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March 1983
Notice
This report has been reviewed in accordance with the U.S. Environmental
Protection Agency's peer and administrative review policies and approved for
presentation and publication. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
-------
March 1983
Foreword
The Ames Sa/mone//a/m\crosoma\ mutagemcity assay has been developed and
used successfully with supporting chemical data by the EPA's National Enforce-
ment Investigations Center in Denver, the Health Effects Research Laboratory, Re-
search Triangle Park, and several EPA Regional Laboratories for air and wastewater
characterization and health effects research However, the method has not been
employed in a uniform manner by all Agency Laboratories, and there is some
question of comparability of data among these laboratories.
Other EPA Regional and research laboratories and Program Offices with
responsibility for toxic and hazardous substances have expressed an immediate
need to apply the Ames test in their activities. The test protocols and guidance
provided here were prepared — in a joint effort between the Environmental
Monitoring Systems Laboratory-Las Vegas, Nevada and the National Enforcement
Investigations Center, Denver, Colorado — to meet that need. The standardization
of Ames testing methods for EPA use is intended to assure, to the extent possible,
that mutagenicity assay data produced by the Agency is valid, defensible, and
comparable with assay data produced by other laboratories.
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March 1983
Table of Contents
Page
Foreword jjj
Figures vi
Ta bles vi i
Acknowledg ments viii
Section
1. Introduction 1-1
2 Summary of the Method 2-1
Plate Assay Method 2-1
Selection of Specific Tests, Test Modifications 2-1
3. Mutagenesis Assay Procedures 3-1
Preliminary Test — Plate-Incorporation Procedure 3-1
Confirmatory Test — Plate-Incorporation
Procedure 3-1
Examining Background Lawn 3-1
Premcubation Assay 3-2
General Recommendations 3-3
4. Salmonella Tester Strains 4-1
Processing, Regeneration and Storage 4-1
Checking Characteristics of Tester Strains 4-1
Standard Mutagens 4-2
S-9 Activity 4-3
5. Quality Control/Quality Assurance 5-1
General Requirements 5-1
Quality Control Testing 5-1
6 Data Analysis, Interpretation and Reports 6-1
Data Analysis 6-1
Interpretation 6-2
Reporting Test Results 6-4
References R. 1
Appendix I — Preparation of Media and Reagents AI-1
Appendix II — Facilities, Equipment, and Supplies /\ll-1
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March 1983
Figures
Number Page
1 Flow diagram — Recommended mutagemcity testing sequence
for complex mixtures or environmental samples 2-2
2 Steps in the Ames plate-incorporation assay procedure 3-2
3 Quality Control Data Form — Cell titer and viability
measurements 5-4
4 Quality Control Data Form — Strain function tests 5-5
5 Quality Control Data Form — Culture media record 5-6
6 Quality Control Data Form — Reagents, standards, and test
materials -. 5-7
7 Quality Control Data Form — Equipment record 5-8
8 Example of graphical presentation of Salmonella mutagenicity
test data (simulated) 6-2
9 HERL IN VITRO System — Results Form 6-4
10 HERL IN VITRO System — Results Continuation Form 6-5
11 HERL IN VITRO System — Sample Identification Form (Interim) . 6-6
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March 1983 vii
Tables
Number Page
1 Recommended Experimental Conditions for Conducting Salmonella
Mutagenicity Assays 3-3
2 Genotype of Five Salmonella Tester Strains Used for General
Screening in Mutagen Testing 4-1
3 Standard Mutagens for Confirming Tester Strain Function 4-3
4 Quality Control Tests for Ames Plate Assay 5-2
5 Statistical Techniques Useful for Evaluating Mutagenicity
Testing Data 6-2
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March 1983
Acknowledgments
The cooperation of the Review Panel in providing input to and comments on the
draft manuscript is gratefully acknowledged. Participants on the Review Panel
included'
Dr. A. Auletta, EPA - Office of Toxic Substances
Dr. W L Belser, University of California, Riverside
Dr. K.W. Brown, Texas A&M University
Dr. D.J. Brusick, Litton Bionetics
Dr. R J. Bull, EPA-HERL/Cm
Dr. L D. Claxton, EPA-HERL/RTP
Dr. V C. Dunkel, FDA/Washington, D C.
Dr. C P Flessel, California Dept of Health Services
Ms. J Gridley, Monsanto Research
Dr D.M. Hanson, Bioassay Systems Corporation
Dr S. R Haworth, EG G Mason Research Institute
Dr M C Henry, Dept of the Army, Fort Detrick, Md.
Dr. T.J. Hughes, Research Triangle Institute
Dr. F.W. Larimer, Oak Ridge National Laboratory
Ms. D Maron, University of California, Berkeley
Ms. N.E McCarroll, Hazleton Laboratories America, Inc
Mr J.R. Meier, EPA-HERL/Cm
Dr. K Mortelmans, SRI-lnternational
Dr. Wm Pelon, Louisiana State University Medical Center
Dr. C.W. Piper, Searle Research and Development
Dr. T. Sugimura, National Cancer Center Research Institute, Tokyo
Ms. S. Toney, Northrup Services, Inc
A very special thanks to Dr Mortelmans, Dr Flessel, and Ms Maron for the many
hours of unselfish effort they contributed to reviewing and resolving the many and
varied comments of the Review Panel Finally, a debt of gratitude is owed to Mr.
William Stang of EPA's National Enforcement Investigations Center, who, prior to
his untimely death in 1980, was a driving force for standardization of mutagenicity
testing procedures within the Agency
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March 1983
1-1
Section 1
The Salmonella /mammalian micro-
some mutagenicity assay commonly
known as the Ames test; Ames et al.
