Introduction:

The Ames test is performed to know the mutagenic properties of various substances that are
capable of causing cancer. This test mainly utilizes two strains S. typhimurium and E. coli which
are very much dependent on amino acids that they dont grow in the absence of those sources.
For instance, in the absence of histidine in media, the bacterial cells do not survive as they
themselves cannot produce amino acid. They can only grow on histidine deficient media when
they get mutated. Spontaneous mutations are caused by mutagens which are usually more,
compared to that of background mutations (Ames, et al, 1975).
The test mutagen discs are pressed in the medium containing minimal which allows reverting
back the mutations in bacteria due to which the colonies are formed. But it has limitations, the
Ames test performed in this experiment utilizes bacteria which are prokaryotic in nature which
helps us to determine mutations in prokaryotes. This limitation can be anyways not taken in to
account as DNA is pretty much similar in all the living organisms as far as chemical nature is
concerned. The other limitation is that it identifies only mutations related to histidine gene in
Salmonella, while many mutations occur in Salmonella when exposed to mutagen. This test has
an additional limitation that it does not determine whether the chemical is a cancer causing agent,
it just helps to find out the mutation causing agent (Cengage, 2012).
Methods and materials:
a) Materials:
For traditional Ames test: Minimal salts plate, soft molten agar 0.005M Biotin and
0.005M Histidine, Salmonella culture and five mutagens: sodium azide, - napthol,
kovacs reagent, red and orange coloring dyes.

For modified Ames test: David Mingoli Salt, D glucose, Bromocresol purple, D Biotin,
L-Histidine, sterile water, 2-NF, test mutagen and medium required for growth.

b) Traditional Ames test:

Minimal salt plates were labeled in duplicates with five different chemical mutagen names
(Sodium azide, -napthol, Kovacs reagent, food coloring dyes red and orange).
Besides, label 2 control plates and also one control plate for entire class which is labeled as
auxotrophic plate and a spontaneous mutation plate.
Add 300ul, 0.005M Biotin and 0.005M histdine along with 100ul of Salmonella culture in sof
top molten agar medium and pour in onto minimal salts agar medium. This procedure is followed
until ten plates are poured and are then set to solidify.
For the next step, gloves were worn and a disc was pressed down on to the agar plate to which
15ul of mutagenic compound was added. (This is a modification done in method performed than
in protocol).
In addition, a minimal salts agar plate was streaked with Salmonella culture which served as an
auxtrophic control (-ve control).
Incubate all the plates at 35C.
c) Modified Ames test:
21.62ml David Mingoli Salt + 4.75ml D glucose+ 2.38ml Bromocresol purple+ 1.19ml D
Biotin+ 0.06ml L-Histidine and this will make up to 30ml

2.5ml of the above made reaction mixture was received by each group in a sterile tube of 50 ml.
Then the volume was made to 20ml by adding 17.5ml of distilled water. This tube was vortexed
to make sure the contents were mixed well.
Then add 200ul into each well for first 24 wells which would serve as blank.
To the remaining 15 ml of reaction mixture, add 5ul of Salmonella overnight. The mixture was
properly mixed and added to the next 24 wells which served as background to detect any
spontaneous mutations.
After this, out of remaining 10ml, half volume was transferred to a new tube. To one of the tubes
test specified volume of test mutagen (- napthol) was added and mixed well. 20ul of this
mixture was then pipetted into each of the next 24 wells.
To other tube containing 5ml of culture, 25ul of 2-NF was added and then mixed well. This will
serve the purpose of positive control. 20ul of the above mixture prepared was then pipette in to
each of the last 24 wells.
The above plate with wells was then sealed using parafilm and then incubated at 35C for 4-6 hrs.
Results:
a) Traditional Ames test:
The results obtained after performing traditional Ames test are expressed in terms of colony
count or distance of colony from center.

Table 1: Mutated colony count of Salmonella and its distance of first colony from the mutagen
disc placed in the center of plate.
S. no

Mutagen used

Average

of

colony Distance from antibiotic

counts

disc

1.

