ANDC DU/Biology Protocols/Ame's Test

=Experiment: To conduct Ames test for screening substances for mutagenicity=

First developed by Dr. Bruce Ames in 1971, the Ames test is a widely used technique for screening potential carcinogens by testing for mutagenesis of bacteria. It relies on the observation that the most common cause of cancer is the occurrence of somatic mutations brought about by DNA damage. Chemicals that damage bacterial DNA, and induce mutations, are also likely to cause mutations in mammalian cells. Doing the assay in bacteria gives much faster and less expensive results than animal studies, with results usually being available in 2 days.

The assay developed by Dr. Ames used auxotrophic his mutants of Salmonella enterica (formerly S. typhimurium), unable to synthesize their own histidine. Since revertant mutations to a wild type prototrophic phenotype grow on minimal media without any histidine supplement, it became possible to select mutants directly, rather than screening for the presence of mutations by replica plating. (The test is actually done using a trace amount of histidine that allows 2-3 generations of growth for auxotrophic mutants before the histidine is depleted. This increases the chance of mutation events occurring by allowing DNA replication to occur in the presence of the potential mutagens, and also allows the toxicity of compounds to be assessed by inhibition of the faint background bacterial growth.)

The Ames test can be done in two different ways: a qualitative "spot test" that allows the rapid screening of multiple compounds for possible mutagenicity, and a quantitative assay using sequential dilutions of the test chemical added to the top agar overlay to find out what concentrations of the test chemical are mutagenic and/or toxic. In this lab protocol, you will be doing spot tests for 8 different chemicals.

Further improvements to the assay include the use of strains mutated for DNA repair and cell wall synthesis (to increase sensitivity to mutagens) and the use of several strains containing different types of mutants (which allow the detection of different types of DNA damage). Enhanced results have also been achieved by the treatment of potential mutagens with liver cell extracts, to duplicate the conversion of compounds that are not mutagenic into carcinogenic compounds by liver cell enzymes (Ames et al, 1973).

In this protocol we will be using his auxotrophs of Escherichia coli K12. (Strain AB 3612 is also a thiamine auxotroph, so the overlays will have to be supplemented with thiamine as well.) The assay will be similar to the original assay, except that the use of the non-pathogenic E. coli strain will be less risky than using the pathogenic S. enterica. The major differences from the standard Ames test protocol will be that a single type of auxotrophic mutant strain will be used, the strain will not be optimized for mutagenesis by multiple mutations, and that the compounds used will not be treated with liver extracts. This means that the assay will actually be determining mutagenicity, rather than carcinogenicity.

Materials:
1) 5 Vogel-Bonner minimal media E plates with 2 % glucose (prewarmed to 37 °C) Composition of Vogel-Bonner minimal medium	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 minimal media top agar overlays (3 ml volume each) Minimal media overlay contains 5-7 g of agar per litre, composition as described above. (melted in microwave, cooled to 50 °C in water bath) 3) 2 LB top agar overlays (3 ml volume each) (melted in microwave, cooled to 50 °C in water bath) Composition of LB medium overlay: LB medium          pH 7.5 Ingredient	per 1000 ml Tryptone	10 g Yeast Extract	5 g NaCl	5 g Glucose	1 g Agar	5 g Add salts in the order indicated. Add agar after adjusting the pH to 7.5 with the help of 1N HCl and 1 M NaOH accordingly.

