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Correction: Microbial Mutagenicity Assay: Ames Test    

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Original research article

A brief version of this protocol appeared in:

We checked the composition of Vogel-Bonner media and other media and salts. There were some unit mistakes. Please replace them with the following (The correct units have been highlighted in yellow). Thank you so much for your help.

Recipes

  1. Vogel-Bonner medium E (50x)
    For Minimal agar (Recipe 9)
    Ingredients
    Per 500 ml
    Warm distilled H2O (40-50 °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
    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
  1. 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 in refrigerator.
  2. 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. 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
  2. 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
  3. 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
  4. 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
  5. 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
  6. 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 (4 mg/ml 0.02 N NaOH)
    1.58 ml
    *Sterile tetracycline solution (4 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.
  7. 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

References

  1. Vijay, U., Gupta, S., Mathur, P., Suravajhala, P. and Bhatnagar, P. (2018). Microbial Mutagenicity Assay: Ames Test. Bio-protocol 8(6): e2763.
Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Vijay, U., Gupta, S., Mathur, P., Suravajhala, P. and Bhatnagar, P. (2018). Correction: Microbial Mutagenicity Assay: Ames Test. Bio-protocol 8(16): e3016. DOI: 10.21769/BioProtoc.3016.
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