Mutation Research, 120 (1983) 201-205 201

Elsevier

A m e s test: Reliable results within 24 h

B. K h o u d o k o r m o f f

Research and Development, Gist-Brocades N. V., P.O. Box 1, Delft (The Netherlands)
(Accepted 31 January 1983)

The principle of the Ames test relies on the scoring of histidine-prototrophic

revertants (his ÷) induced by (back)mutation in histidine-deficient mutant strains of
S a l m o n e l l a t y p h i m u r i u m , TA1535, TA1537, TA1538, TA98 and TA100 (Ames et
al., 1975) after exposure to the chemical compound under investigation. The actual
test may roughly be subdivided into 3 phases. Under appropriate conditions of pH,
temperature, concentration of dissolved compound, etc. some chemicals lead to the
formation of unstable electrophilic radicals (phase I). These may originate from
directly reacting mutagens or, more commonly, from (pro)mutagens, which require
metabolic activation for the generation of such radicals. The Ames test incorporates
the so-called $9 mix (microsomal fraction of rat-liver homogenate together with the
essential co-factors) as a source of mammalian metabolic activation. It is essential
that most of the activity of $9 mix be expressed before the results of the test are
read. In the liquid pre-incubation test, this is reached within from 30 min at 37°C
(Frantz and Mailing, 1975) to 50-60 min at 25°C (Yahagi et al., 1977), whereas in
soft-agar overlay $9 mix activity is virtually exhausted within about 6 h (Nohmi et
al., 1981).
The electrophilic radicals penetrate the cell of the test organism and interact with
the DNA (phase II); any mutated state is expressed during the subsequent nuclear
replication and cellular division(s), which is rendered possible in the Ames test by
the addition of traces of histidine to the top agar layer. Phases I and II are com-
pleted within hours. The third phase, incubation for 48-72 h is required simply to
allow the mutated cell to form a colony of visible size. By optimizing the conditions
of growth in phase III - without affecting the previous phases - the duration of
testing may, conceivably, be substantially shortened. Recently, indeed, Arimoto et
al. (1981) reported that the addition, at 1 mg/1, of a mixture of 16 amino acids to
the Ames basic medium (Vogel and Bonner, 1956) permitted them to shorten the
incubation time from the usual 48-72 h to 24 h. Histidine was left out owing to the
criteria of selection, and cysteine because it had been reported to influence the fre-
quency of mutagenesis (Rosin and Stich, 1978; Negishi and Hayatsu, 1979).

0165-7992/83/$ 03.00 © 1983 Elsevier Science Publishers B.V.

202

In the system devised by Arimoto et al. (1981), the test culture, after exposure,
was grown in the presence, at 1 mg/l, of a mixture of 16 amino acids, i.e. 1/16 of
a mg, or 0.0625 mg/l, of each amino acid. On a molar basis, these authors have
thus tested the addition of a pool of on average about 0.5 mM of each amino acid
(ranging from 0.833 mM glycine to 0.306 mM tryptophan).
We have recently confirmed these results, using a mixture of 0.5 mM of each of
the 16 amino acids (glycine, alanine, serine, threonine, proline, valine, leucine,
isoleucine, aspartic acid, lysine, glutamic acid, methionine, phenylalanine, arginine,
tyrosine and tryptophan), and observed visible colonies after only 22-24 h of in-
cubation at 37°C (Khoudokormoff, 1982). Preliminary experiments also revealed
that most of the amino acids do not affect the growth rate specifically. More exten-
sive studies showed that: (a) the addition of threonine, methionine and glutamine
and/or asparagine (or their acids) is a prerequisite for growth of visible colonies
within 24 h of incubation; (b) methionine can be only partially replaced by cysteine;
(c) arginine stimulates the formation of larger colonies, but is not essential; (d) the
addition of phenylalanine to the 3 or 4 'essential' amino acids delays accelerated
growth, the phenomenon being reversed, on an equimolar basis, by tryptophan but
not by tyrosine; (e) the results were not substantially affected when 4 mM i.o. 8 mM
of the pooled 16 amino acids, or 1 mM i.o. 0.5 mM of each of the essential amino
acids were added. Finally, in the presence of 8 mM of a pool of all amino acids ex-
cept threonine and methionine, no early growth could be observed and incubation
had to proceed for 48 h.
Routine short-term testing of known and potentially mutagenic chemical com-
pounds could successfully be carried out by plating on the basic-medium agar
enriched with 0.5 mM of each of threonine, methionine, glutamic and aspartic
acids, and reading the plates after 24 h of incubation at 37°C; the results did not
differ from those obtained under standard conditions of the Ames test after 48 h.
Thus, e.g., the compounds given in Table 1 were scored as mutagens within 24 h.