4) 1.5 ml of 0.105 mg/ml histidine, 1.00 µg/ml thiamine solution (filter sterilized) 5) overnight culture of Escherichia coli strain AB 3612 (centrifuged and resuspended in an equal volume of sterile saline) 6) sterile dH2O (~ 3 ml) 7) dimethyl sulphoxide (DMSO) (~ 2 ml) 8) 10 mg/ml histidine solution (filter sterilized) 9) 20 sterile filter paper disks 10) Eppendorf tubes (~ 4) 11) 2 syringe filters 12) 2 one-ml syringes 13) p10 tips, p100 tips, p1000 tips

Procedure:
Week 1

1. Add 330 µl of histidine/thiamine solution and 100.0 µl of E. coli to each minimal media overlay, blend by phage mixing (Because top agar is a viscous solution, standard vortexing tends to introduce air bubbles into the media, which may be difficult to distinguish from bacterial colonies. To reduce the number of air bubbles resulting from mixing, molten agar solutions should be mixed using an appropriate technique.  The exact technique to use will depend on the type of tube the samples are prepared in. A technique called “phage mixing” has been developed for samples in standard glass test tubes.  This is done by holding a tube with the cap in the palm of the hand, and the fingers firmly holding on to the tube.  The tube is held at a 45° angle, and rotated in a circle 4-6 times in 1-2 seconds. This technique allows for a gentle but thorough mixing, with minimal bubbles being introduced.) and prepare pour plates using 3 of the minimal media plates. (Leave the plates at 37 °C until just before use.) Label the plates to distinguish them from the LB overlay plates. 2. Prepare 2 LB overlay pour plates without any added bacteria or histidine/thiamine solution. 3. Once the plates have solidified (at room temperature), divide the plates into 4 equal sections by drawing lines on the bottom of the plate, and aseptically place a sterile filter paper disk in the centre of each section of the plate. 4. Choose 2 substances that you wish to test for mutagenicity. (These should be different from the chemicals prepared by other groups – check the sign-up list on the board in the lab.) If the substance is in solid form, weigh out 10 mg on the analytical balance, and then dissolve it in 1.000 ml dH2O. (If the substance will not dissolve in dH2O, dissolve it in 1.000 ml DMSO. If the substance will not dissolve in either solvent, let it settle out, draw off the solvent using a p1000, and transfer to a clean sterile Eppendorf tube.)  If the substance is in liquid form already, proceed to the next step. Note the concentration of the original solution (if known). Because these compounds are suspected carcinogens, they should be handled with gloves. 5. If your samples have been dissolved in dH2O, filter sterilize each of the solutions using a syringe filter, and transfer to a fresh sterile Eppendorf tube. This will be demonstrated in the lab. (If your sample has been dissolved in DMSO, it should already be sterile, and will not need to be filtered.) Label your tubes with the substance used and the concentration. 6. Choose 8 different substances from among the solutions prepared, and add 10 µl of each sterile solution to a different filter paper disk on the minimal media overlay plates. Do not move the filter paper disc once the solutions have been added. Label the plates with the substances used and all other relevant data. 7. Add 10 µl of the same solutions to 8 filter paper disks on the LB overlay plates. Label the plates with the substances used and all other relevant data. 8. On the remaining minimal media overlay plate, add 10 µl sterile dH2O to one disk, 10 µl DMSO to the second disk, 10 µl of 10 mg/ml histidine solution to the third disk, and nothing to the filter paper disk. Label the plates with the substances used and all other relevant data. 9. Place inverted plates on tray by incubator. These will be incubated at 37°C for 2 days, and then refrigerated until next week.

Week 2

Examine your plates and see if there are elevated levels of revertant mutants in a circle surrounding your filter paper disks. If there is a zone of inhibited background growth around any of the filter paper disks, measure the size of it. Compare your results with the control plates.

Report
Short report required.

Calculate the actual amounts of each substance added to each disk. (If initial concentration of liquid is not known, or substance will not dissolve in H2O or chloroform, note this in your results.) Note whether the substance is mutagenic or not at the level used. If colonies appeared on your control plates, note how this affects the interpretation of your test plates. Use the known mechanisms for different mutagens to work out what sort of mutation event produced the original auxotrophic mutant.

References:
Ames, B.N., Lee, F.D., and Durston, W.E. (1973). An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proceedings of the National Academy of Sciences USA, 70: 782-786.