(1975) has proven to be reliable for,
identification of a large number of
mutagenic and potentially carcinogenic
substances This test offers a means of
obtaining dose-responsive data with a
wide variety of environmental samples
The general applicability of the Ames
test has already been demonstrated as a
prescreen for potential genetic hazards
of complex environmental effluents or
products, eg , tobacco smoke conden-
sates, natural products, hair dyes, soot
from city air, fly ash, synthetic fuel oils,
aqueous wastes and diesel particulates
(Epler et al 1978, Claxton et al 1981)
Recent applications (e g , Donnelly and
Brown, 1 981) of the procedures to solid
wastes and waste leachates are further
evidence of the broad-spectrum utility
of the Ames test for screening and
environmental monitoring
The purpose of this document is to
provide an interim standardized Ames
test procedure to be used by Agency,
State, and contract laboratories con-
ducting mutagenicity testing under
EPA's hazardous waste monitoring
program A supplement to this docu-
ment — that will address sample
preparation or chemical fractionation
procedures for use with the mutagenicity
assay — is planned A program is
currently underway to provide collabo-
rative testing and evaluation of the test
protocol contained in this document
Section 1
Introduction
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March 1983
2-1
Section 2
Section 2
Summary of the Method
Plate Assay Method
The test system developed by Ames
and his coworkers has been widely used
as a rapid-screening procedure for the
determination of mutagenic and poten-
tial carcinogenic hazards of pure
chemicals, complex environmental
mixtures, and commercial products.
Detailed experimental procedures have
been provided in the "methods paper"
by Ames etal. (1975)' and supplements.
An excellent review of the requirements
for applying the Ames test is presented
by de Serres and Shelby (1979).
These papers are "must reading" as
the minimum introduction necessary to
carry out the procedures described in
this document.
The Ames test involves the use of five
standard tester strains of Salmonella
typhimurium containing a specific
mutation in the histidme operon. These
genetically altered strains cannot grow
in the absence of histidine; when they
are placed in a histidme-free medium,
only those cells that revert spontaneously
to histidme-independence are able to
form colonies. The range of spontaneous
reverse mutation values for each strain
is relatively constant However, if a
chemical mutagen is added to the
medium the mutation value is increased
significantly.
The sensitivity of the Salmonella
tester strains has been enhanced by the
introduction of two additional mutations,
namely uvrB and rfa. The deletion
mutation covering the uwflgene results
in elimination of the accurate DNA
repair system. Because this deletion
also includes the biotin gene, the cells
require the addition of biotin to grow.
The rfa (deep rough) character allows
increased cell permeability and greater
penetration of chemical mutagens or
large chemical molecules such as crystal
violet into the bacterial cell, due to
partial loss of the lipopolysacchande
(LPS) barrier of the cell surface.
Certain mutagens are directly active
in the system while others require
activation by mammalian microsomes
(e.g., rat-liver enzymes) added to the test
system Generally, these are obtained
from Aroclor 1254-induced rats, using
the 9000-g supernatant (S-9) of the
homogenized rat liver. These micro-
somes contain enzymes which perform
'and as revised (Marson and Ames, Mutation
Research, in press)
metabolic conversions mimicking those
of mammalian organs in vivo. Metabolic
activation of test materials with these
enzymes improves the correlation
between mutagenesis in this in vitro
bacterial test system and carcinogenesis
in mammals.
In conducting the test, a tester strain
is added to soft agar containing a low
level of histidine and an excess of biotin
along with varying amounts of the test
substance. This mixture is overlaid on
mimimal agar plates, and the plates are
incubated for at least 48 hours at 37°C
The bacteria undergo several divisions
before the trace amounts of histidine
are used up and thus form a light film of
background growth (lawn) on the plate.
In many cases, this growth is necessary
for mutagensis to occur. Revertants to
histidine independence (his*), induced
by the mutagenic chemical(s), continue
to grow in the absence of histidine to
form visible colonies on the plate. These
colonies are examined after 48 and 72
hours of incubation. Although visible
colonies can usually be obtained with
48-hour incubation at 37°C, the toxicity
of some chemicals may delay the
appearance of revertants When this is
suspected, plates should be incubated
for 72 hours (de Serres and Shelby,
1979). Counts of revertant colonies on
these test plates are compared to
counts (spontaneous revertants) of
the same strain on control plates
containing all components but the test
substance (solvent controls). The assay
is quantitated with respect to doses
(amount of chemical added per plate) of
mutagen.
The plate assay method described
here is basically as used by Ames, and
incorporates recommendations of de
Serres and Shelby (1979) and Belser et
al., (1981), designed to make the test
more quantitative.
The desired result of a mutagenicity
test is a definitive "positive" or a
"negative" with respect to the test
system employed. Since a positive dose-
response is a primary criterion for our
identification of a chemical or mixture
as mutagenic, the terminal tests selected
are all dose-response assays and the
screening tests have dose-response
potential under the proper conditions.
As a result, it is possible to demonstrate
and confirm mutagenicity with only two
consecutive tests whenever a dose-
response can be demonstrated in the
preliminary test (see Figure 1). Were we
to adopt a rangefmding preliminary test
solely for toxicity, a minimum of three
consecutive tests would be required to
obtain confirmation of any test result
(positive or negative) noted.
Selection of Specific Tests,
Test Modifications
The plate-incorporation procedure
described by Ames et al. (1975) is well
tested and widely used for testing pure
compounds and complex mixtures.
Perhaps the most widely used and
successful modification of the plate-
incorporation assay is the preincubation
method described by Yahagi et al.
(1977). Preincubation techniques have
been shown to enhance the mutagenic
potentials of many chemicals (Sugimura
and Nagao, 1980). These techniques
may be the method of choice for groups
of compounds such as the nitrosamines
(Yahagi et al 1977), pyrrolizidine
alkaloids (Yamanaka et al. 1979), and
quinoline derivatives (Nagao et al.
1977). Indeed, the preincubation method
is now used routinely in some labora-
tories and is recommended for use in
cases where results from the standard
plate assay are inconclusive (de Serres
and Shelby, 1979).
Modifications of the liquid suspension
assay described by Mailing (1971) are
receiving some attention as possible
alternatives for testing samples which
have proven too cytotoxic to successfully
test with standard plate-incorporation
procedures
Among qualitative screening proce-
dures suggested for rapidly determining
the most appropriate tester strain(s),
cytotoxicity of the test material, and/or
effects of metabolic activation are the
spot test (Ames etal 1975), the well test
(Pellizan 1978), and micro-well tech-
nique (Loveday, unpublished protocol)
and the toxicity screening procedure
reported by Waleh et al. All represent
efforts to simplify and reduce the testing
requirements for obtaining basic
screening information.
