000020 , 10 μl)
  4. Vortex mixer (Labnet International, catalog number: S0100 )
  5. Hot water bath (Daiki Sciences, catalog number: KBLee2001 )
  6. Autoclave (TSC)
  7. Automatic Colony counter (Sonar)
  8. Refrigerator centrifuge (Thermo Fisher Scientific, Thermo ScientificTM, model: Heraeus Biofuge Primo R )
  9. pH meter (Labindia Analytical Instruments, model: PICO pH Meter , catalog number: PC13330101)
  10. Tissue tearor (Bio Spec Products, catalog number: 985370-04 )

Procedure

  1. Before performing the experiment, inoculate a single fresh colony of standard strains of S. typhimurium TA 98, 100 and 102, in oxoid nutrient broth-2 and incubate for 10-12 h at 37 °C in an incubator shaker at 120 rpm to ensure sufficient aeration for 1 x 109 bacterial cells. Each strain of S. typhimurium is grown separately in Erlenmeyer flasks (10 ml).
  2. Prepare fresh mutagen for each experiment (see Recipes).
    Negative control: Autoclaved distilled water
    Positive controls for TA 98, TA 100 and TA 102 without S9 metabolic activation (S9 mix): sodium azide (1 μg/ml) 2-nitrofluorine (1 μg/ml) and mitomycin (0.125 μg/ml)
    For TA 98, TA 100 and TA 102 with S9 metabolic activation (S9 mix): 2-Anthramine (2 μg/ml)
  3. Preparation of minimal glucose agar (MGA) plates: Mix the medium of minimal glucose agar plates (Recipe 9) and pour 25 ml into each Petri dish. Prepare the plates freshly before use.
  4. Label all minimal glucose agar plates and Eppendorf tubes prior to experiment.
  5. To the 2 ml sterile Eppendorf tubes, add the following each:
    1. 0.1 ml fresh culture of Salmonella strains
    2. 0.2 ml of His/Bio solution
    3. 0.5 ml sodium phosphate buffer (absence of S9 mix) or 0.5 ml S9 (presence of S9 mix)
    4. 0.1 ml of test sample or 0.1 ml of positive or negative control
    5. Make up to 1 ml with autoclaved distilled water.
  6. Mix the contents of Eppendorf tubes and pour onto Petri plates and spread using L-shaped spreader on the surface of MGA plates. Cover the Petri plates with sterile aluminum foil to protect the testing sample from photo reactive substances.
  7. After incubation of 48 h at 37 °C, spontaneous revertants colonies appear and are clearly visible with unaided eyes. All plates are run in triplicates.
  8. Revertants form a uniform lawn of auxotrophic bacteria on the surface the background of medium.

Data analysis

Non-statistical analysis
The most widely used method for non-statistical analysis of result in Ames test is ‘two-fold rule’ described by Mortelmans and Zeiger (2000) and Morino-Caniello and Piegorsch (1996). On the basis that the increase in the number of revertant colonies, the concentration of the tested sample goes up (dose-dependent manner), mutagenicity ratio (MR) is calculated first by counting the number of revertant colonies per plate and then calculating the MR as described by Maron and Ames (1983) using the formula below (see Sample data below for results):


Sample data
Medical liquid waste was collected from different health care premises of Jaipur city. Salmonella mutagenicity test was performed on all the samples in their crude natural state using the plate incorporation procedure described by Maron and Ames, 1983. The results of Salmonella mutagenicity assay was analyzed through Mutagenicity Ratio method and shown in Table 4.

Table 4. Mutagenicity ratios of S. typhimurium strains TA98, TA100 and TA102 treated with waste water from different health premises (Vijay, 2014)

+Mutagenicity Ratio > 2.0 imply mutagenic, -Ratio < 2.0 imply non-mutagenic

Conclusion
The Ames test is a widely accepted bacterial assay to detect the mutagenicity in pathogenic bacteria. In this protocol, although we have shown the step wise methodology to perform Ames assay applicable for three strains, this method can be used for studying all compounds to infer mutagenicity. Whereas the Ames assay experiments involve sterile measures, care must be taken in ensuring the sample/plasmid is not contaminated. The improved methods to detect the genotoxicity of compounds help us troubleshoot methods for studying the compounds tested in clinical trials.

Notes

Sterilization (safety considerations while working with Salmonella)

  1. As S. typhimurium is a pathogenic bacterium, it is prudent to use precautionary measures every time and apply standard biosafety guidelines such as using plugged pipettes, proper sterilization by 70% ethanol and autoclaving all contaminated material.
  2. Handling of chemicals and strains should be done in biosafety cabinet. Before and after the use, cabinet must be sterilized using 70% ethanol and exposed to 15 min UV.
  3. Care must be taken to protect from chemical exposure by wearing gowns, eye glasses and gloves.
  4. Before discarding, all contaminated material (e.g., test tubes, pipettes and pipette tips, gowns and gloves) should be properly autoclaved.


Limitations

Ames assay consists of Salmonella typhimurium strains and so it is not a perfect model for human. Mice liver S9 hepatic fraction is used to minimize the mammalian metabolic activations formed in the hepatic system so that the mutagenicity of metabolites can be assessed. There are several differences between human and mice metabolism which can affect the mutagenicity of testing substances. Major disadvantages of fluctuation test is slower and slightly more laborious than Ames protocol. The test is primarily used for testing aqueous samples containing low levels of mutagen and therefore, this test is well adapted for evaluating the mutagenicity of wastewater samples.

