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
