Lab 5:  Ames test

 

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 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, 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 lab 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. 

 

Several different chemicals will be provided in the lab, but you are also welcome to bring in your own choices (e.g. pesticides, tobacco, household products) for testing.  If you are looking for ideas, (McCann et al, 1975) has a list of 300 substances tested by the Ames lab.  Note that since the test selects against auxotrophic mutants for amino acid synthesis, addition of histidine-containing solutions will result in invalid results.  Because of this, substances with a high protein content (meat, dairy, or legume products) are not suitable for testing using this technique.

 

Protocol

 

Materials (per group of 3-4)

 

Week 1

 

5 Vogel-Bonner minimal media E plates with 2 % glucose

(prewarmed to 37 °C)

3 minimal media top agar overlays (3 ml volume each)

(melted in microwave, cooled to 50 °C in water bath)

 2 LB top agar overlays (3 ml volume each)

(melted in microwave, cooled to 50 °C in water bath)

1.5 ml of 0.105 mg/ml histidine, 1.00 µg/ml thiamine solution

(filter sterilized)

overnight culture of Escherichia coli strain AB 3612

(centrifuged and resuspended in an equal volume of sterile saline)

sterile dH2O     (~ 3 ml)

dimethyl sulphoxide (DMSO)  (~ 2 ml)

10 mg/ml histidine solution       (filter sterilized)

20 sterile filter paper disks

eppendorf tubes (~4)

2 syringe filters

2 one-ml syringes

p10 tips, p100 tips, p1000 tips

 

Methods

 

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 (see Lab 1 and aseptic techniques handouts), 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, and place it in the designated rack at the front bench.

 

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   (On reserve in library)

 

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.

 

McCann, J. and Ames, B.N.  (1976).  Detection of carcinogens as mutagens in the Salmonella/microsome test:  Assay of 300 chemicals:  Discussion.  Proceedings of the National Academy of Sciences USA, 73:  950-954.

 

McCann, J., Choi, E., Yamasaki, E., and Ames, B.N.  (1975).  Detection of carcinogens as mutagens in the Salmonella/microsome test:  Assay of 300 chemicals. Proceedings of the National Academy of Sciences USA, 72:  5135-5139.

 

Materials Safety Data Sheets (on web)

 

http://www.fishersci.ca/homepage4.nsf/(waSearch)?openagent&lang=E&DB=msds2.nsf