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Section 2
22
March 1983
Test Material
Screening
Quantitation
Confirmation'
Known to Contain
Specific Chemical
Classes (see text),
Non-Diffusible or
Labile Components
Preliminary
Test-
Premcubation
Screen
4
Dose-Response Test-
Premcubation Assay
Inconclusive2
Extract or
Fraction of
Sample in
Appropriate
Solvent
Preliminary Screen-
Plate Incorporation
Assay Procedure
//
Dose-Response Test-
Assay Procedure
Repeat
Moderate to High Cytotoxic/ty3
Further Fraction -
ation or Cleanup
of Sample
Extract
i
Consider Optional
Tests
Spot Test,
Well Tests
Suspension
Assay4
Qualitative
Information
Only
Dose-Response Test
Suspension Assay
Repeat
'Optional, if positive preliminary test result was confirmed (repeated) by the first "dose-response test "
2Results of preliminary test suggest mutagenic activity but do not meet all criteria for a positive test
3Mutagenic activity cannot be resolved from cytotox/c/ty by dilution alone
"Not as well defined as plate incorporation and preincubatlon assays, often difficult to perform and interpret satisfactorily
Figure 1. Flow diagram - recommended mutagenicity testing sequence for complex mixtures or environmental samples
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March 1983
3-1
Section 3
Section 3
Mutagenesis Assay Procedures
Preliminary Test — Plate-
Incorporation Procedure
a Prepare Master Plates as described
in Section 4 On the day prior to
performance of the assay, select
single isolated well-grown colonies
from the Master Plate for each strain
and inoculate into nutrient broth.
Use tubes or erlenmeyer flasks about
five times the volume of the nutrient
broth.
b. Incubate cultures with continuous
gentle (eg., 120 rpm) agitation
overnight for 14-18 hours at 37°C.
c. Prepare top agar as outlined in
Appendix I. Melt prepared top agar in
an autoclave for 3 minutes at 121 °C,
or melt the agar in a microwave
oven. Mamtainthe molten top agar in
a 45°C waterbath or warming oven
d. Prepare a histidme/biotin solution
as outlined in Appendix I. Place the
histidme/biotin solution in a water-
bath and warm to 45°C. Add 20 ml of
this solution to 200 ml of top agar.
e Prewarm minimal agar plates at
37°C for 24 hours before inoculation.
This tests for sterility of the medium
The warm plates also aid in uniformly
distributing the top-agar overlay
f Insert the required number of sterile
13 x 100 mm culture tubes into
heating block or waterbath preheated
to 45°C Pipet 2 ml molten top agar
supplemented with histidine/biotm
solution into each tube
The next steps in the plate incorpora-
tion assay procedure are depicted in
Figure 2.
Perform the following steps under an
appropriate biological cabinet or a
laminar-flow hood; wear fully fastened
laboratory coat with solid front and
surgical gloves.
g. Add 0.1 ml of fresh nutrient broth
culture (from Step 'b') of the desired
test strain (approximately 10s cells)
to each tube (positive and negative/
solvent controls and each test dose
level in appropriate replication) to be
tested. Fresh broth cultures should
be kept in an ice bath while preparing
the plates. Remember that upon
inoculation of the top agar the
organisms must not remain at 45°C
for more than about 10 minutes.
h. Add 50 /j\ of a solution of the test
material in DMSO1 to each tube of
top agar inoculated with culture. A
general guide is to span up to a 3-log
dose range, with 1/3 to 1/2-log
intervals between doses, for screen-
ing. Suggested concentrations of test
material include 100, 30, 10, 3, 1
and 0 3 mg/ml for each test strain to
yield effective doses of approximately
5, 1 5, 0.5, 0 15, 0.05 and 0.015 mg
test material per plate, respectively.
Prepare a set of six tubes for each
combination of strain and dose.
i. To two of the tubes from each set
prepared in 'h', add 0.5 ml of "LowS-
9 Mix" (see Appendix I for prepara-
tion of S-9 mixes). To the next two
tubes from each set add 0.5 ml of
"High S-9 Mix" in the same manner.
Remove tubes in each case prior to
adding S-9 mix and do not return to
heating block or 45°C waterbath. The
S-9 mix should not be exposed to the
45°C temperature for longer than a
few seconds. To the final pair of
tubes, substitute 0.5 ml of 0.2 M
sodium phosphate buffer solution
(see Appendix I) so that volume
conditions for the "activated" and
non-activated tests will be compa-
rable.
j. Use a mechanical Vortex mixer to
thoroughly mix the materials; mix
gently for not more than three
seconds, taking care to prevent
bubble formation in the top agar
k. Pour the contents of each tube onto
the center of a prewarmed minimal
agar plate. Gently tilt and rotate each
plate to spread the top agar uniformly
over the surface of the minimal agar
Uniform distribution can be facilitated
by placing the plate on a level table
and gently vibrating the table surface,
e.g., with a mechanical vibrating
device (Belser et al. 1981), but this is
not mandatory.
I. Cover each plate and place on a flat
surface until the agar sets (several
minutes). Then incubate the plates in
the dark at 37°C for 48 to 72 hours in
an inverted position.2 Observe the
10r other appropriate solvent, if 50 fj\ of solvent will
not solubilize the test material, increase the
solvent level uniformly for all dose levels Do not
exceed 100 fj\ of DMSO (Belser et al, 1981) See
Maron, et al (1981) for information on compatibility
of solvents with the Ames test
2lf sample is known or suspected to contain
significant quantities of volatile chemicals, seal
plates in individual plastic bags or place into a
dessicator prior to incubating
number of revertant colonies shortly
after removal from the incubator at
48 and 72 hours.3 If unable to
perform plate counts immediately,
storage for up to 2 days at 4°C is
acceptable. If satisfactory colony
development has occurred by 48
hours, further incubation (i.e , to 72
hours) may be unnecessary. How-
ever, until the investigator is confi-
dent that he/she can consistently
distinguish those tests requiring
extended incubation, it is recom-
mended that counts be made at both
48 and 72 hours.