Recipes

  1. Vogel-Bonner medium E (50x)
    For Minimal agar (Recipe 9)
    Ingredients
    Per 500 ml
    Warm distilled H2O (45 °C)
    335 ml
    Magnesium sulfate (MgSO4·7H2O)
    5 mg
    Citric acid monohydrate
    50 mg
    Potassium phosphate, dibasic (anhydrous) (K2HPO4)
    250 mg
    Sodium ammonium phosphate (NaNH4HPO4·4H2O)
    87.5 mg
    1. Salts are added to the warm water in a flask. Place the flask on a hot plate
    2. After each salt dissolves entirely, transfer the solution into glass bottles and autoclave for 20 min at 121 °C
    3. When the solution gets cool, cap the bottle tightly
    4. Store the solution at 4 °C
  2. 0.5 mM histidine/biotin solution
    For mutagenic bioassay
    Ingredients
    Per 125 ml
    D-Biotin (F.W. 247.3)
    15.45 mg
    L-Histidine·HCl (F.W. 191.7)
    12.0 mg
    Distilled H2O
    125 ml
    Dissolve the biotin in hot distilled water. The solution is autoclaved for 20 min, at 121 °C and then stored at 4 °C
  3. Salt solution (1.65 M KCl + 0.4 M MgCl2)
    For S9 hepatic fraction
    Ingredients
    Per 250 ml
    Potassium chloride (KCl)
    30.75 mg
    Magnesium chloride (MgCl2·6H2O)
    20.35 mg
    Distilled H2O to final concentration of
    250 ml
    All the components are dissolved in water. The solution is autoclaved for 20 min, at 121 °C and then stored at 4 °C
  4. 0.2 M sodium phosphate buffer, pH 7.4
    For S9 hepatic fraction 
    Ingredients
    Per 250 ml
    0.2 M sodium dihydrogen phosphate (NaH2PO4·H2O)
    30 ml (6.9 mg/250 ml)
    0.2 M disodium hydrogen phosphate (Na2HPO4)
    220 ml (7.1 mg/250 ml)
    Adjust pH to 7.4. Sterilize the buffer by autoclaving for 20 min at 121 °C
  5. 1 M nicotinamide adenine dinucleotide phosphate (NADP) solution
    For S9 hepatic fraction
    Ingredients
    Per 2.5 ml
    NADP
    191.5 mg
    Sterile distilled H2O
    2.5 ml
    NADP is dissolved in the distilled water and mixed by vortexing. Tubes are placed in an ice bath. The solution can be used for up to six months
  6. 1 M glucose-6-phosphate
    For S9 hepatic fraction
    Ingredients
    Per 5 ml
    Glucose-6-phosphate (G-6-P)
    1.41 mg
    Sterile distilled H2O
    5 ml
    Glucose-6-phosphate is dissolved in the 5 ml distilled water and mixed by vortexing. Tubes are placed in an ice bath. The solution can be used for up to six months
  7. Ampicillin solution (4 mg/ml)
    Used in tests of ampicillin resistance
    Master plates for R-factor strains
    Ingredients
    Per 500 ml
    Ampicillin trihydrate
    0.4 mg
    Sodium hydroxide (0.02 N)
    50 ml
    Ampicillin trihydrate is dissolved in the 50 ml of NaOH (0.02 N) and mixed by vortexing. Tubes are placed in an ice bath
  8. Crystal violet solution (0.1%)
    Used in tests for crystal violet sensitivity (to confirm rfa mutation)
    Ingredients
    Per 500 ml
    Crystal violet
    0.05 mg
    Distilled H2O
    50 ml
  9. Minimal glucose plates
    Used in Mutagenic bioassay
    Ingredients
    Per 500 ml
    Agar
    7.5 mg
    Distilled H2O
    465 ml
    50x VB salts (Recipe 1)
    10 ml
    40% glucose
    25 ml
    Add agar in 465 ml of distilled water and autoclave for 20 min, at 121 °C. After cooling, add the salts and glucose gently
  10. Histidine/Biotin plates (Master plates for non R-factor strains)
    Used in tests for histidine requirement 
    Ingredients
    Per 500 ml
    Agar
    7.5 mg
    Distilled H2O
    457 ml
    50x VB salts
    10 ml
    40% glucose
    25 ml
    Sterile histidine (2 g per 400 ml H2O)
    5 ml
    Sterile 0.5 mM biotin
    3 ml
    Dissolve agar in the given concentration in distilled water. Autoclave each solution separately for 20 min. After cooling of solution, add each salt gently
  11. Ampicillin and tetracycline* plates
    Master plates for the cultivation of strains containing the plasmids pKM101 and pAQ1*
    Ingredients
    Per 500 ml
    Agar
    7.5 mg
    Distilled H2O
    405 ml
    50x VB salts
    10 ml
    40% glucose
    25 ml
    Sterile histidine (2 g per 400 ml H2O)
    5 ml
    Sterile 0.5 mM biotin
    3 ml
    Sterile ampicillin solution (8 mg/ml 0.02 N NaOH)
    1.58 ml
    *Sterile tetracycline solution (8 mg/ml 0.02 N HCl)
    0.125 ml
    Dissolve agar in the given concentration in distilled water. Autoclave each solution separately for 20 min. After cooling of solution, add each salt gently
    *Note: TA 102 is resistant to tetracycline. The shelf life of the plates is two weeks at 4 °C.
  12. Nutrient agar plates
    Used in tests for genotypes [Crystal violet sensitivity (rfa) and UV sensitivity (AuvrB)] and viability of bacteria
    Ingredients
    Per 500 ml
    Nutrient agar
    7.5 mg
    Distilled H2O
    500 ml
    Dissolve agar in the given concentration in distilled water. Autoclave separately for 20 min. Pour the cooled solution into the Petri plates
  13. S9 mix (Rat Liver Microsomal Enzymes + Cofactors)
    Ingredients
    Standard S9 mix Per 25 ml
    Mice liver
    1.0 ml (2%)
    MgCl2-KCl salts
    0.5 ml
    1 M glucose-6-phosphate
    0.125 ml
    0.1 M NADP
    1.0 ml
    0.2 M phosphate buffer, pH 7.4
    12.5 ml
    Sterile distilled H2O
    9.86 ml
    Note: Add each ingredient in the reverse order listed above (First water, and then phosphate buffer…). Avoid refreezing the S9 mix.
  14. Sodium azide
    Used in Mutagenicity assay 
    Ingredients
    Per ml
    Sodium azide
    10 µg
    Autoclave distilled H2O
    990 µl (to make a total volume of 1 ml)
    Working concentrations are prepared by taking 1, 2, 4 µl of 10 mg/ml
  15. 2-Nitrofluorine
    Used in Mutagenicity assay 
    Ingredients
    Per ml
    2-Nitrofluroine
    10 µg
    Autoclave distilled H2O
    990 µl (to make a total volume of 1 ml)
    Working concentrations are prepared by taking 1, 2, 4 µl of 10 mg/ml
  16. Mitomycin
    Used in Mutagenicity assay 
    Ingredients
    Per ml
    Mitomycin
    10 µg
    Autoclave distilled H2O
    990 µl (to make a total volume of 1 ml)
    Working concentrations are prepared by taking 1, 2, 4 µl of 10 mg/ml
  17. 2-Anthramine
    Used in Mutagenicity assay 
    Ingredients
    Per ml
    2-Anthramine
    10 µg
    Autoclave distilled H2O
    990 µl (to make a total volume of 1 ml)
    Working concentrations are prepared by taking 1, 2, 4 µl of 10 mg/ml

Acknowledgments

Urvashi Vijay would like to thank the Department of Zoology, The IIS University, Jaipur where the work was carried out. The financial help received by IISU Fellowship 2012/9389 to Urvashi Vijay is gratefully acknowledged.
Conflict of interests: None declared.
Authors contributions: Urvashi Vijay, Sonal Gupta, Priyanka Mathur carried out the protocol and the methods under the guidance of Pradeep Bhatnagar. Prashanth Suravajhala re-reviewed the works and proofread the manuscript before all authors approving it.

