The Ames test is a bacterial mutagenicity assay used to identify potential carcinogens. It uses strains of Salmonella typhimurium bacteria that are unable to synthesize the amino acid histidine and contain mutations in DNA repair systems. When these bacteria are exposed to a mutagen, some bacteria will experience a reverse mutation that allows them to synthesize histidine. These revertant bacteria will form colonies on a histidine-deficient growth medium. The Ames test is a quick and inexpensive method to screen chemicals compared to animal carcinogenicity studies. It has helped identify many carcinogens and led to the regulation of harmful substances. However, it also has limitations as bacteria differ from human metabolism and some known carcinogens are

1. AMES TEST
Vivek Giri
Msc Microbiology
2nd semester
P. G. Department of Biosciences

2. Contents
• Introduction
• General procedure
• Strains used by Ames
• Use of liver homogenate
• General procedure
• Importance of Ames test
• Limitations of Ames test

3. Introduction
• Mutagens are agents which causes mutation.
• Many chemical mutagens are found to be carcinogenic
• The Ames test (Salmonella typhimurium reverse mutation assay) is
a bacterial short-term test for identification of carcinogens using
mutagenicity in bacteria as an end point.
• All carcinogens are mutagens but some mutagens are not
carcinogens.
• The Ames test was described by Bruce Ames and his group at the
University of California, Berkeley in a series of papers in the 1970s

4. General procedure
Bacteria (Salmonella typhimurium) is used as a test organism – cheap and easy to culture.
Auxotrophic mutant strain of Salmonella typhimurium is used which lacks genes for histidine
biosynthesis and a mutation for excision repair system.
Cells in which a back mutation occurs due to the test compound grows and produce colony.
Small amount of histidine is added for initial growth
Rat liver extract is added to the medium which provides activation by cellular enzymes.
It permit identification of substances that are not directly mutagenic (or carcinogenic) but are
converted into mutagens by the enzymatic reactions that take place in the liver of animal

6. Strains used by Ames
The deletion through the uvrB region of the chromosome
eliminates the excision repair system for DNA.
The gal and rfa (deep rough) mutations eliminate, to
different extents, the polysaccharide side chain of the LPS
that coats the bacterial surface, making the bacteria more
permeable and completely nonpathogenic.
The TA1535 set (TA1535, TA1536, TA1537, TA1538), which
is rfa and uvrB, is recommended for general testing for
mutagens and carcinogens in vitro, as it is the most
sensitive to mutagenesis.
The TA1975 set is used for examining the effect of repair
on mutagenesis and killing (in comparison to the TA1535
set).
The TA1950'set and TA1530-set are less sensitive to
mutagens in vitro, but may be required for use in the host
mediated assay in which Salmonella strains are incubated
in the peritoneum of a mouse or rat

7. Use of liver homogenates
The active forms of carcinogens such as aflatoxin, polycyclic hydrocarbons, dimethyl nitrosamine,
and various aromatic amines are formed by mammalian metabolism, in particular by the TPNH-
dependent microsomal enzymes of liver.
The principal limitation of any bacterial system for detecting carcinogens as mutagens is that
bacteria do not duplicate mammalian metabolism in activating carcinogens.
Mammalian-liver homogenates have been used by Garner et al., to activate aflatoxin B1 to a
compound lethal to our bacterial tester strain lacking excision repair, by Malling to activate
dimethyl nitrosamine to a compound that reverts one of our bacterial tester strains, and by Slater
et al., to activate dimethyl nitrosamine to a compound lethal for bacteria lacking polymerase I.

8. Preparation of liver homogenate
Preparation of liver homogenate
Male rats (Sprague-Dawley/Bio-1 strain, Horton Animal Laboratories) were
maintained on Purina laboratory chow. A week before they were killed, their
drinking water was made 0.1% in sodium phenobarbital . The rats (250-500 g)
were killed by a blow to the head and cervical dislocation; the liver was
removed and placed in a sterile, ice-cold beaker.
A portion of human liver was obtained from an autopsy of a 77-year-old man
who had died 7 hour earlier of heart failure.
Source of liver

9. Preparation of Liver Homogenate Fraction "S-9“
The liver (rat livers
were 10-25 g each)
was washed in an
equal volume of
0.15 M KCl, minced
with sterile scissors
in three volumes of
0.15 M KCl (3 ml/g
of wet liver), and
homogenized with a
Potter-Elvehjem
apparatus with a
Teflon pestle.
The homogenate was
centrifuged (Sorvall
RC2-B) for 10 min at
9000 X g, and the
supernatant, which we
call the S-9 fraction,
was decanted and
saved.
1 ml of S-9
fraction
contained
microsomes from
250 mg of wet
liver
The fresh S-9
fractions (rat and
human) were
distributed in 2-ml
portions in small
plastic tubes (2-ml
liquid nitrogen
storage tubes/4-
Shore-USA, La Jolla,
Calif.), quickly
frozen in dry ice,
and stored at -80°
in a Revco freezer.

10. Test procedure
Addition of S-9 mix to
the top agar the S-9 Mix
contains per ml: 0.3 ml
of S-9 fraction, 8 mM
MgCl2, 33 mM KCl, 5
mM glucose-6-
phosphate, 4 mM TPN
(triphosphopyridine
nucleotide), and 100
mM sodium phosphate
(pH 7.4).
To 2 ml of molten top
agar at 45⁰C are added
0.1 ml of the bacterial
tester strain culture (2
to 3 X 109/ml), up to
0.1 ml of a solution
(Me2SO or water) of the
compound to be tested,
and 0.5 ml of S-9 Mix
Then the tube is rotated
quickly and the contents
are poured on the agar
plate. The additions and
pouring should take less
than a minute. The
colonies on the plates (his+
revertants) are counted
after a 2-day incubation at
37°.

11. Ref 2

12. Ref 3

13. Ref 1

14. Fluctuation method

15. Importance of Ames test
Compared to the carcinogenic assays on mice and rats (takes few years) the Ames test is quick
and inexpensive.
Used in wide variety of fields such as pharmaceutical, chemical, fertilizer, cosmetics, food
and many more industries for testing chemicals for mutagenicity and carcinogenicity.
In 1972-77 a compound known as Tris-BP was used as a flame retardant in children’s polyester
pyjamas which was later found to be a potential mutagen in Salmonella and Drosophila
And it also interacts with human DNA and damages mammalian chromosomes.
Before its use was discontinued, more than 50 million children were exposed to the chemical.

16. Limitations of Ames test
Salmonella typhimurium is not a perfect model for humans as it is a prokaryote.
The sensitivity for some of the known carcinogen is less in the Ames test.

17. References
1. Ames, B. N. (1971) in Chemical Mutagens: Principles and Methods for their Detection,
ed. Hollaender, A. (Plenum Press, New York), Vol. 1, pp. 267-282.
2. Ames, Bruce N., Frank D. Lee, and William E. Durston. 1973. “An Improved Bacterial
Test System for the Detection and Classification of Mutagens and Carcinogens.”
Proceedings of the National Academy of Sciences, USA 70: 782
3. Ames, Bruce N., William E. Durston, Edith Yamasaki, and Frank D. Lee. 1973.
“Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for
Activation and Bacteria for Detection.” Proceedings of the National Academy of
Sciences, USA 70: 2281–85.
4. Harlt, D. L. (2014). Essential Genetics: A Genomics Perspective. Jones & Bartlett
Learning
5. Brooker, P. D., & Robert J. Brooker, P. D. (2014). Genetics: Analysis and Principles.
McGraw-Hill Education

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