Confirmatory Test — Plate-
Incorporation Procedure
If the preliminary screen shows
increased numbers of revertants over
solvent controls or a positive dose-
response relationship, repeat all steps
of the initial test at least in duplicate
using additional doses of test material,
with the most active strains and
conditions. For example, if the 1 mg
dose shows mutagenic activity, doses of
choice might include 0 25, 0.5, 0.75, 1,
1.5, 2.5, and 4.0 mg to establish a dose-
response relationship Proper test doses
must be selected independently for
each sample tested.
Metabolic activation with S-9 mix, at
the appropriate level, should be used if
those were the conditions under which
the initial "positives" were detected.
If the preliminary screen does not
suggest any mutagenic activity, repeat
the test conditions of the preliminary
screen but increase the maximum dose
level, if necessary, to 5 to 10 mg per
plate or that which is clearly cytotoxic.
With mixed test materials, one or more
components may precipitate on the
plate at relatively low concentrations. If
the precipitate does not interfere with
the scoring of plates, this kind of
precipitate should not restrict the upper
limit of dose concentration.
Examining Background Lawn
Use a dissecting microscope to
examine the background lawn.
The background growth or lawn is a
result of the trace amount of histidine
present in the top agar. If the test
3Toxicity of some chemicals may delay the
appearance of revertant colonies beyond 48
hours, in which case incubation should be
extended to 72 hours (de Serres and Shelby,
1979) For the same reason, Belser (1981)
recommends a 63-hour incubation period
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Section 3
3-2
March 1983
Test Substance Solvent Salmonella H'gh or 0.2 M Sodium Phos-
in Appropriate Solvent Alone Test Strain phate Buffer (pH 7 4)
S-9 Mix
Combine
Mix
0
f
Pour
Level
(fa
2 ml Molten (45°C) top agar fwith
Trace of Histidine and Biotinj
"I Mix Gently to A void
Entrapping Bubbles
Vortex-Type Mixer
Pour Top Agar Mixture into
Center of Plate, Tilt and Swirl to
Distribute
Minimal Agar Plate
fPrewarmed to 37°C)
Top Agar Mixture
Z~J^ Minimal Agar .
Cover, Vibrate on a Flat, Level
Surface to Uniformly Level the
Top Agar, and Allow to Cool
Incubate
Incubator
Invert and Incubate at 48 to 72
Hours at 37 ± 0.5°C.
Count
Colony Counter
Count the Number of Histidme-
Independent {Revertant)
Colonies.
Figure 2. Steps in the Ames plate-incorporation assay procedure.
substance is toxic, massive cell death
will occur and the background lawn will
be sparse or absent compared tocontrol
plates Because more histidme is
available to the survivors, they will
undergo more cell divisions and may
give rise to visible colonies. These
colonies can be mistaken for revertants;
consequently, the presence or absence
of a normal lawn must be noted and
recorded.
To verify the histidme independence
of resulting colonies, selected control
and test plates should be replica plated
onto plates containing minimal agar
with biotm. Revertant colonies will grow
on the minimal agar; phenocopy colonies
will not. Replica plating satisfies the
question as to whether a statistically
adequate number of colonies (especially
where the plate count is high) has been
checked. Replica plating of the two
plates showing highest plate counts
(highest revertant levels) should be
adequate to evaluate the histidine
independence of the colonies.
AII positive mutagenie responses
should be verified by a repeatable assay.
Preincubation Assay
A most widely used modification to
the standard Ames test is the prein-
cubation assay initially described by
Yahagi et al (1977). This procedure is
useful for certain types of chemicals
(e.g , nitrosammes), or in cases where
results of the standard plate assay are
inconclusive. Its use as part of the
screening assay has been recommended
(de Serres and Shelby, 1979). Prein-
cubation tests are performed at least in
duplicate The following preincubation
conditions are those recommended by
Sugimura and Nagao (1 980)
a. Prepare Master Plates as described
in Appendix I. On the day prior to
performance of the assay, select
single isolated well-grown colonies
from the Master Plate for each strain
and inoculate into nutrient broth.
Use tubes or erlenmeyer flasks about
five times the volume of the nutrient
broth.
b. Incubate cultures with continuous
gentle (eg, 120 rpm) agitation
overnight for 14-18 hours at 37°C.
c. Prepare top agar as outlined in
Appendix I. Melt prepared top agar in
an autoclave for 3 minutes at 121 °C,
or melt the agar in a microwave oven.
Maintain the molten top agar in a
45°C waterbath or warming oven.
d. Prepare a histidine/biotin solution
as outlined in Appendix I. Place the
histidine/biotin solution in a water-
bath and warm to 45°C. Add 20 ml of
this solution to 200 ml of top agar.
Perform the following steps under a
biological cabinet or laminar-flow hood;
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March 1983
3-3
Section 3
wear fully fastened laboratory coat with
solid front and surgical gloves
e. Add appropriate concentrations of
test material to duplicate sterile 13 x
100 ml test tubes which have been
placed in an ice bath (seePre/immary
Test — Plate Incorporation Procedure,
Step 'h')
f Inoculate each tube with 0 1 ml
overnight broth culture of thedesired
test strain.
g. Add 0 5 ml of either High S-9 Mix,
Low S-9 Mix, or 0.2 M phosphate
buffer solution to each tube con-
taining sample extract and culture
h Quickly mix the contents of each tube
with a Vortex mixer and incubate
with continuous moderate agitation
at 37°Cfor 15 minutes.1
i. Add 2 ml of molten top agar with
trace amounts of histidme and
excess biotm (from Step 'd') to each
tube, and vortex each tube for not
more than 3 seconds to thoroughly
mix the materials, taking care to
avoid entramment of bubbles in the
agar
j Pour the contents of each tube onto
the center of a prewarmed Minimal
Agar plate Gently tilt and rotate
each plate to spread the top agar
uniformly over the surface of the
Minimal Agar. Uniform distribution
can be facilitated by placing the plate
on a level table and gently vibrating
'Although 1 5- or 20-mmute incubation periods are
widely used, optimal conditions for a given sample
may require up to 1 hour or more
the table surface, e g , with a
mechanical vibrating device (Belser
et al 1981), although this is not
mandatory.
k Cover each plate and place on a flat
surface until the agar sets (several
minutes). Then incubate the plates in
the dark at 37°C for 48 to 72 hours in
an inverted position (see footnotes 2
and 3, page 22) Countthenumberof
revertant colonies shortly after re-
moval from the incubator
I If increased numbers of revertant
colonies are noted, repeat the above
steps at least in duplicate (preferably
in triplicate) with the most active
strains using additional dosage
levels of test materials (see Confirm-
atory Test — Plate Incorporation
Procedure)
m. If the preliminary test does not
suggest any mutagenic activity,
repeat the test conditions but increase
the maximum dose level, if necessary,
to 5 to 10 mg per plate or that which
is clearly cytotoxic. With mixed test
materials, one or more components
may precipitate on the plate at
relatively low concentrations If
precipitate does not interfere with
the scoring of plates, this kind of
precipitate should not restrict the
upper limit of dose concentration.