Alpha-napthol

53.5

4.5mm

2.

Sodium azide

48

2mm

3.

Kovacs reagent

52.5

4.5mm

4.

Red food dye

48

7.5mm

5.

Orange food dye

56.5

3.5mm

Average colony counts obtained for Alpha napthol and sodium azide are 53.5 and 48 colonies/
plate respectively. Kovacs reagent gave a colony count of 52.5 colonies/plate, while the colony
count for Red food dye was found to be 48/ plate. 56.5 colonies were found to be mutated due to
Orange food dye. The distance from mutagenic disc was 4.5mm for both alpha-napthol, whereas
sodium azide plate had first colony at about 2mm from the mutagenic disc. Red orange food dyes
plate first colony from disc was found at 7.5mm and 3.5mm respectively.
These results were then plotted on a bar graph which is represented below.

60

Test mutagen Vs colony count/plate & distance from antibiotic in mm

50
40
30

colony count per plate

distance from disc in mm

20
10
0
alpha
napthol

Sodium
azide

Kovacs
reagent

red food orange

dye
food dye

Test mutagen

Figure 1: Different mutagenic agents and their mutagenic activity in terms of colony counts per
plate and distance of first colony from antibiotic disc.
B) Modified Ames test:
Negative control, background, sample mutagen and positive control (2-NF) were pipette into 24
wells each and were incubated at 35C for 4-6 days. The results obtained after that are as follows:
1. Negative control: all wells were found to be blue in color indicating no growth.
2. Background: 1 among the 24 wells showed yellow color indicating one mutated well.
3. Alpha-napthol (sample assigned): the solution immediately became yellow after addition
of sample to the tube and remained yellow after incubation for 4-6 days.
4. Positive control: two wells with yellow color were observed indicating growth in only
two wells.

Discussion:
The mutagen with less concentration will have a low zone of inhibition when compared to that of
highly mutagenic compound. This is because of the highly mutagenic compound is so strong that
it leads to toxicity and kills the bacteria very near to it instead of mutating it. At a distance where
it reaches a lower concentration may start producing mutation, but may have lesser colonies in a
given area range. So, due to the limitation in the area of the plate taken, it might be not justifiable
to compare the colony counts of various mutagenic compounds (Dutkiewicz, 2002). Therefore,
considering the distance from the antibiotic disc on minimal plate, red food dye is the strongest
mutagen (7.5mm), while Sodium azide is least mutagenic agent.
The negative control wells were found to be blue in color with no mutations which shows that
there was no contamination in the chemical mix used. Besides, there was only one background
mutation observed out of 24 wells where no mutagenic agent was used. This result is apt because
spontaneous mutations are very rare. Positive control used should show yellow color in all wells,
but had only two wells with yellow color. This might be due to the low concentration of the
mutagen used which was not sufficient enough to revert back the mutations in Salmonella or the
strain might be resistant to 2-NF. Sample specified for test was alpha- napthol which turned the
5ml Salmonella culture in chemical mixture to yellow immediately after adding. This might be
due to the presence of any acidic agent in the mutagen or due to any acidic contaminant turning
the solution to yellow even before incubation. The false positives may be because of the amino
acids that may be present in the medium (CDER, 2012).Hence, it was difficult to decide about
the mutagenic capability of alpha-napthol.

References:
Ames, B., et al, 1975. AMES TEST: Bacterial Reverse Mutation Assay taken from GenPharm
Tox,
http://www.genpharmtox.de/downloads/AssaySheetAMESTEST.pdf.
Cengage, 2002. Ames test from Encyclopedia of Public Health, 2002 retrieved from
http://www.enotes.com/ames-test-reference/ames-test
Dutkiewicz, R., et al, 2002. Comparison of the Ames test and a newly developed assay for
detection of mutagenic pollution of marine environments. From US national Library of
Medicine Library of , 2002 Aug 26,519(1-2)67-74.
US department of Drug and Health services, CDER & CBER, 2012. Guidance for Industry
S2(R1) Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for
Human Use. June, 2012, ICH.