I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
I
N
V
I
T
T
O
X
O
N
-
L
I
N
E
|
Protocol no. 30
THE AMES TEST
Reverse Mutation in Histidine-requiring Strains
of Salmonella typhimurium (complies with OECD Guideline 471) This procedure
evaluates the mutagenic potential of test chemicals by their effect on
five histidine requiring strains of the bacterium, Salmonella typhimurium
in the absence and presence of a rat liver metabolising system.
CONTACT
INVITTOX 34 Stoney Street Nottingham NG1 1NB
UK Tel: England - 0602 584740 Fax: England - 0602 503570
RATIONALE
The objective of this assay is to evaluate the
mutagenic potential of test chemicals by studying their effect on one or
more histidine requiring strains of Salmonella typhimurium in the absence
and presence of a liver metabolising system. When the cultures are exposed
to a mutagen some of the bacteria undergo genetic changes due to chemical
interactions resulting in reversion of the bacteria to a non-histidine-requiring
state. The reverted bacteria will then grow in the absence of exogenous
histidine thus providing an indication of the potential of the chemical
to cause mutation. Multiple tester strains are necessary because different
strains are mutated by a different class (or different classes) of compound.
Other types of bacteria can also be used, e.g. tryptophan requiring strains
of Escherichia coli. The basis of the test is very similar, the only difference
being that the bacteria have a requirement for a different amino acid.
BASIC PROCEDURE
Nutrient broth is inoculated with the appropriate
Salmonella strain (TA98, TA100, TA1535, TA1537, TA102) and incubated overnight.
A dose rangefinder for the test chemical is carried out using strain TA100
only over a wide dose range. Bacterial culture, test chemical and S9 mix
(or co-factor solution) are mixed with soft agar and then added to minimal
agar plates. The plates are incubated, inverted in the dark, for 48-72
hours. After this time the number of revertant colonies are counted. A
further two mutation experiments are carried out, with doses chosen on
the basis of the rangefinder. The number of colonies are counted from both
experiments and the mean is calculated for the individual plate counts
for each dose within an experiment. Statistical analysis of the counts
are carried out, and the results for mutagenicity are assessed.
CRITICAL ASSESSMENT
The assay is a rapid, reliable and economical
method for screening compounds of potential genetic activity at the nucleotide
level. It can be used in the evaluation of many types of substances including
solids, liquids, gases, and highly toxic substances. The results of several
independent large-scale studies have shown that there is a good correlation
between mutagenicity in Salmonella and carcinogenicity in mammals. A large
database has been accumulated with this assay, confirming its ability to
detect genetically active compounds of most chemical classes with an efficiency
of about 80%. The results obtained may be combined and correlated with
the results of other tests (e.g. in vitro mammalian cell assays and in
vitro cytogenetic assays) to evaluate the possible mutagenic risk of a
test chemical. The following bacterial strains, amongst others, are used
in this assay: Type of mutation in Strain the histidine gene TA98 frame-shift
TA100 base-pair substitution TA1535 base-pair substitution TA1537 frame-shift
TA102 base-pair substitution The two standard testing strains (TA1535 and
TA1537) are used in combination with the strains, TA98, TA100 and TA102,
which include the plasmid pKM101 as well as their particular histidine
gene mutation. The plasmid derivatives (TA98, TA100 and TA102) have an
increased sensitivity to certain mutagens as the pKM101 plasmid codes for
an error-prone DNA repair system. Strain TA102 has its histidine gene mutation
located on a multicopy plasmid, pAQ1. This strain is particularly sensitive
to the activity of oxidative mutagens and cross-linking agents. In addition
to the histidine mutation, most of the tester strains possess two additional
mutations that greatly increase their sensitivity to mutagens: one causes
the loss of the excision repair system (this mutation is not present in
strain TA102) and the other, loss of the lipopolysaccharide barrier that
coats the surface of the bacteria. Mutational events are rare, therefore
it is essential that large populations of bacteria are used in mutagenicity
testing. Maximum sensitivity is achieved by plating around 108 bacteria
although there are conflicting opinions as to what is the optimum concentration.
If fewer bacteria are plated, the number of spontaneous revertants may
be normal but the number of induced revertants may be low for a particular
concentration of mutagen. Some substances cannot be tested using the standard
Ames test because their physical nature precludes it. However, the basic
procedure can be adapted:- Volatile materials can be tested at reduced
temperatures or at raised pressure. Alternatively, chemicals which are
volatile, or relatively water insoluble, can be tested in Petri dishes
containing the relevant tester strain plus the S9 mix in soft agar overlay.
The plates are then exposed to known concentrations (v/v) of the compound
in air. Substances with low solubilities or diffusion rates can be tested
but produce results which are qualitative rather than quantitative in nature.