General Recommendations
Table 1 provides recommended con-
ditions for conducting the preliminary
and confirmatory (dose-response) tests
using conventional plate procedure and
premcubation assays.
Table 1. Recommended Experimental Conditions for Conducting Salmonella Mutagenicity Assays
Test
Designation
Prelim/nary {PR)
Test
Confirmatory
(Dose-Response)
Test'
Preincubation
(PI) Screen
Confirmatory
Preincubation
(Dose-Response)
Assay
Assay Type
Plate
incorporation
Plate
incorporation
Preincubation
and plate
incorporation
Preincubation
and plate
incorporation
Test
Strains
TA1535
TA1537
TAT 538
TA98
TA100
Strains
most
active in
PR Test
TA98
TAT 00
TA1535
TAT 537
TA1538
Strains
most
active in
PI Screen
S-9
Activation
High, Low
and no S-9
for each
strain
Conditions
active in PR
Test
High, Low
and no S-9
for each
strain
Conditions
active in
PI Screen
Plates per Test
Condition
Recommended
Dose Regimen Minimum
Minimum of 5 doses over
2- to 3-log range
6-8 doses: bracket dose
level showing highest
activity in PR Screen,
space closely (e.g., 8
doses over 1/2 -log range)
Minimum of 5 doses over
2- to 3-log range
6-8 doses; bracket dose
level showing highest
activity in PI Screen;
space closely
2
2
2
2
'For negative results, repeat conditions of preliminary tests or retest material using Preincubation Screen.
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March 1983
4-1
Section 4
Section 4
Salmonella Test Strains
The five standard Salmonella tester
strains used in the assay are described
in the table below. TA1535 has a
missense mutation at the hisG46 locus
and is used to detect mutagens that
cause base-pair substitutions (e g.,
replacement of one nucleotide base pair
in DNA by another). StrainsTA1537and
TA1538 carry a frameshift mutation,
hisC3076 and hisD3052. respectively,
and are used to detect chemicals that
cause frameshift mutations.
Strains TA100 and TA98 are derived
from TA1535 and TA1538, respectively,
and contain the resistance transfer
factor plasmid (R-factor pKM101). The
R-factor increases sensitivity to certain
mutagens, possibly through error-prone
repair, confers resistance to the antibiotic
ampicillin, and makes strain TA100
sensitive to some frameshift mutagens
(Table 2). For example, TA1535 will not
detect aflatoxin B, or benzo-a-pyrene,
while TA100 will; TA1538 is not
sensitive to sterigmatocystin or benzyl
chloride, while strain TA98 is.
The five Salmonella strains used in
the Ames test may be obtained from Dr.
Bruce N Ames, Biochemistry Depart-
ment, University of California, Berkeley,
California 94720. The bacteria are
impregnated onto small absorbent discs
and are mailed upon written request
(together with the latest "Supplement
to the Methods Paper").
Processing, Regeneration
and Storage
a. Upon receipt of new tester strains,
streak each disc onto an individual
nutrient agar plate and drop each
disc into individual tubes or flasks
(approximately 50 ml volume) con-
taining 10 ml of sterile nutrient
broth. Close tubes or flasks with
closure that allows gaseous exchange,
and shake gently at about 120 rpm.
As soon as possible, return to the
plates you streaked and cross-
streak them with a sterile loop to
distribute the cells and facilitate
obtaining individual colonies. These
plates are designated for convenience
"Reserve Plates." Incubate plates at
37°Cfor 24 hours. Place in refrigera-
tor at 4°C. If strains check out (Part C,
below) satisfactorily, discard "Re-
serve Plates." If a tester strain does
not check out (i.e , for spontaneous
revertant value, genetic markers,
and mutagenic activity using a
standard mutagen), pick 4 or 5
isolated colonies from the "Reserve
Plate" to prepare overnight nutrient-
broth cultures and repeat the check-
out procedures. Use the isolated
culture which best demonstrates the
overall desirable characteristics of
the strain to prepare frozen perma-
nent stocks.
b. Incubate nutrient-broth cultures of
each tester strain with gentle shaking
at 37°C for 14 to 18 hours.
c Pipette 0.8 ml of nutrient-broth
culture into the desired number of 2-
ml, sterile, glass vials with teflon-
lined screwcaps. Add 70 fj\ of
dimethyl sulfoxide (DMSO). Consider
one set of irozen permanents as
"Routine Use" and the other, "Master
Copy" frozen permanents.
Quick-freeze vials in dry ice and store
in freezer at -80°C or lower until
needed.
Master Plates
To avoid frequently opening frozen
permanents, "Master Plates" can be
used as the source of inoculum for
overnight cultures used in assays.
To prepare Master Plates, use Minimal
Agar plates to which histidmeandbiotm
(and ampicillin for TA98 and TA100)
have been added 1 to 2 days prior to
inoculation. Spread 0.1 ml of sterile 0 1
M L-histidine and 0.1 ml of 0.5 mM
biotin on the surface of each Minimal
Agar plate and label properly with a
waterproof marker pen. For those
Master Plates that will be used to
maintain TA98 and TA100 cultures, add
Table 2.