TABLE I
Compound Solvent Concentration $9 mix a Test strain(s)
0zg) in 50-#l/dish
Sodium azide water 1 - TA100, TA1535
2-Aminoacridine methanol 25 - TA1537
2-Nitrofluorene methanol 5 - TA98, TAt538
2-Aminofluorene methanol 5 + TA98, TA1538
Ethidium bromide methanol 50 + TA98, TA1538
B-Naphthoflavone methanol 65 + TA1535
2-Aminoanthracene methanol 0.125 + |
Benzo[a]pyrene methanol 50 + / all 5 strains
20-Methylcholanthrene methanol 10 +
aS9 mix was prepared according to Ames et al. (1975), by using rat-liver homogenate from /3-
naphthoflavone and phenobarbital i.p. pretreated animals, as described by Matsushima et al. (1976).
 203

24 h
48h
2oo-
o

o~ 150-
o
c~
cn 100
>,
(J
g 50
~r
u_
106 1107 1C)8 1'09
t ~Cell population per plate
® 60
_~
o. ~ _ _ _ _ ~ h I 2.2 x 10e cells I/~
=,.~ so ~ . . . . .~4
448 hhl 1.0 x 109 cells 11~
~ 40

> 30
0=
~ • ~
20 ...... "~ J "J

_c 10
t
(~ 110 2; 3'0
410 510 6'0 7'0 8'0 9101(~0
~Conc. 9 AAC#g/plate
•o . . '-. . ..24
o48 hh t 4.2 x 10 e cells
1300 -4 ~48 hh I 4.7 x 107 cells
=24
® •
- o. . . . . , ,He
24 h
hi 1.9 x 10a cells // •
.-/"//'

.~~>~~°1"500900700100- =~=
. . . . " ~. . ~'S
. . ~. .' ' '.' " . f ."/'"/
. ~ JJ

o= 300
'10
t 100 -'A- ____e_-.e-

6 o11 o12 0'3 o'4 o'5

Conc. 2 AAN#g/ptate
Figs. 1-3. Influence of the cell population (per plate) of the test organism. Fig. 1. Frequency of spon-
taneously reverting mutants, strain TAI00. Fig. 2. Frequency of revertants induced by 9-aminoacridine
(9 AAC), strain TA1537. Fig. 3. Frequency of reversion induced by 2-aminoanthracene (2 AAN) in the
presence of $9 mix, strain TA100.
204

The frequency of spontaneous revertants was usually slightly higher after 48 h

than after 24 h; the frequency of induced revertants of all 5 strains visible after 24
h hardly rose on further standing (less than 10°70), and was in good agreement with
the number of colonies observed in similar experiments under standard conditions
of the Ames test. However, whenever a tested chemical is suspected of toxicity to
the test organism, more prolonged incubation may be advisable - as has also been
suggested in the 'classical' Ames test (de Serres and Shelby, 1979).
Several authors have reported a non-linear correlation between the number of col-
onies appearing on the agar and the number of cells incubated per dish, in the Ames
test (e.g. Ellenberger and Mohn, 1975; Bartsch et al., 1976; Fumero et al., 1981).
More recently, Salmeen and Durisin (1981) showed, both on theoretical and ex-
perimental grounds, that the slope of dose-response curves depends on the initial
inoculum per dish: the higher the inoculum size, the steeper the slope, whereas the
number of spontaneous mutants depends only weakly on the population of in-
oculated cells.
We have investigated the influence of the population of cells of the test organism
on the number of spontaneous and induced revertant colonies observed after 24 h
of incubation of the amino-acid-enriched medium. Figs. 1-3 illustrate the pattern
of response of the spontaneous mutation rate (TA100) and of the induced rate of
reversion under the influence of a direct mutagen (9-aminoacridine on TA1537), and
of a mutagen requiring metabolic activation (2-aminoanthracene on TA100), respec-
tively. From these results it appears that, whether the frequency is scored after 24
or 48 h, a 200-fold increase of the inoculated population per plate hardly leads to
a 2-4-fold higher number of spontaneously appearing revertant colonies, which ful-
ly agrees with previous observations after 48 h, on unsupplemented basic medium
agar (e.g. Salmeen and Durisin, 1981). The frequency of induced revertants in-
creases linearly with the concentration of the mutagen, the slope of the curve being
less steep with a lower inoculum size. The fair similarity of the results obtained after
24 and 48 h of incubation is particularly striking when the test on amino-acid-
enriched medium is carried out resorting to an inoculum population of 1-4 x 108
cells per dish, which corresponds to the generally recommended conditions of
testing in the Ames test (de Serres and Shelby, 1979).

References

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Arimoto, S., K. Negishi and H. Hayatsu (1981) A modification of the Ames test procedures accelerated
growth of the his + revertants, Mutation Res., 91, 407-411.
Bartsch, H., A. Camus and C. Malaveille(1976) Comparative mutagenicity of N-nitrosamines in a semi-
solid and in a liquid incubation systemin the presenceof rat or human tissue fractions, Mutation Res.,
37, 149-162.
 205

de Serres, F.J., and M.D. Shelby (1979) Recommendations on data production and analysis using the
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