Bactericidal substances may be studied using the liquid suspension assay
method. For most mutagens there is a linear dose-response over a certain
concentration range. The number of revertants per plate reported for any
mutagen should be taken from this region of the curve. Excessive bacterial
killing by a mutagen causes a decrease in the number of revertants on the
plate. If only one concentration was tested on the downhill part of the
curve it would be easy to obtain a misleading result on the quantitative
activity of a particular mutagen. Over several years, a large data base
of results has been accumulated which has confirmed its ability to detect
genetically active compounds of most chemical classes with an efficiency
of 80%. Background lawn The agar contains a trace of histidine which allows
all the bacteria to undergo several divisions thus producing a faint background
lawn of bacteria. DNA replication is necessary in many cases for mutagenesis
to occur and therefore the background lawn provides a good indicator of
the inhibition of growth caused by the test chemical. Colonies appearing
on a plate that has no background lawn may not be revertants but colonies
that arise due to the surviving bacteria that live off the histidine present
in the agar and therefore should not be scored. To check whether colonies
are true revertants they should be streaked out onto minimal plates containing
biotin but no histidine. True revertants will be able to grow, whereas
microcolonies arising due to toxicity will not. Furthermore, if the background
lawn is thin compared to the control, it indicates toxicity of the chemical
to the bacteria. If the amount of histidine in the agar is increased, mutagenesis
may be enhanced but will also cause a heavy growth of the background lawn
obscuring the revertants. Spontaneous reversion Spontaneous reversion (to
histidine independence) should be measured routinely and can be expressed
as the number of spontaneous revertants per plate. The number of spontaneous
revertants is dependent on two populations: i) pre-existing mutants in
the inoculum; and ii) mutants arising following divisions on the plate.
The amount of histidine present is the limiting factor because the more
bacteria plated the fewer divisions are possible, and thus fewer "plate"
mutants will arise. Each tester strain should revert spontaneously at a
frequency that is characteristic of the strain. The number of spontaneous
revertants per plate should be completely independent of the initial number
of bacterial cells plated within the limits of 105-108 cells (i.e. the
histidine concentration within the agar is constant and, therefore, the
background level of bacteria should also be constant). The number of spontaneous
revertants varies between experiments, therefore it is recommended that
at least three control plates are included for each strain in a mutagenicity
assay. This is especially important when the mutagen is weak. Any deviation
from a laboratories historical range (as a guide, without S9:- TA98, 10-50;
TA100, 60-220; TA1535, 5-50; TA1537, 1-25; TA102, 250-500; but these may
differ slightly in the presence of S9) is an indication that the genetic
characteristics of the strain may have altered and this should be tested.
Each laboratory should establish its own historical range as these ranges
are likely to vary from laboratory to laboratory. Abnormally high spontaneous
reversion may indicate contamination or the accumulation of back mutations
by repeated sub-culturing. If this occurs the strain should be recovered
by re-isolation from the frozen master copy. Spontaneous reversions are
influenced by histidine concentration and, consequently, fluctuations in
the histidine content of the agar will be reflected in fluctuations of
the number of spontaneous revertants on the plate. Consistently high spontaneous
values not attributable to the histidine concentration can sometimes be
traced to mutagens in the environment of the bacteria. Ethylene oxide (used
to sterilise plastic ware) is a potent mutagen for TA1535 and TA100. Residues
of ethylene oxide in the plates can greatly increase the number of revertant
colonies of these strains. It is essential that any plastic ware used is
not sterilised using this reagent. Checking for strain characteristics
Histidine requirement The His- character of the tester strains should be
confirmed by demonstrating the histidine requirement for growth on selective
agar plates. The uvrB deletion extends through the biotin gene, hence the
need for biotin in those strains possessing the uvrB mutation. These essential
components can be added to minimal glucose agar before the test plates
are poured or they can be applied to the surface of minimal glucose agar
plates and incorporated into the plates using a glass spreader. A sample
of the cultures is streaked across a biotin control plate and then a histidine-biotin
plate. The plates are incubated overnight at 37°C and examined for growth
on the histidine-biotin plates. There should be no growth on the control
plates. The bio gene cannot be reverted, therefore it is not necessary
to test for biotin requirement. Ampicillin resistance This factor should
be checked routinely because the ampicillin resistant R factor is somewhat
unstable and can be lost from the bacteria. A small amount of ampicillin
solution should be streaked out on the surface of a nutrient agar plate
and allowed to dry. Cross-streak the culture to be tested against the ampicillin.