References

  1. Ames, B. N. (1971). The detection of chemical mutagens with enteric bacteria. In: Hollaender, A. (Ed.). Chemical Mutagens, Principles and Methods for Their Detection vol. 1. Plenum pp: 851-863.
  2. Ames, B. N., Durston, W. E., Yamasaki, E. and Lee, F. D. (1973a). Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A 70(8): 2281-2285.
  3. Ames, B. N., Lee, F. D. and Durston, W. E. (1973b). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A 70(3): 782-786.
  4. Ames, B. N., McCann, J. and Yamasaki, E. (1975a). Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat Res 31(6): 347-364.
  5. Ames, B. N., Yamasaki, E., Mc Cann, J. (1975b). Detection of carcinogens in the Salmonella/microsome test. Assay of 300 chemicals. Proc Natl Acad Sci U S A 72: 5135-5139.
  6. Barnes, W., Tuley, E and Eisenstadt, E. (1982). Base sequence analysis of His+ revertants of the hisG46 missense mutations in Salmonella typhimurium. Enviorn Mutagen 4:297.
  7. Bernstein, L. S., Kaldor, J., McCann, J. and Pike, M. C. (1982). An empirical approach to the statistical analysis of mutagenesis data from the Salmonella test. Mutat Res 97: 267-281.
  8. Brusick, D. J., Simmon, V. F., Rosenkranz, H. S., Ray, V. A. and Stafford, R. S. (1980). An evaluation of the Escherichia coli WP2 and WP2 uvrA reverse mutation assay. Mutat Res 76(2): 169-190.
  9. Czyz, A., Szpilewska, H., Dutkiewicz, R., Kowalska, W. A., Biniewska-Godlewska, A., and Wegrzyn, G. (2002). Comparison of the Ames test and a newly developed assay for detection of mutagenic pollution of marine environments. Mutat Res 519: 67-74.
  10. DeFlora, S., Zanacchi, P., Camoirano, S., Bennicelli, S., Badolati, G. S. (1984). Genotoxic activity and potency of 135 compounds in the Ames reversion test and in a bacterial DNA-repair test. Mutat Res 133: 161-198.
  11. Fluckiger-Isler. S., Baumeister, M., Braun, K., Gervais, V., Hasler-Nguyen, N., Reimann, R., Van Gompel, J., Wunderlich, H. G. and Engelhardt, G. (2004). Assessment of the performance of the Ames II assay: a collaborative study with 19 coded compounds. Mutat Res 558: 181-197.
  12. Frantz, C. N and Malling, H. V. (1975). The quantitive microsomal mutagenesis assay method. Mutat Res 31: 365-380.
  13. Garner, R. C., Miller, E. C. and Miller, J. A. (1972). Liver microsomal metabolism of aflatoxin B 1 to a reactive derivative toxic to Salmonella typhimurium TA 1530. Cancer Res 32(10): 2058-2066.
  14. Gupta, P., Mathur, N., Bhatnagar, P., Nagar, P. and Srivastava, S. (2009). Genotoxicity evaluation of hospital wastewaters. Ecotoxicol Environ Saf 72(7): 1925-1932.
  15. Hayes, A. W. (1982). Principles and methods of toxicology. Raven Press pp: 238-241.
  16. Henderson, L., Albertini, S. and Aardema, M. (2000). Thresholds in genotoxicity responses. Mutat Res 464(1): 123-128.
  17. Levin, D. E., Yamasaki, E. and Ames, B. N. (1982). A new Salmonella tester strain, TA97, for the detection of frameshift mutagens. A run of cytosines as a mutational hot-spot. Mutat Res 94(2): 315-330.
  18. Maron, D. and Ames, B. N. (1983). Revised methods for the Salmonella mutagenicity test. Mutat Res 113: 173-215.
  19. Maron, D., Katzenellenbogen, J. and Ames, B. N. (1981). Compatibility of organic solvents with the Salmonella/microsome test. Mutat Res 88: 343-350.
  20. Mathur, N., Bhatnagar, P. and Bakre, P. (2005). Assessing mutagenicity of textile dyes from Pali (Rajasthan) using Ames Bioassay. Appl Ecol Env Res 4: 111-118.
  21. Morino-Caniello, N. F. and Piegorsch, W. W. (1996). The Ames test: the two-fold rule revisited. Mutat Res 369: 23-31.
  22. Mortelmans, K. and Stocker, B. A. D. (1979). Segregation of the mutator property of plasmid R46 from its ultraviolet-protection properties. Mol Gen Genet 167: 317-327.
  23. Mortelmans, K. and Zeiger, E. (2000). The Ames Salmonella/microsome mutagenicity assay. Mutat Res 455: 29-60.
  24. Prival, M. J., Bell, S. J., Mitchell, V. D., Peiperl, M. D. and Vaughan, V. L. (1984). Mutagenicity of benzidine and benzidine-congener dyes and selected monoazo dyes in a modified Salmonella assay. Mutat Res 136(1): 33-47.
  25. Shanabruch, W. G. and Walker, G. C. (1980). Localization of the plasmid (pKMIOl) gene(s) involved in recA+/ex4+-dependent mutagenesis. Mol Gen Genet 179: 289-297.
  26. Simmon, V. F., Kauhanen, K. and Tardiff, R. G. (1977). Mutagenic activitiesof chemicals identified in drinking water. In: Progress in Genetic Toxicology. 249-258.
  27. Venitt, S. and Bosworth, D. (1983). The development of anaerobic methods for bacterial mutation assays: aerobic and anaerobic fluctuations tests of human faecal extracts and reference mutagens. Carcinogenesis 4: 339-345.
  28. Vijay, U., Bhatnagar, P. and Mathur, P. (2014). Mutagenicity Evaluation of Health Center wastewater with the mutant TA 100 and TA 102 strains of Salmonella typhimurium. Inventi Impact: Pharm Biotech & Microbio 4: 195-198.
  29. Vijay, U. (2014). Physico-chemical characterization and toxicological evaluation of liquid effluents generated by health care establishments of Jaipur. The IIS University. Thesis
  30. Walker, G. C. and Dobson, P. P. (1979). Mutagenesis and repair deficiencies of Escherichia coli umuC mutants are suppressed by the plasmid pKM101. Mol Gen Genet 172: 17-24.
  31. Wilcox, P., Naidoo, A., Wedd, D. J. and Gatehouse, D. G. (1990). Comparison of Salmonella typhimurium TA102 with Escherichia coli WP2 tester strains. Mutagenesis 5(3): 285-291.
  32. Zeiger, E. (1985). The Salmonella mutagenicity assay for identification of presumptive carcinogens. In: Milman, H. A. and Weisburger, E. K. (Eds). Handbook of Carcinogen Testing. Noyes Publishers pp: 83-99.