Strain
TA 1535
TA100
TA 1537
TA 1538
TA98
Genotype of Five Salmonella Tester Strains Used for General
Screening in Mutagen Testing
Mutation Repair LPS R-Factor
Missense Mutation hisG46
Missense Mutation hisG46
Frameshift Mutation hisC3076
Frameshift Mutation hisD3052
Frameshift Mutation hisD3052
uvrB
uvrB
uvrB
uvrB
uvrB
rfa
rfa
rfa
rfa
rfa
pKM101
pKM101
0.1 ml of 7 mg/ml ampicillin to each
plate to help retain the R factor. Allow
the histidme, biotin and ampicillin
solutions to be absorbed into the agar
for 24 to 48 hours before inoculation
Streak each prepared Minimal Agar
plate with overnight nutrient-broth
culture of each tester strain Incubate
for 2 days at 37°C and store in a
refrigerator at 4°C Plates may be used
for up to 4 weeks.
Optional Method of Processing
Tester Strains
Based on the modifications to the
Ames test described by Belser et al
(1981) the following changes may be
adopted.
Preparation of Initial Fresh Nutrient
Broth Cultures:
• Use 30 ml of sterile nutrient broth
in a 150 ml erlenmeyer flask to
prepare initial cultures.
• Adjust the bacterial count of the
fresh cultures to 1 to 2 x 108
cells/ml using a spectrophotometer
at 550 nm or a Coulter counter
(Salmeen and Durisin, 1981) and
standard curves developed within
the laboratory to relate instru-
mental measurements to plate
counts of serial culture dilutions
Add DMSO to a final concentration
of 8 percent.
• Prepare frozen permanents, each
consisting of 0 5 ml of overnight
nutrient-broth culture. Quick-
freeze, e.g , i n a tray of fragmented
dry ice and store at -80°C.
• To conduct a mutagenicity assay
use entire 0.5 ml of frozen culture
to prepare a fresh nutrient-broth
culture that is then used as inocu-
lum for the assay (i.e. to add to top
agar, etc.).
Checking Characteristics of
Tester Strains
Spontaneous Reversion Count
Spontaneous reversion (SR) count
tests should be performed upon receipt
of the tester strains and in triplicate
whenever a mutagenicity test is per-
formed. Results should be maintained
as part of the permanent quality control
record. These values serve as an
indicator of strain activity and as a
-------
Section 4
4-2
March 1983
negative control for solvent controls
used in the mutagenicity test If the SR
values for the negative and solvent
controls are significantly different, the
test data should be suspect SR tests are
conducted as follows
a Prepare top agar (Appendix I) Melt
prepared top agar in autoclave for
three minutes at 121 °C, or melt the
agar in a microwave oven Maintain
the molten top agar at 45°C in a
waterbath or warming oven
b Prepare histidme/biotm solution
(Appendix I) Warm histidme/biotin
solution in 45°C waterbath Add 20
ml histidme/biotm solution per 200
ml top agar
c Warm the Minimal Agar platesmthe
incubator at 37°C for 24 hours This
facilitates the formation of a uniform
layer of top agar after pouring, and it
verifies the sterility of the Minimal
Agar plates
d Place the required number of sterile,
capped, 13 x 100 mm culture tubes
into a heating block or waterbath
preheated to 45°C Pipet 2 ml
molten top agar (with trace of biotin
and histidme) into each tube
The following steps should be per-
formed in an appropriate hood or glove
box.
e For each strain, add 01 ml of fresh
nutrient-broth culture containing 1 x
108 cells to each of three pairs of
tubes The organisms should remain
at 45°C no longer than about 2
minutes to avoid excessive cell
death
f When performed as part of a muta-
genicity assay, addO 5 ml of the High
S-9 Mix to each of the first tubes, add
0 5 ml of the Low S-9 Mix to each of
the second pair of tubes, and add 0 5
ml of 0 2M phosphate buffer (pH 7 4)
to each of the third pair of tubes
g Mix the material in each tube with a
vortex mixer (gently, to prevent
bubbleformation mthe topagar) The
add it ion and mixing in of the S-9 Mix
should be carried out in the shortest
practicable time (seconds) to avoid
mactivation of the enzymes.
h Pour the contents of each tube onto
the surface of a prewarmed Minimal
Agar plate Gently tilt and rotate each
plate to spread the top agar over the
SL rface of the mini ma I agar. Place on
a level dry surface, vibrate gently to
uniformly distribute the top agar (a
mechanical vibrating device, e g., a
vibrating table [Belser et al. 1981]
may assist in obtaining uniform
distribution), and allow to harden.
