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The Salmonella Mutagenicity Assay


LPI/CCP protocol for Salmonella Mutagenicity Test
1. Equipment and materials  
1.1 Autoclave, autoclave wraps and tapes
1.2 Laminar flow hood
1.3 Gas burner (optional)
1.4 0.2 µ filter discs and syringes
1.5 Antiseptic wipers
1.6 Various sizes of sterile culture flasks, tubes, bottles and petri dishes
1.7 Fresh cultures for Salmonella tester strains (see Protocol for Growing Salmonella Tester Strains)
1.8 37°C incubator
1.9 Dry heating block
1.10 Vortex mixer
1.11 Test compound
1.12 Standard positive control mutagens
1.13 Top agar
1.14 Rat liver S9
1.15 Minimal glucose agar plates
1.16 Marker pens
1.17 Automatic colony counter
1.18 Other general laboratory items e.g. microwave oven, centrifuge, latex gloves, thermo-protective gloves, safety goggles…
 
2. Chemicals, reagents and media
2.1 0.5 mM histidin/0.5 mM biotin solution for the top agar in mutagenicity test
    Ingredient per 250 ml  
    D-Biotin (F.W. 244.3) 30.5 mg    
    L-histidine.HCl.H2O
(F.W. 209.63) 		26.2 mg    
    dd H2O 250 ml    
  Dissolve biotin in hot water first, filter sterilize and store at 4°C in glass bottle.
   
2.2 Vogel-Bonner medium E (50 x strength stock) for minimal agar base
    Ingredient per 1000 ml    
    Warm dd H2O (45°C) 670 ml    
    MgSO4.7H2O 10 g    
    Citric acid monohydrate 100 g    
    K2HPO4 500 g    
    NaHNH4 PO4.4H2O 175 g    
  Add salts in the order indicated to warm water in a 2-liter flask placed on magnetic stirring hot plate. Allow each salt to dissolve completely before adding the next. Adjust the volume to 1 liter. Distribute into two 1-liter glass bottles. Autoclave, loosely capper, for 20 min at 121°C. When the solutions have cooled, tighten the caps.
   
2.3 40% glucose (autoclaved sterile)
    Ingredient per 1000 ml    
    glucose 400 g    
    dd H2O to 1000 ml    
   
2.4 Minimal glucose plates for mutagenicity test
  Ingredient per 1000 ml  
    Agar 15 g    
    dd H2O 930 ml    
    VB medium E stock
(autoclaved sterile) 		20 ml    
    40% glucose
(autoclaved sterile) 		50 ml*    
 

Add agar to dd H2O in a 2-liter flask. Place a large magnetic stirring bar inside for later mixing. Autoclave at 121°C for 20 min using slow exhaust. When the solution has cooled slighted, add sterile VB medium E stock and sterile 40% glucose. Stir the mix thoroughly. Pour 30 ml into each plate.

*Note: For TA97a, reduce the glucose level to 0.4% (i.e. 10 ml of 40% per 1000 ml). Glucose at 2% retards colony growth.
   
2.5 Top Agar for mutagenicity test
  Ingredient per 1000 ml  
    Agar 6 g    
    NaCl 5 g    
    dd H2O to 1000 ml    
  Microwave to dissolve the agar. Mix thoroughly and make 100 ml aliquots to 250 ml glass bottles with screw caps. Autoclave at 121°C for 20 min with loosened caps. Slow exhaust. Cool the agar and tighten the caps.
   
2.6 Potassium/magnesium solution
  Ingredient per 500 ml  
    KCl (1.65 M) 61.5 g    
    MgCl2 6H2O (0.4 M) 40.7 g    
    dd H2O to 500 ml    
  Dissolve the salts. Autoclave at 121°C for 20 min. When the solution has cooled slighted, tighten cap. Store at RT or 4°C.
   
2.7 0.2 M Sodium phosphate buffer, pH 7.4
  Ingredient per 500 ml  
    NaH2PO4 12.0 g    
    Na2HPO4.2H2O 17.8 g    
    dd H2O to 500 ml    
  Dissolve the salts with 450 ml dd H2O. Adjust pH with 0.2 M NaH2PO4 or 0.2 M Na2HPO4 to 7.4. Add dd H2O to the final volume of 500 ml.
Autoclave at 121°C for 20 min. When the solution has cooled slighted, tighten cap. Store at RT or 4°C.
   
2.8 Fresh 80 mM NADP (Triphosphopyridine nucleotide, sodium salt, hydrate)
  Ingredient per 1 ml  
    NADP (F.W. 765.39) ~61.23 mg    
    Sterile dd H2O ~1 ml*    
  *It is better to weigh a batch of dry aliquots of NADP in small sterile glass tubes with screw caps. Wrap the tubes with metal foil and mark the NADP weight outside. Store them in a desiccator at -20°C. Add dd H2O according to the exact weight of NADP upon use. This is one of the expensive reagents used in the SMA – make up stock solutions as required and not in excess.
   
2.9 Fresh 120 mM D-Glucose-6-phosphate (G-6-P, monosodium salt)
    Ingredient per 1 ml  
    G-6-P (F.W. 304) ~36.48 mg    
    Sterile dd H2O ~1 ml*    
  *Prepare as for NADP above.
   
2.10 Fresh High S-9 mix for 40 plates (with cofactors)*
    Ingredient per
20 ml 		final
concentration
    Sterile dd H2O 5.6 ml      
    0.2 M phosphate buffer,
pH 7.4 		10.0 ml 0.1 M    
    80 mM NADP 1.0 ml 4 mM    
    120 mM G-6-P 1.0 ml 6 mM    
    K, Mg salts solution 0.4 ml 33 mM – 8 mM    
    Rat liver S-9
(Aroclor-1254 induced)		 2.0 ml 10% v/v    
  *Prepare immediately before use in ice bucket in above order. Keep the ingredients chilled in preparation. Add in S9 fraction last. Discard any leftover for S9 or S9 mix. Never re-use S9 or S9 mix.
   
3. Operation procedures
3.1 Prepare serial concentrations of the test chemical, and solutions for negative and positive controls.
3.2 Dissolve a top agar aliquot (100 ml)* in microwave oven. Handle with care while the content is hot.
  *Calculate the aliquots size according to the total number of plates needed for a test. Duplicate or triplicate plates are needed for each dose levels and controls.
3.3 Add 1/10 volume (10 ml) of 0.5 mM histidine/0.5 mM biotin solution to the molten top agar. Mix thoroughly by swirling.
3.4 Dispense 2 ml of molten top agar to each of culture tubes held at 45°C in a heating block. Cap the tubes.
3.5 Prepare S9 mix.
3.6 Add 0.1 ml of a fresh overnight culture of the test strain, 0.1 ml or less of the test compound/positive control mutagens, and 0.5 ml of PBS (PBS can be added with 0.5 mM bio/his, or optionally 0.5 ml of cold S9 mix) to each agar tube. Both control with and control without S9 mix are necessary.
3.7 Vortex the mix for 3 sec at low speed.
3.8 Pour the mix on a minimal glucose agar plate. Quickly tilt and rotate to spread the top agar evenly on the plate. Operation time from step 3.6 to the end of this step should not take more than 20 seconds.
3.9 Place the plate on a level place and allow several minutes for hardening.
3.10 If photosensitive chemicals are used, cover the plates promptly with suitable material.
3.11 Incubate the invert plates at 37°C in a dark vented incubator.
3.12 After 48 to 72 hr incubation, check the background lawn of the plates and score the revertant colonies using the automatic colony counter.
3.13 Record or save results according to the laboratory protocol for recoding results.