简介

微生物致突变性艾姆斯试验是一种细菌生物测定法,用于体外评估各种环境致癌物和毒素的致突变性。 虽然艾姆斯试验可用于鉴定菌株中存在的回复突变,但它也可用于检测环境样品如药物,染料,试剂,化妆品,废水,农药和易溶于水的其他物质的致突变性 液体悬浮液。 我们提供在实验室进行艾姆斯测试的协议。

【背景】微生物艾姆斯试验是一种简单,快速和强大的细菌试验,由不同的菌株和鼠伤寒沙门氏菌/ E的应用组成。用于确定致突变潜力(Levin等人,1982; Gupta等人,2009)。 1975年,艾姆斯和他的追随者对传统的艾姆斯检测方案进行了标准化,并于20世纪80年代进行了再次评估(Maron and Ames,1983)。替换已有突变的新突变的诱导允许恢复基因功能。新形成的突变细胞允许在没有组氨酸的情况下生长并形成菌落,因此该测试也被称为“逆转测定法”(Ames,1971)。虽然传统的艾姆斯测试对于初始监测致突变化合物来说非常费力且费时,但是通过使其更便利,液体悬浮液的小型化显着地影响了可用性。标准剂量(2μl,5μl,10μl,50μl和100μl)用于评估从低浓度到高浓度的致突变性(Hayes,1982)。小鼠肝脏已被用作制备匀浆9,000xg(S9肝脏级分)的组织,而在
遗传方法:沙门氏菌/ E。大肠杆菌测试菌株:在Ames测定中已使用几种鼠伤寒沙门氏菌菌株,其需要组氨酸合成来评估致突变性。在组氨酸操纵子中,每个测试菌株含有不同的突变。除了组氨酸突变之外,鼠伤寒沙门氏菌的标准测试菌株还含有其他突变,这些突变大大增强了它们检测突变的能力(图1)。其中一种突变( rfa )引起脂多糖屏障的部分丧失,其覆盖细菌表面并增加对大分子例如苯并[a]芘的渗透性,从而不允许穿透正常的细胞壁(Mortelman和Zeiger,2000)。存在于测试样品中的诱变剂在基本培养基上产生诱导的回复物(不存在组氨酸)。它们进一步用于观察先前突变的菌株中的回复突变(不能在没有组氨酸的培养基中生长)。另一种突变(uvrB)是一种缺失突变,其中编码DNA切除修复系统的基因缺失导致检测许多诱变剂的敏感性逐渐增加(Ames等人, 1973a)。这种突变背后的原因是删除切除生长素生长素的这些细菌的uvrB基因的缺失。标准菌株如TA 97,TA 98,TA 100和TA 102含有R因子质粒pKM101。这些R-因子菌株被许多诱变剂回复,这些诱变剂被非R-因子亲本菌株轻微检测或根本检测不到(Ames等人,1975a)。

“”src
图1.用于评估沙门氏菌菌株中致突变性的遗传学方法<从
https://en.wikipedia.org/wiki/Ames_test

许多研究(Ames等人,1975b; Levin等人,1982)揭示TA 1535和TA 1538菌株中质粒pKM101的发展导致补充其他同基因菌株例如TA 98,TA 100,TA 104和TA 102.在TA 100和TA 1535中的他的G46突变编码组氨酸生物合成的第一种酶(hisG)(艾姆斯等人,1975b)。这种由DNA序列分析确定的突变取代脯氨酸(-GGG-)代替野生型生物体中的亮氨酸(-GAG-)(Barnes等,1982)。测试菌株TA TA1535及其存在于TA 100中的R因子衍生物,检测通常在这些G-C对之一处引起碱基对取代的诱变剂。 TA 1538和TA 98中的hisD3052突变位于编码组蛋白脱氢酶的hisD基因中。 TA1538及其R-因子衍生物TA98检测重复序列中的各种移码突变,作为导致移码突变的“热点”(Walker和Dobson,1979; Shanabruch和Walker,1980)(表1)。表1.用于诱变测试的<沙门氏菌菌株的基因型


Levin等人(1982)描述了称为TA102的标准菌株鼠伤寒沙门氏菌细菌,其被用于评估一些化合物与核苷酸AT反应的作用。含有核苷酸AT的测试菌株TA102,存在于带有质粒pAQ1的hisG基因中。存在某些由TA102检测到但不由TA1535,TA1537,TA1538,TA98和TA100检测到的致突变剂(Wilcox等人,1990)。在进行实验之前,准备一套新的新鲜菌株;并对基因型进行评估(R因子,His,rfa和 uvrB 突变)。对于这些,我们引用读者许多优秀的评论(Walker,1979; Czyz等人,2002; Fluckiger-Isler等人,2004)。

在生物反应中以活性形式存在的某些致癌物质易于被细胞色素P450催化。代谢活化系统在沙门氏菌中不存在,并且为了提高细菌测试系统的潜力,使用瑞士白化小鼠的肝提取物。这在将致癌物转化为亲电子化学物质方面起着丰富的作用,所述亲电化学物质被掺入以检测体内诱变剂和致癌物(Garner等人,1972; Ames等人。,1973a)。粗制肝匀浆为9,000xg S9级分,含有游离内质网,微粒体,可溶性酶和一些S9浓度为10%的辅因子(Franz和Malling,1975)。加氧酶需要烟酰胺腺嘌呤二核苷酸磷酸(NADP)的还原形式,其通过葡萄糖-6-磷酸脱氢酶和还原型NADP的作用通常原位被用作测定法中的辅因子(Prival 等人,1984; Henderson等人,2000)。虽然水被认为是阴性对照,但是用于TA98,TA100和TA102的没有S9代谢活化的叠氮化钠,2-硝基氟和丝裂霉素和用S9肝脏级分的2-蒽胺作为进行试验的阳性对照(表2 )。在进行实验之前,必须准备新的解决方案。

表2.具有和不具有S9代谢活化的阳性对照(DeFlora et al。,1984)


自发逆转控制:每种沙门氏菌都含有特定的突变体范围。溶剂的选择显示了对自发突变体频率范围的影响(Maron和Ames,1983)(表3)。研究实验室中回复物的范围各不相同。肉眼可见的自发回复物(图2)。

表3.自发回复物控制各种菌株类型和回复物数量的值(Mortelmans and Stocker,1979)


“”src
图2.在不同浓度的沙门氏菌致突变性测定中,从健康中心加入废水后获得的自发回复菌落,即, 2μl,10μl,50μl,100μl(Vijay,2014)