i Incubate the plates at 37°C for 48
hours in an inverted position
j Examine the background lawn for
proper growth, with and without a
dissecting microscope
k Count the number of spontaneous
revertant colonies and record Deter-
mine whether or not the number of
revertant colonies per plate is within
an acceptable range
Expected Spontaneous Revertants for
Each Strain
Strain
TA1535
TA1537
TA1538
TA98
TA100
A/o of Colonies
10-35
3-15
15-35
30-50
120-200
From Supplement to the Methods
Paper (Methods Paper is Ames
et al 1975), revised February
1981
Note Revertant values maybe slightly
higher or lower on plates con-
taining S-9 mixes Each labora-
tory should establish an accept-
able range of spontaneous
revertants per plate to determine
if the strains are responding
propefly R-factor loss is usually
indicated by one or more of the
following: ampicillin sensitivity;
reduced SR values, and increased
sensitivity to uv radiation
Histidine Requirement (Check as
part of each assay)
a Prepare two minimal agar plates by
coating each plate with 01 ml of
sterile 0 5 mM biotm Spread 01ml
of sterile 0 1 M histidme onto one of
the plates
b. Apply a single cross-streak of broth
culture to each plate with each test
strain Incubate plates at 37°C for 24
hours
Each strain should show growth on
the plate containing histidme; no
growth should occur on the plate
containing only biotm
Crystal-violet (rfa character)/
Ampicillin Sensitivity (R-factor)
(Check as part of each assay)
a Pipet 0 1 ml fresh nutrient-broth
culture into 2 ml top agar (with trace
of histidme and biotm) Pour mixture
onto a Nutrient Agar plate and allow
to solidify Using sterile tweezers,
place a filter-paper disc (with 10/^gof
crystal-violet) off-center on the plate
b Placea 10/ug ampicillm-impregnated
disc (Difco 6363 "Dispens-o-Disc")
off-center, opposite the crystal-violet
disc Use a separate plate for each
test strain Incubate 24 hours at
37°C
All test strains should have a zone of
growth inhibition (approximately 14 mm
diameter) around the crystal-violet,
indicating the presence of the rfa
mutation Strains TA1 535, TA1 537 and
TA1 538 should show a zone of growth
inhibition around the ampicillin disc
Strains TA98 and TA100 (containing
the R-factor) should not be inhibited by
the ampicillin
uv Sensitivity (uvrB -deletion) (Check
each strain at least monthly)
The following procedure is suggested
as an option to that described in Ames
et al (1975) Cross-streak each test
strain on a separate nutrient agar plate
Divide each plate into four approximately
equal zones by marking three parallel
lines on the bottom of the plates with a
waterproof marker Remove lid, maskall
but one of the zones, and irradiate the
exposed zone undera 15-wattgermicidal
lamp at a distance of 33 cm (13 in) for 3
seconds Move the mask to expose
both the previously irradiated zone and
the next (adjacent) zone Repeat the 3-
second irradiation. Move mask again so
that only the final zone is shielded and
repeat 3-second irradiation At this
point zones 1 -4 will have been exposed
to 9, 6, 3 and 0 sec irradiation,
respectively (Note The output of a uv
light tube diminishes with use This may
require compensatory exposure adjust-
ment) Use caution to avoid looking into
the uv lamp or exposing skin surfaces to
any unnecessary radiation Immediately
cover and place plate in the dark to
prevent photoreactivation
Incubate all plates at 37°C for 18-24
hours All strains should retain the
uvrB deletion No growth should appear
in any of the zones exposed to 9-
second irradiation Plates containing
TA1535, TA1537, and TA1538 should
show no growth at 6-second exposures
either, although slight growth may be
visible with TA98 and TA100 strains
Slight to moderate growth is likely in
zones exposed to only 3-second irradia-
tion All unexposed zones should show
active growth The intermediate growth
at 3 seconds and marginal growth at 6
seconds provide a rough baseline —for
the relative resistance of the tester
strains —.that should be compared on a
regular basis to detect changes in test-
strain resistance
Standard Mutagens
Standard mutagens (positive controls)
shall be included with each assay to
confirm proper tester-strain mutagenic
activity and specificity Standard muta-
gens may be purchased in diluted
quantities from a commercial laboratory
Some mutagenic materials (eg, 2-
Anthramme) are unstable while others
-------
March 1983
4-3
Section 4
(e g , sodium azide) may be useableforG
months or more When in doubt, make
up standard mutagens fresh each time
they are needed if adequate high-
hazard facilities are available Dose-
response curves should be established
for each standard mutagen This curve
is used to monitor and possibly adjust
the mutagen concentration over time
Each laboratory shall use positive
controls at concentrations yielding
reproducible counts to confirm the
reversion properties or mutagenic
activity of each strain The standard
mutagens are also used to evaluate the
metabolic activity of newly prepared S-9
mixtures It is recommended that dose
levels of standard mutagens be selected
(from the dose-response curves) that
generally yield reversion values more
than five times the SR value for TA1 537
and more than three times the SR value
forTA1535,TA1538, TA98andTA100
Table 3 contains a listing of mutagens
found to be useful in plate-incorporation
assays for confirming activity of the
strains indicated opposite them Often
the choice of controls maybe influenced
by the suspected chemical characteristics
of the sample
S-9 Activity
Since the protein content, and hence
the enzymatic activity, of S-9 batches
can vary widely, it is recommended that
each batch of S-9 be tested with
standard mutagens and tester stramsof
known characteristics The following
procedure may be used to determine,
and adjust if necessary, the activity of a
specific batch of S-9
Prepare S-9 mix with four different
levels (20%, 10%, 5% a nd 2%) of S-9
preparation
Conduct plate-incorporation assays
with strain TA1538, using a single
dose level of 25 /jg of 2-ammofluo-
rene (as the standard mutagen) and
each of the four S-9 mixes
Concurrently repeat assays using a
single dose level of 5 /jg benzo-a-
pyrene (as a second standard muta-
gen)
Construct dose response curves, for
each set of data, plotting S-9 per-
centage against resulting plate
counts
Compare response levels to those
made with previous batches (or
preparations) of S-9
Adjust, if required, the percentageof
S-9 added to the High S-9 and Low
S-9 mixes to yield desired revertant
levels based upon the response
curves obtained with the standard
mutagens
Table 3. Standard Mutagens for Confirming Tester Strain Function
Mutagen^
Amount/
Plate2
RLE
(S-9)
Test Strains Positive
TA1535
JAT 537
TA1538
TA98
TA100
Sodium azide 1 pg
N-Methyl-N'-mtro-N- 5 fig
nitrosoguanidme
Methylmethanesulfonate 25 /j/
(1 -50 dilution)
2-nitrofluorene 10 ug
X
X
X
X
X
X
9-aminoacridme
Daunomycin HCI
Hycanthone
2-anthramme
Benzo(a)pyrene
2-ammofluorene
Dimethylbenzanthracene
150 fjg3
50 /jg
25m?
2/jg3
5ng3
25 ng
Wfjg
— —
— —
X X
X —
X —
•^
X
—
X
X
—
~
—
—
X
X
X
~
X
X
X
X
X
~
—
—
X
X
X
X
^All chemical solids are dissolved(1 mg/ml) in DMSO except for Daunomycin and sodium azide, which are dissolved'(1mg/'ml)
in distilled water Many of the standard mutagens are available in diluted form from Nanogens International, P 0 Box 1025,
Watsonv/l/e, CA 95076 Additional standard mutagens and sources for obtaining them are provided in Ames ft'981)
2Recommended starting levels, modify concentration as necessary to determine acceptable levels (that reproducibly yield
expected results with each strain)
^Values obtained from McCann, et al (1975)
-------
March 1983
5-1
Section 5
Section 5
Quality Control/Quality Assurance
General Requirements
Quality control refers to those pro-
cedures that are implemented by the
lexicologist, microbiologist, staff mem-
bers, and supervisor to reduce the
variability and bias associated with data
generated by their bioanalytical testing
programs, and to increase the reliability
of the test methods used The end result
of a carefully administered quality
control program will, hopefully, be
testing data of known accuracy and
precision. Necessary steps in conducting
a successful testing program include:
• Selection of an appropriate ex-
perimental design,
• Rigid adherence to appropriate
test protocols and standard oper-
ating procedures,
• Assurance of test and data securi-
ty;
• Careful interpretation and review
of test data; and
• Accurate reporting of test results.
