Materials and Reagents

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 )
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 )
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

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

Procedure

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).
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)
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.
Label all minimal glucose agar plates and Eppendorf tubes prior to experiment.
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.
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.
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.
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)

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.
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.
Care must be taken to protect from chemical exposure by wearing gowns, eye glasses and gloves.
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

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 g
Citric acid monohydrate	50 g
Potassium phosphate, dibasic (anhydrous) (K₂HPO₄)	250 g
Sodium ammonium phosphate (NaNH₄HPO₄·4H₂O)	87.5 g

Salts are added to the warm water in a flask. Place the flask on a hot plate
After each salt dissolves entirely, transfer the solution into glass bottles and autoclave for 20 min at 121 °C
When the solution gets cool, cap the bottle tightly
Store the solution at 4 °C

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
Salt solution (1.65 M KCl + 0.4 M MgCl₂)
For S9 hepatic fraction
Ingredients
Per 250 ml
Potassium chloride (KCl)
30.75 g
Magnesium chloride (MgCl₂·6H₂O)
20.35 g
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
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 g/250 ml)
0.2 M disodium hydrogen phosphate (Na₂HPO₄)
220 ml (7.1 g/250 ml)
Adjust pH to 7.4. Sterilize the buffer by autoclaving for 20 min at 121 °C
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
1 M glucose-6-phosphate
For S9 hepatic fraction
Ingredients
Per 5 ml
Glucose-6-phosphate (G-6-P)
1.41 g
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
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 g
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
Crystal violet solution (0.1%)
Used in tests for crystal violet sensitivity (to confirm rfa mutation)
Ingredients
Per 500 ml
Crystal violet
0.05 g
Distilled H₂O
50 ml
Minimal glucose plates
Used in Mutagenic bioassay
Ingredients
Per 500 ml
Agar
7.5 g
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
Histidine/Biotin plates (Master plates for non R-factor strains)
Used in tests for histidine requirement
Ingredients
Per 500 ml
Agar
7.5 g
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

Ampicillin and tetracycline* plates
Master plates for the cultivation of strains containing the plasmids pKM101 and pAQ1*

Ingredients	Per 500 ml
Agar	7.5 g
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.

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 g
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
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.
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
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
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
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.

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