关键字:诱变作用, 致癌作用, 沙门氏菌菌株, 基因突变, 回复突变体

材料和试剂

  1. 物料
    1. 提示(1,000μl,200μl,10μl)(Tarsons)
    2. 无菌培养皿(HiMedia Laboratories,目录号:PW001)
    3. 锥形瓶和烧杯(SchottDuran,10毫升,250毫升,500毫升)
    4. Eppendorf管(Tarsons,1.5毫升,2.0毫升)
    5. 金属环固定器(金属环Ch-2,HiMedia实验室,目录编号:LA012)
    6. L形撒布机(HiMedia Laboratories,目录号:PW1085)

  2. 诱变剂
    1. 叠氮化钠(HiMedia Laboratories,目录号:GRM1038)
    2. 4-硝基喹啉氧化物(Sigma-Aldrich,目录号:N8141)
    3. 2-氨基芴(Sigma-Aldrich,目录号:A55500)
    4. 苯并[a]芘(Sigma-Aldrich,目录号:B1760)
    5. 丝裂霉素C(Roche Diagnostics,目录号:10107409001)
    6. 2,4,7-三硝基-9-芴酮(Accustandard,目录号:R-033S)
    7. 4-硝基 - 苯二胺(Sigma-Aldrich,目录号:108898)

  3. 试剂

    1. 2号Oxoid营养肉汤(Sigma-Aldrich,目录号:70123) 注意:此产品已停产。
    2. 70%乙醇
    3. 硫酸镁七水合物(MgSO 4·7H 2 O)(HiMedia Laboratories,目录号:RM683)
    4. 柠檬酸一水合物(HiMedia Laboratories,目录号:GRM1008)
    5. 磷酸氢二钾(K 2 HPO 4)(无水)(Merck,目录号:61788005001730)
    6. 磷酸四氢铵(NaNH 4 HPO 4·4H 2 O)(Sigma-Aldrich,目录号:S9506)
    7. D-生物素(HiMedia Laboratories,目录号:TC096)
    8. L-组氨酸(HiMedia Laboratories,目录号:TC076)
    9. 盐酸(HCI)(HiMedia Laboratories,目录号:AS003)
    10. 氯化钾(KCl)(Merck,目录号:61753305001730)
    11. 氯化镁六水合物(MgCl 2·6H 2 O)(HiMedia Laboratories,目录号:MB040)
    12. 磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)(Merck,目录号:1063700050)
    13. 磷酸氢二钠(Na 2 HPO 4)(HiMedia Laboratories,目录号:TC051)
    14. NADP(钠盐)(HiMedia Laboratories,目录号:RM392)
    15. D-葡萄糖-6-磷酸盐(单钠盐)(Sigma-Aldrich,目录号:G7879)
    16. 氨苄青霉素三水合物(Sigma-Aldrich,目录号:A6140)
    17. 氢氧化钠(NaOH)(Merck,目录号:106462)
    18. 结晶紫(Sigma-Aldrich,目录号:C6158)
    19. 琼脂(Himedia Laboratories,目录号:RM026)
    20. 营养肉汤(HiMedia实验室,目录号:M002)
    21. 四环素(Sigma-Aldrich,目录号:87128)
    22. 二甲基亚砜(HiMedia Laboratories,目录号:TC185)
    23. Vogel-Bonner中型E(50x)(见食谱)
    24. 0.5 mM组氨酸/生物素溶液(见食谱)
    25. 盐溶液(1.65M KCl + 0.4M MgCl 2)(见食谱)
    26. 0.2 M磷酸钠缓冲液,pH 7.4(见食谱)
    27. 1 M烟酰胺腺嘌呤二核苷酸磷酸(NADP)溶液(见食谱)
    28. 1 M葡萄糖-6-磷酸盐(见食谱)
    29. 氨苄青霉素溶液(4毫克/毫升)(见食谱)
    30. 结晶紫溶液(0.1%)(见食谱)
    31. 最小的葡萄糖板(见食谱)
    32. 组氨酸/生物素平板(见食谱)
    33. 氨苄青霉素和四环素*板(见食谱)
    34. 营养琼脂平板(见食谱)
    35. S9混合物(大鼠肝微粒体酶+辅因子)(见食谱)
    36. 叠氮化钠(见食谱)
    37. 丝裂霉素(见食谱)
    38. 2-Anthramine(见食谱)

设备

  1. 轨道振荡培养箱(Remi,型号:RIS-24(BL))

  2. 层流罩(生物安全柜)(Deepak Meditech Pvt Ltd.,Steri clean)
  3. 移液器(Eppendorf,型号:Research plus plus,目录号:3120000062,1000μl;目录号:3120000046,200μl;目录号:3120000020,10μl)。
  4. 涡旋混合器(Labnet International,目录号:S0100)
  5. 热水浴(Daiki Sciences,目录号:KBLee2001)
  6. 高压灭菌器(TSC)
  7. 自动菌落计数器(声纳)
  8. 冰箱离心机(Thermo Fisher Scientific,Thermo Scientific TM,型号:Heraeus Biofuge Primo R)
  9. pH计(Labindia Analytical Instruments,型号:PICO pH计,目录号:PC13330101)
  10. 组织分娩器(Bio Spec Products,产品目录号:985370-04)

程序

  1. 在进行实验之前,接种一株标准品系的新鲜菌落。鼠伤寒沙门氏菌TA 98,100和102在牛痘营养肉汤-2中培养10-12小时,并在37℃下在培养摇床中在120rpm下培养以确保充足的1×10 -9 /细菌细胞。每个S的菌株。
    在锥形瓶(10毫升)中单独生长鼠伤寒杆菌
  2. 为每个实验准备新鲜的诱变剂(见食谱)。
    阴性对照:蒸压蒸馏水
    没有S9代谢活化(S9混合)的TA 98,TA 100和TA 102的阳性对照:叠氮钠(1μg/ ml)2-硝基氟(1μg/ ml)和丝裂霉素(0.125μg/ ml)
    对于具有S9代谢活化(S9混合)的TA 98,TA 100和TA 102:2-Anthramine(2μg/ ml)
  3. 最小葡萄糖琼脂(MGA)平板的制备:混合最小葡萄糖琼脂平板的培养基(配方9)并将25ml倒入每个培养皿中。使用前准备新鲜的盘子。

  4. 在实验前标记所有最小葡萄糖琼脂平板和Eppendorf管。
  5. 向2毫升无菌Eppendorf管中添加以下各项:
    1. 0.1ml新鲜培养的沙门氏菌菌株
    2. 0.2毫升His / Bio解决方案
    3. 0.5ml磷酸钠缓冲液(不含S9混合物)或0.5ml S9(存在S9混合物)
    4. 0.1毫升的测试样品或0.1毫升的阳性或阴性对照