Cultures should be incubated for 12-24 hours at 37°C. Strains which do
not contain the R factor (TA1535 and TA1537) will show a zone of growth
inhibition around the ampicillin streak, whereas R factor-containing strains
will not. N.B. Ampicillin resistance is a convenient marker enabling testing
for the presence of the plasmid but it has nothing to do with the increased
sensitivity of the strains to reversion by mutagens. rfa character The
rfa character of the tester strains is confirmed by determining crystal
violet sensitivity. The rfa mutation permits large molecules, such as crystal
violet, to enter and kill the bacteria. Fresh culture is added to top agar,
vortexed, and poured onto a nutrient agar plate. Crystal violet is added
to the centre of sterile filter paper and transferred to the seeded plates
and the disc is pressed lightly to embed it slightly. The plate is incubated
inverted. A clear zone of inhibition appears around the disc, indicating
the presence of the rfa mutation. uvrB mutation The uvrB deletion is quite
stable and is not easily lost from bacteria but its presence can be easily
confirmed by demonstrating UV sensitivity. Streak the strain across a nutrient
agar plate and cover half the plate with cardboard (half of each bacterial
streak should be covered). The plate is irradiated for 6 seconds in the
case of Non R factor strains (TA1535 and TA1537) and for 8 seconds in the
case of R- factor strains (TA98 and TA100). Strains with the uvrB deletion
will only grow on the un-irradiated side of the plate. Strain TA102 does
not possess the uvrB deletion and is a useful control culture for these
tests. S9 fraction For optimal mutagenesis with a particular compound,
the amount of S9 per plate is critical. Too much as well as too little
S9 can drastically lower the sensitivity. Each new batch of S9 prepared
should be routinely checked with several compounds (e.g. benzo[a]pyrene,
2-acetylaminofluorene, 2-aminofluorene, aflatoxin B1).
TEST STATUS
Validated
REFERENCES
- Ames, B.N., McCann, J. & Yamasaki, E. (1975)
Methods for detecting carcinogens and mutagens with Salmonella/mammalian-microsome
mutagenicity test. Mutation Res. 31, 347-364.
- Booth, S.C., Welch, A.M. & Garner, R.C. (1980)
Some factors affecting mutant numbers in the Salmonella/microsome assay.
Carcinogenesis, 1, 911-923.
- Garner, R.C. (1979) Carcinogen prediction in
the laboratory: a personal view. Proc. Roy. Soc. Ser. B, 205, 121-134.
- Gatehouse, D.G., Wilcox, P., Forster, R., Rowland,
I. & Callender, R.D. (1990) Bacterial mutation assays. In "Basic
Mutagenicity Tests. UKEMS Recommended Procedures." Report of the UKEMS
Sub-committee on Guidelines for Mutagenicity Testing. Ed D.J. Kirkland.
Cambridge University Press.
- Jagannath, D.R. & Brusick, D.J. (1983) Mutagens
and Carcinogens in Bacteria. In A Guide to General Toxicology (eds. Homburger,
F., Hayes, J.A. & Pelikan, E.W.), S. Karger AG, P.O. Box, CH-4009 Basel,
Switzerland, pp 344-364.
- Mahon, G.A.T., Green, M.H.L., Middleton, B.,
Mitchell, I. de G., Robinson, W.D. & Tweats, D.J. (1989) Analysis of
data from microbial colony assays. In: Statistical evaluation of mutagenicity
test data. Report of the UKEMS Sub-committee on Guidelines for Mutagenicity
Testing, Part III. (ed. D.J. Kirkland). Cambridge University Press.
- Maron, D.M. & Ames, B.N. (1983) Revised methods
for the Salmonella mutagenicity test. Mutation Res., 113, 173-215.
- De Serres, F.J. & Shelby, M.D. (1979) Recommendations
on data production and analysis using the Salmonella/microsome mutagenicity
assay. Mutation Res., 64, 159-165.
- Sugimura, T., Sato, S., Nagoa, M., Yahagi, T.,
Matsushima, T., Seino, Y., Takeuchi, M. & Kawachi, T. (1976) Overlapping
of carcinogens and mutagens. In: Fundamentals of Cancer Prevention (eds.
Magee, P.N., Takayama, S., Sugimura, T. & Matsushima, T.) University
of Tokyo Press/Baltimore University Park Press, pp 191-215.
- Venitt, S., Forester, R. & Longstaff, E.
(1983) Bacterial Mutation Assays. In Report of the UKEMS Sub-Committee
on Guidelines for Mutagenicity Testing. Part 1. Basic Test Battery. Ed.
B.J. Dean. United Kingdom Environmental Mutagen Society, Swansea, pp5-40.
IP-30 © January 1992
|
PLEASE NOTE THAT TO OBTAIN DETAILS OF THE PROTOCOL YOU SHOULD REGISTER 
