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Hi,
We have recently started standardising the Ames Test for mutagenicity
and have run into the following problems:
1. In certain experiments we see unusually high number of revertants
in our negative controls.
2. We set up the experiment in triplicates and sometimes see no
revertant or lawn growth in one of the replicates.
3. What should be the fold increase over negative control for a
positive test?
4. Some of the positive mutagens do not show more that 5-fold
increase over negative controls when tested at concentrations
reported in literature.
5. In the last 2-3 experiments we have seen overgrowth of the lawn.
We have individually tested the various components of the assay but
have not been able to figure out the cause.
If anyone has any suggestions or a trouble shooting guide for Ames
test, we would appreciate it.
Regards
Tejal
Dr. Tejal Choksi
Scientist-1
Dept. Cell & Molecular Biology
Torrent Research Centre
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Good Morning,
Regarding your questions concerning the Ames Test for mutagenicity.
1. In certain experiments we see unusually high number of revertants
in our negative controls.
It's Normal. It could be one of the incidences of the global (in)
stability of number of plasmid (around 30 copy of) transfected in the
strains. The real point of discussion is the frequencies of this
extra-variation related to an evolution of the quality of the strains.
2. We set up the experiment in triplicates and sometimes see no
revertants or lawn growth in one of the replicates.
I don't know. Check carefully your experimental process.
3. What should be the fold increase over negative control for a
positive test?
Generally, the factor used is 2 or 3, regarding the strains
considered (but we speak about clear positive). Basically, your
historical data (including the frequencies of extra-variation) are
the real and the more significant reference, because it represents
the "normal variation in your standard condition of test". Ames test
is a truncated (incomplete) test. Your historical data is a based for
discussion and conclusion.
4. Some of the positive mutagens do not show more that 5-fold
increase over negative controls when tested at concentrations
reported in literature.
Ames is based on the measurement of the NON-LETHAL mutagenic effect.
Generally, when it's the case (not show more that 5-fold increase),
it's related to a strong cytotoxicity also due to lethal mutagenic
effect.
5. In the last 2-3 experiments we have seen overgrowth of the lawn.
We have individually tested the various components of the assay but
have not been able to figure out the cause.
Many reasons are possible, mainly the conditions of culture.
I hope that you will be able to find solutions.
Best regards
Yann Courbebaisse
Pharmacien
Responsable des operations
EUROSAFE
Parc d'activites de la Breteche
Batiment B1
35760 SAINT-GREGOIRE
tel : +33 (0)2 99 23 63 56 (ligne directe)
tel : +33 (0)2 99 23 46 46 (standard)
fax : +33 (0)2 23 25 29 74
mail : yann.courbebaisse.aaa.eurosafe.fr
web : www.eurosafe.fr
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