    5. 用高压灭菌蒸馏水补充1毫升
  6. 将Eppendorf管的内容物混合并倒入Petri板中,并使用L形扩散器涂布在MGA板的表面上。
    使用无菌铝箔覆盖培养皿,以保护测试样品不受光反应物质影响。
  7. 在37℃孵育48小时后,出现自发回复菌落,并且用肉眼清楚可见。所有板块都重复运行三次。

  8. 回归物在表面背景下形成一个统一的营养缺陷型细菌草坪。

数据分析

非统计分析
对于Ames检验结果的非统计分析最广泛使用的方法是Mortelmans和Zeiger(2000)和Morino-Caniello和Piegorsch(1996)描述的“双重规则”。根据回复菌落数量的增加,受试样品浓度升高(剂量依赖性方式),首先通过计算每个平板的回复菌落数目计算致突变性比率(MR),然后计算MR如Maron和Ames(1983)所述,使用以下公式(见下面的样本数据获得结果):


示例数据
医疗液体废物是从斋浦尔市的不同医疗保健场所收集的。使用Maron和Ames,1983年描述的平板掺入程序对所有样品进行沙门氏菌致突变性测试。通过诱变性比率法分析沙门氏菌致突变性测定的结果,并在表中显示4.

表4.使用来自不同健康场所的废水处理的鼠伤寒沙门氏菌菌株TA98,TA100和TA102的致突变性比率(Vijay,2014)

+致突变性比率&gt; 2.0意味着诱变,-Ratio < 2.0意味着非诱变

结论
艾姆斯试验是一种广泛接受的细菌检测法,用于检测致病菌的致突变性。在该协议中,尽管我们已经显示了用于执行适用于三种菌株的艾姆斯试验的逐步方法,但是该方法可以用于研究所有化合物以推断诱变性。鉴于艾姆斯分析实验涉及无菌措施,必须小心确保样品/质粒未被污染。检测化合物遗传毒性的改进方法有助于我们诊断研究临床试验中所用化合物的方法。

笔记

灭菌(与沙门氏菌处理时的安全考虑)

  1. 作为 S。鼠伤寒沙门氏菌是一种致病菌,每次使用预防措施都是谨慎的,并应用标准的生物安全指导方针,例如使用堵塞式移液器,70%乙醇进行适当的灭菌以及对所有污染物质进行高压灭菌。
  2. 化学品和菌株的处理应在生物安全柜中进行。使用前后,柜子必须使用70%乙醇灭菌,并暴露于15分钟的紫外线。

  3. 穿着长袍,眼镜和手套,必须注意防止化学品暴露
  4. 在丢弃之前,应对所有受污染的材料(例如,试管,移液器和移液器吸头,长袍和手套)进行适当的高压灭菌。


的限制

艾姆斯试验由鼠伤寒沙门氏菌菌株组成,因此它不是人类的完美模型。小鼠肝脏S9肝脏级分用于最小化在肝脏系统中形成的哺乳动物代谢激活,从而可以评估代谢物的致突变性。人和小鼠新陈代谢之间有几个不同之处,可能会影响检测物质的致突变性。波动测试的主要缺点比Ames协议更慢,更费力。该测试主要用于测试含有低水平诱变剂的含水样品,因此,该测试适用于评估废水样品的致突变性。

