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_Ames Test _
By: George Cusmai
Ames test Objective: A mutagen is a chemical that mutates or alters DNA.
Many times, mutagens may be carcinogens as they may induce cancer. Dr. Bruce
Ames developed a method by using specific mutant strains of the bacteria
Salmonella typhimurium to detect mutagens and possible carcinogens. This lab
will test various potential mutagens using Dr. Bruce Ames procedure and
methods. Introduction: Salmonella typhimurium is a prototrophic bacterium,
which simply means that it can survive on its own as long as it has a source
of energy, a carbon source, and inorganic salts. For the experiment, the
auxotrophic form of salmonella is used. This mutated version cannot synthesize
the amino acid histidine. Growth of this type of bacteria will only grow if
hidtidine is added to the median or if the mutation occurs which corrects the
original mutation. The latter is known as a back mutation or a reversion,
where the mutation causes the bacteria to return to its wild type state. This
type of mutation is either brought about by normal replication errors, or by
mutagenesis, due to the action of a mutagen. This experiment will use four
different strains of the bacteria salmonella (see table 1). Each strain has a
unique mutation, but they all lack the ability to produce histidine (His-).
This test is applied to measure the ability of chemicals, which may be
potential mutagens, that convert the His- bacteria to His+, hence producing
twice the number of colonies in a His- environment as compared to the number
that occur spontaneously. Summary: Several petri dishes were prepared each
containing 1ml of melted soft agar, 60 ml of a certain type of bacteria, and
20 ml of a potential mutagen. The dishes were incubated for 72 hours and
finally observed. Table 2 gives the overall results of the potential mutagens.
From these results, it was determined that the cough drop in bacteria TA98
(see table 1) had produced more than two times the number of colonies formed
when compared to both the negative (water) and positive (sodium azide)
controls. Therefore, the cough drop was used to further test this chemical as
a potential mutagen. Ten more petri dishes were set up each one with a
different concentration of potential mutagen (cough drop) to water. They were
set up using TA98 from of salmonella. Two petir dishes were prepared
containing 100% of the cough drop, another two containing a water, another two
containing a mixture of 50/50 cough drop to water, another two containing a
mixture of 40/60 cough drop to water, and another containing 70/30 cough drop
to water respectively. The results are observed in table 3, which shows a
breakdown of how the colonies developed. Ironically, the 100% cough drop
containing gel did not produce far more colonies than did the negative control
as would have been expected when comparing these results with table 2. The
same is observed in the different concentrations of cough drop to water. The
positive and negative controls help to compare and contrast in order to
determine whether a certain chemical is a possible mutagen or not. If medians,
which contained histidine, were used, colonies would have grown for all
chemicals tested including the negative control and would appear as a positive
test as a mutagen. Conclusion: Overall, the Ames test does help to determine
whether chemicals are mutagens or not. The first part of the experiment,
yielding results for table 2 gave a good indication on what a potential
mutagen may be. Yet, the further evaluation of that particular chemical, which
results can be found on table 3, did not produce results, which would
establish that particular chemical as a mutagen. Table 1: Mutant types of
Salmonella used for testing different chemicals Mutated Salmonella
Type Description of Mutation TA97a Frameshift mutation (deletion) in the hisC
gene TA98 Frameshift mutation (insertion) in the hisD gene TA100 Base
substitution in the hisG gene TA102 Nonsense mutation in the hisG gene Table
2: Results for testing possible mutagens Potential
Mutagen TA97a TA98 TA100 TA102 Sodium azide 3 300+ 25 53 Water 37 10 20 35
Dye 38 3 20 17 Sunny Delight 6 1 25 14 Sugar free cookies 32 45 40 48 Cough
drops 36 400+ 25 62 Table 3:Results for testing the cough drop as a potential
mutagen Petri Dishes Containing Mutagen Number of colonies 1 (100% cough
drop) 98 6 (100% cough drop) 105 2 (water) 78 7 (water) 65 3 (50/50 cough
drop/water) 80 8 (50/50 cough drop/water) 60 4 (40/60 cough drop/water) 74 9
(40/60 cough drop/water) 50 5 (70/30 cough drop/water) 43 10 (70/30 cough
drop/water) 50
Word Count: 770
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