Ames test

Ames test

 Introduction

Ames test is used to measure the mutagenic potential of a chemical and also check can cause mutation in the genome of bacteria. The bacterial reverse mutation assay detects point mutations, both frame shifts and/or base pair substitutions. Strains of Salmonella typhimurium auxotroph is unable to synthesis histidine When this histidine (his-) dependent cells are exposed to the minimal media with trace amount of histidine and biotin only those cells which revert to (his+) independence are able to form colonies. The trace amount of histidine in the media allows all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. The his+ revertants are readily discernable as colonies against the limited background growth of the his-cells. By using several different tester strains, base pair substitution mutations and frameshift mutations can be detected. Spontaneous reversions occur with each of the strain, which will be considered as background level. Mutagenic compounds cause an increase in the number of revertant colonies relative to the background level.

Aim:

To check the mutagenic potential of chemicals by observing whether they cause revert mutations in sample bacteria.

Principle:

Ames test uses several strains of bacteria (Salmonella, E.coli) that carry a particular mutation. Point mutations are made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied. But culturing His- Salmonella is in a media containing certain chemicals, causes mutation in histidine encoding gene, such that they regain the ability to synthesize histidine (His+). This is to say that when a mutagenic event occurs, base substitutions or frameshifts within the gene can cause a reversion to amino acid prototrophy. This is the reverse mutation. These reverted bacteria will then grow in histidine- or tryptophan-deficient media, respectively.

A sample’s mutagenic potential is assessed by exposing amino acid-requiring organisms to varying concentrations of chemical and selecting for the reversion event. Media lacking the specific amino acid are used for this selection which allow only those cells that have undergone the reversion to histidine / tryptophan prototrophy to survive and grow. If the test sample causes this reversion, it is a mutagen.

Procedure:

I ) Isolate or procure an auxotrophic strain of Salmonella typhimurium for histidine (ie. His-ve) or mutant strain of E. coli for tryptophan. (ie. Trp-ve)

II) Prepare a test suspension of his-ve Salmonella typhimurium or E.coli in a plain buffer with test chemical (eg. 2-aminofluorene). Also add a small amount of histidine or tryptophan

III) Also prepare a control suspension of His-ve Salmonella typhimurium or E.coli but without test chemicals.

IV)  Incubate the suspensions at 37°C for 20 minutes

V) Prepare the two agar plate and spread the suspension on agar plate.

VI)  Incubate the plates at 37°C for 48 hours.

VII) After48 hours count the number of colonies in each plate.

Result Interpretation:

• The mutagenicity of chemicals is proportional to number of colonies observed.
• If there is a large number of colonies on the test plate in comparison to control, then such chemical are said to be mutagens.
• Very few numbers of colonies can be seen on control plate also. This may be due to spontaneous point mutation on hisidine or tryptopan encoding gene.

Left: Control without mutagenic chemical

Right: The colonies occurred on his+ or trp + containing media reveals reverse mutation. It results the chemical possess a mutagenic poential