食谱

  1. Vogel-Bonner中型E(50x)
    用于最小琼脂(配方9)
    成分
    每500毫升
    温热蒸馏H 2 O(45°C)
    335毫升
    硫酸镁(MgSO 4·7H 2 O)
    5毫克
    柠檬酸一水物
    50毫克
    磷酸钾,二元(无水)(K 2 HPO 4)
    250毫克
    磷酸铵钠(NaNH 4 HPO 4·4H 2 O)
    87.5毫克
    1. 将盐加入烧瓶中的温水中。将烧瓶放在热板上
    2. 每种盐完全溶解后,将溶液转移到玻璃瓶中并在121℃高压灭菌20分钟。
    3. 当溶液变凉时,紧紧盖住瓶子
    4. 将溶液储存在4°C
  2. 0.5 mM组氨酸/生物素溶液
    用于诱变生物分析
    成分
    每125毫升
    D-Biotin(F.W. 247.3)
    15.45毫克
    L-组氨酸·HCl(F.W.191.7)
    12.0毫克
    蒸馏水 O O
    125毫升
    将生物素溶于热蒸馏水中。该溶液在121℃高压灭菌20分钟,然后在4℃下保存。
  3. 盐溶液(1.65M KCl + 0.4M MgCl 2)
    对于S9肝脏部分
    成分
    每250毫升
    氯化钾(KCl)
    30.75毫克
    氯化镁(MgCl 2·6H 2 O)
    20.35毫克
    蒸馏H 2 O至终浓度为
    250毫升
    所有组分都溶于水中。该溶液在121℃高压灭菌20分钟,然后在4℃下保存。
  4. 0.2 M磷酸钠缓冲液,pH 7.4
    对于S9肝脏部分
    成分
    每250毫升
    0.2M磷酸二氢钠(NaH 2 PO 4·2H 2 O)
    30毫升(6.9毫克/ 250毫升)
    0.2M磷酸氢二钠(Na 2 HPO 4)
    220毫升(7.1毫克/ 250毫升)
    调整pH值到7.4。
    在121℃高压灭菌20分钟以消毒缓冲液
  5. 1 M烟酰胺腺嘌呤二核苷酸磷酸(NADP)溶液
    对于S9肝脏部分
    成分
    每2.5毫升
    NADP
    191.5毫克
    无菌蒸馏H 2 O
    2.5毫升
    将NADP溶解在蒸馏水中并通过涡旋混合。管放置在冰浴中。该解决方案可以使用长达六个月
  6. 1 M葡萄糖-6-磷酸
    对于S9肝脏部分
    成分
    每5毫升
    葡萄糖-6-磷酸(G-6-P)
    1.41毫克
    无菌蒸馏H 2 O
    5毫升
    将葡萄糖-6-磷酸溶于5ml蒸馏水中并通过涡旋混合。管放置在冰浴中。该解决方案可以使用长达六个月
  7. 氨苄青霉素溶液(4毫克/毫升)
    用于耐氨苄西林的试验
    R因子菌株的主板
    成分
    每500毫升
    氨苄西林三水合物
    0.4毫克
    氢氧化钠(0.02 N)
    50毫升
    将氨苄青霉素三水合物溶于50ml NaOH(0.02N)中并通过涡旋混合。管被放置在冰浴
  8. 结晶紫溶液(0.1%)
    用于结晶紫灵敏度测试(确认rfa突变)
    成分
    每500毫升
    结晶紫
    0.05毫克
    蒸馏水 O O
    50毫升
  9. 最小的葡萄糖平板
    用于诱变生物测定
    成分
    每500毫升
    琼脂
    7.5毫克
    蒸馏水 O O
    465毫升
    50倍VB盐(配方1)
    10毫升
    40%葡萄糖
    25毫升
    在465ml蒸馏水中加入琼脂,并在121℃高压灭菌20分钟。冷却后,轻轻加入盐和葡萄糖
  10. 组氨酸/生物素平板(非R因子菌株的主平板)
    用于测试组氨酸需求量
    成分
    每500毫升
    琼脂
    7.5毫克
    蒸馏水 O O
    457毫升
    50倍VB盐
    10毫升
    40%葡萄糖
    25毫升
    无菌组氨酸(每400毫升H 2 O 2克)
    5毫升
    无菌0.5 mM生物素
    3毫升
    将琼脂溶于蒸馏水中的给定浓度。分别高压灭菌每个溶液20分钟。溶液冷却后,轻轻加入每种盐
  11. 氨苄青霉素和四环素*平板
    用于培养含有质粒pKM101和pAQ1 *的菌株的主盘
    成分
    每500毫升
    琼脂
    7.5毫克
    蒸馏水 O O
    405毫升
    50倍VB盐
    10毫升
    40%葡萄糖
    25毫升
    无菌组氨酸(每400毫升H 2 O 2克)
    5毫升
    无菌0.5 mM生物素
    3毫升
    无菌氨苄青霉素溶液(8 mg / ml 0.02 N NaOH)
    1.58毫升
    *无菌四环素溶液(8 mg / ml 0.02 N HCl)
    0.125毫升
    将琼脂溶于蒸馏水中的给定浓度。分别高压灭菌每个溶液20分钟。溶液冷却后,轻轻加入每种盐
    注意:TA 102对四环素有抗性。 4°C时板的保质期为两周。
  12. 营养琼脂平板
    用于检测基因型[结晶紫灵敏度(rfa)和紫外敏感度(AuvrB)]和细菌存活率
    成分
    每500毫升
    营养琼脂
    7.5毫克
    蒸馏水 O O
    500毫升
    将琼脂溶于蒸馏水中的给定浓度。分开高压灭菌20分钟。将冷却后的溶液倒入培养皿中
  13. S9混合(大鼠肝微粒体酶+辅因子)
    成分
    标准S9混合每25毫升
    小鼠肝脏
    1.0毫升(2%)
    MgCl 2 -KCl盐
    0.5毫升
    1 M葡萄糖-6-磷酸
    0.125毫升
    0.1 M NADP
    1.0毫升
    0.2 M磷酸盐缓冲液,pH 7.4
    12.5毫升
    无菌蒸馏H 2 O
    9.86毫升
    注意:按照上面列出的相反顺序加入每种成分(先加水,然后加磷酸盐缓冲液...)。避免重新凝固S9混合物。
  14. 叠氮钠
    用于诱变试验
    成分
    每毫升
    叠氮钠
    10微克
    高压灭菌器蒸馏H 2 O
    990μl(使总体积为1 ml)
    通过服用1,2,4μl10 mg / ml
    制备工作浓度
  15. 2-Nitrofluorine
    用于诱变试验
    成分
    每毫升
    2-Nitrofluroine
    10微克
    高压灭菌器蒸馏H 2 O
    990μl(使总体积为1 ml)
    通过服用1,2,4μl10 mg / ml
    制备工作浓度
  16. 丝裂霉素
    用于诱变试验
    成分
    每毫升
    丝裂霉素
    10微克
    高压灭菌器蒸馏H 2 O
    990μl(使总体积为1 ml)
    通过服用1,2,4μl10 mg / ml
    制备工作浓度
  17. 2-Anthramine
    用于诱变试验
    成分
    每毫升
    2-Anthramine
    10微克
    高压灭菌器蒸馏H 2 O
    990μl(使总体积为1 ml)
    通过服用1,2,4μl10 mg / ml
    制备工作浓度

    致谢

    Urvashi Vijay想感谢工作进行的斋浦尔IIS大学动物学系。感谢由2012/9389年度国际友好协会奖学金获得给Urvashi Vijay的资助。
    利益冲突:没有声明。
    作者贡献: Urvashi Vijay,Sonal Gupta,Priyanka Mathur在Pradeep Bhatnagar的指导下执行协议和方法。 Prashanth Suravajhala在所有作者批准之前重新审查了作品并对原稿进行了校对。

    参考

    1. 埃姆斯,B.N。(1971)。 用肠内细菌检测化学诱变剂在: Hollaender,A.(编辑)。化学诱变剂,其检测原理和方法vol。 1. Plenum pp:851-863。
    2. Ames,B. N.,Durston,W. E.,Yamasaki,E.和Lee,F. D.(1973a)。 致癌物是致突变物:一种简单的测试系统,将肝匀浆用于活化和细菌检测。美国国立科学院美国科学院 70(8):2281-2285。
    3. Ames,B. N.,Lee,F. D.和Durston,W. E.(1973b)。 改进的细菌检测系统,用于检测和分类诱变剂和致癌物。 Proc Natl Acad Sci USA 70(3):782-786。
    4. Ames,B. N.,McCann,J.和Yamasaki,E.(1975a)。 检测具有沙门氏菌/哺乳动物 - 微粒体致突变性的致癌物和致突变物的方法测试。 Mutat Res 31(6):347-364。
    5. Ames,B. N.,Yamasaki,E.,Mc Cann,J。(1975b)。 检测沙门氏菌 /微粒体测试中的致癌物质。分析300种化学物质。 Proc Natl Acad Sci U S A 72:5135-5139。
    6. Barnes,W.,Tuley,E和Eisenstadt,E。(1982)。 对鼠伤寒沙门氏菌hisG46错义突变His +回复突变体进行碱基序列分析。 Enviorn Mutagen 4:297。
    7. Bernstein,L.S.,Kaldor,J.,McCann,J。和Pike,M.C。(1982)。 沙门氏菌测试诱变数据统计分析的经验方法。 Mutat Res 97:267-281。
    8. Brusick,D.J.,Simmon,V.F.,Rosenkranz,H.S.,Ray,V.A。和Stafford,R.S。(1980)。 对大肠埃希氏大肠杆菌WP2和WP2 uvrA回复突变试验的评估。 Mutat Res 76(2):169-190。
    9. Czyz,A.,Szpilewska,H.,Dutkiewicz,R.,Kowalska,W.A.,Biniewska-Godlewska,A。和Wegrzyn,G。(2002)。 比较艾姆斯试验和新开发的检测海洋环境致突变污染的检测方法。 a> Mutat Res 519:67-74。
    10. DeFlora,S.,Zanacchi,P.,Camoirano,S.,Bennicelli,S.,Badolati,G. S.(1984)。 艾姆斯回复试验和细菌DNA修复试验中135种化合物的基因毒性活性和效力。 Mutat Res 133:161-198。
    11. Fluckiger-Isler的。 S.,Baumeister,M.,Braun,K.,Gervais,V.,Hasler-Nguyen,N.,Reimann,R.,Van Gompel,J.,Wunderlich,H.G。和Engelhardt,G.(2004)。 评估Ames II检测的性能:与19种编码化合物进行合作研究 Mutat Res 558:181-197。
    12. Frantz,C.N和Malling,H.V。(1975)。 定量微粒体诱变测定法。 Mutat Res 31 :365-380。
    13. Garner,R.C.,Miller,E.C。和Miller,J.A。(1972)。 黄曲霉毒素B 1肝微粒体代谢为对鼠伤寒沙门氏菌有毒的活性衍生物 TA 1530. Cancer Res 32(10):2058-2066。
    14. Gupta,P.,Mathur,N.,Bhatnagar,P.,Nagar,P.和Srivastava,S.(2009)。 医院废水的遗传毒性评估 环境毒素环境安全检测 72 (7):1925-1932。
    15. Hayes,A.W。(1982)。 原则与方法毒理学。Raven Press pp:238-241。
    16. Henderson,L.,Albertini,S.和Aardema,M。(2000)。 遗传毒性反应的阈值。 Mutat Res 464(1) ):123-128。
    17. Levin,D.E.,Yamasaki,E.和Ames,B.N。(1982)。 一种新的沙门氏菌检测菌株TA97,用于检测移码突变诱变剂。一系列胞嘧啶作为突变热点。 Mutat Res 94(2):315-330。
    18. Maron,D.和Ames,B.N。(1983)。 修订的<沙门氏菌致突变性测试方法 Mutat Res 113:173-215。
    19. Maron,D.,Katzenellenbogen,J。和Ames,B.N。(1981)。 有机溶剂与沙门氏菌 /微粒体测试的相容性 Mutat Res 88:343-350。
    20. Mathur,N.,Bhatnagar,P.和Bakre,P.(2005)。 评估巴利纺织染料的致突变性(拉贾斯坦邦)使用Ames Bioassay。 Appl Ecol Env Res 4:111-118。
    21. Morino-Caniello,N.F。和Piegorsch,W.W。(1996)。 艾姆斯测试:重新审视双重规则 Mutat Res < 369:23-31。
    22. Mortelmans,K.和Stocker,B. A. D.(1979)。 将质粒R46的突变体特性从其紫外线防护特性中分离出来 Mol Gen Gene t 167:317-327。
    23. Mortelmans,K.和Zeiger,E。(2000)。 艾姆沙门氏菌/微粒体致突变性测定。 Mutat Res 455:29-60。
    24. Prival,M.J.,Bell,S.J.,Mitchell,V.D.,Peiperl,M.D。和Vaughan,V.L。(1984)。 联苯胺和联苯胺同类染料和选定的单偶氮染料在修饰的沙门氏菌中的致突变性< assay。 Mutat Res 136(1):33-47。
    25. Shanabruch,W.G。和Walker,G.C。(1980)。参与recA + / ex4 + - 依赖性诱变的质粒(pKMIO1)基因的定位。
    26. Simmon,V.F.,Kauhanen,K。和Tardiff,R.G。(1977)。 饮用水中化学物质的致突变活动在:进展中在遗传毒理学。 249-258。
    27. Venitt,S.和Bosworth,D。(1983)。 细菌突变检测的无氧方法的发展:人体粪便的有氧和无氧波动测试提取物和参考诱变剂。致癌作用 4:339-345。
    28. Vijay,U.,Bhatnagar,P.和Mathur,P。(2014)。 突变TA 100和TA对健康中心废水的致突变性评估102株伤寒沙门氏菌(Salmonella typhimurium)。 Inventi Impact:Pharm Biotech&amp; Microbio 4:195-198。
    29. 维杰,美国(2014年)。斋浦尔卫生保健机构产生的液体流出物的物理化学表征和毒理学评估。 IIS大学。论文
    30. Walker,G.C。和Dobson,P.P。(1979)。 大肠杆菌umuC 突变体的突变和修复缺陷被质粒所抑制pKM101。
    31. Wilcox,P.,Naidoo,A.,Wedd,D.J。和Gatehouse,D.G。(1990)。 鼠伤寒沙门氏菌 TA102与大肠杆菌的比较 > WP2 tester strains。 Mutagenesis 5(3):285-291。
    32. Zeiger,E。(1985)。用于鉴定推定致癌物的沙门氏菌致突变性测定。在:Milman,H.A。和Weisburger,E.K。(Eds)中。致癌物测试手册。 Noyes Publishers pp:83-99。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vijay, U., Gupta, S., Mathur, P., Suravajhala, P. and Bhatnagar, P. (2018). Microbial Mutagenicity Assay: Ames Test. Bio-protocol 8(6): e2763. DOI: 10.21769/BioProtoc.2763.
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Ajay Kumar
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow
Dear Prashanth
Thank you for your response. Please check the Composition of Vogel Bonner Medium. The right composition is:
1. MgSo4.7h20-10.0gm
2. Citric Acid Monohydrate-100gm
3 K2HPo4-500gm
4- NaHNH4Po4.4H20-175gm

Distilled Water to make vol. one litre.

The Composition of Agar used for solidification is 1-2%, Generally 1.5%, So the Amount would be 15 Gram for 1 litre and 7.5 gm for half litre. Please check your composition.

BTW, Please let me Know this is review paper?
Please make the necessary changes.
7/13/2018 10:00:08 PM Reply
Ajay Kumar
CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow
Dear Sir/Mam
I have read your article in bio-protocol and found so many mistakes in the protocol. Please go through the composition of Vogel Bonner Medium Recipe 1 in your protocol.
1) All the constituents listed are in mg (miligram), but actually it is in grams.
2) the composition Agar mentioned in recipe 9, 10 and 11 in mg but actually it is in grams.
3) Please check all the compositions carefully.
There is so many blunder mistakes, which can make experiment non-reproducible.
Peer review is necessary.
7/9/2018 12:01:09 AM Reply
Prashanth Suravajhala
Department of Biotechnology and Bioinformatics, Birla Institute of Scientific Research, India

Dear Ajay

Apologies if you could not reproduce the protocol. Can you please clarify in details while our PhD fellow Urvashi who benchmarked all these recipes get back to you ASAP?

BTW, this protocol was peer-reviewed by three experts and reviewers and after careful check, it was published.

Regards
Prash for authors

7/9/2018 12:21:40 AM