The first requirement of good science,
that results can be verified by other
investigators at other times and places,
is a sufficient imperative to adopt and
routinely follow a standardized proce-
dure At the same time, it is vital to have
a mechanism to enable significant
improvements in testing methodology
to be incorporated into the standardized
protocols without unnecessary delay so
that the user community can benefit
from these advances The principal
element of the mechanism is a substan-
tial data base to support the recom-
mended changes (improvements).
Security of the testing operation and
of the resulting data mustbeprovidedto
minimize the loss of irreplaceable
testing data Adherence to standard
"good laboratory practices" will help a
great deal. Restriction of test-area access
to authorized personnel only must be
rigorously enforced. This is important
from a safety as well as a security
standpoint. Responsibilities for control
of data records must be clearly defined.
Where sample volumes permit, "library
samples" (aliquots of the sample
material) should be maintained under
proper storage conditions, for the
duration of the experiment, to allow
reruns of samples yielding ambiguous
or questionable test data.
Maintenance of the integrity of test
data depends upon'control over the
performance of the experiment, adher-
ence to details of the measurement
process, and careful handling of the
data Errors can arise during handling
of the data due to transcription, clerical,
or typing mistakes; as a result of the use
of different statistical methods at
different times, computer mistakes or
omissions, inclusion of the wrong data,
omission of parts of the original data;
differences in observational results (as
between two microbiologists), and
changes in interpretation of the data.
At no point in the testing process is
the skill and experience of the analyst/
researcher more important than in the
interpretation of the test data Until
such time as the analyst has acquired
extensive experience in evaluating test
data and drawing appropriate inferences
therefrom, he should make maximum
use of external laboratories for review
and confirmation of his findings A
formalized program of data exchange
for independent analysis is of great
mutual value to the collaborating
parties. Participation in mterlaboratory
studies with known and unknown
sample materials (see Quality Assur-
ance) is also of great benefit in establish-
ing a laboratory's competence and in
supplementing mtralaboratory perform-
ance evaluation procedures
Reports of test results, whether in the
public literature or as proprietary
submissions to a user group, should
either include sufficient data (i.e , all
tests, negative control, and positive
control data) that the interpretations
made by the investigator can be inde-
pendently evaluated or such data
should be readily available upon request.
The use of a statistician or statistical
staff experienced in the analysis and
interpretation of biotestmg data is
highly recommended. Results of tests or
studies should routinely be subjected to
a review system prior to the preparation
and/or publication of reports
Quality Control Testing
Table 4 presents tests that are
considered vital in a mutagenicity
testing program to confirm the condition,
genetic integrity, and responsiveness of
the test organisms; the sterility of media
and test additives; and the characteristics
of the colonies resulting from the plate
assay Also included in Table 4 are
indications as to when the tests should
be performed and how test results may
be used to indicate suitable conditions
for going forward with the mutagenicity
assay. The tests indicated should be
performed in addition to the strain
function confirmation tests presented
earlier (i e., uv radiation, ampicillm, and
crystal-violet sensitivities, and the
histidme requirement test)
Sterility of minimal or nutrient agar
plates (Test SP) is determined by
incubating all plates at 37°C for the 24
hours prior to running a QC test series
or assay Any plates showing growth
(contamination) after the incubation
period should be discarded and the
source of contamination identified if
possible To determine the sterility of
solvents, reagents, standard mutagens
or of the activation mixture (S-9), spread
0 1 ml aliquots of the component in
question in 2 ml of top agar onto
nutrient agar plates by gently tilting and
rotating the plates Incubate the plates
at 37°C for 24 hours, inspect the plates
for microbial growth with and without
the aid of a dissecting microscope.
Discard contaminated materials or, if
appropriate, restenlize.
Solvent and positive control tests (CS,
PN and PA) are all performed concur-
rently with the mutagenicity assay, and
colony counts are performed after the
48- or, if indicated, 72-hour incubation
period. In solvent control tests a 50 p\
addition of pure DMSO (or alternate
solvent) substitutes for the 50 /j\ doses
of test chemical or sample solutions (in
the corresponding solvent) in the
experiment. The data yielded by these
"zero-dose" tests provide the sponta-
neous reversion value against which
other dose-level data are compared in
determining whether or not the sample
material is considered positive for
mutagenicity by this assay.
It is a useful practice — and one that
should be mandatory in laboratories
that have not yet developed an adequate
data base of their own on spontaneous
revertant values for each strain —to run
a negative control (spontaneous rever-
sion test; see Section 6) concurrently
with each mutagenicity test. The
negative control has neither test
solution nor the equivalent volume of
corresponding solvent added, whereas
the solvent control incorporates a
volume of solvent, equal to the total
volume of test solution, mtothe top agar
before pouring the overlay. Comparison
of concurrent negative and solvent
controls may detect either mutagenicity
-------
Section 5
5-2
March 1983
Table 4. Quality Control
Test
Designation
SP
SH
Tests for Ames Plate Assay
Type
Sterility Check
(minimal or
nutrient agar
plates)
Sterility Check
(Histidine/biotin
solution) Plate
on nutrient agar
When Performed
All plates are
incubated at 37°C
overnight prior to the
assay
Perform in duplicate
e "•£/? time histidine/
biotm stock solution
is prepared.
Expected Results
No growth
(Discard any
plates showing
growth).
No growth
(If growth,
prepare and
filter sterilize
or autoclave
fresh stock
solution; recheck
sterility)
ss
SA
SM
CN
CS
PN
Sterility Check
(Sample materi-
al). Plate on
nutrient agar at
highest test dose
level prepared in
appropriate
so/vent.
Sterility Check
(S-9 mix, w/and
w/out S-9)
Plate on nutrient
agar plates.
Sterility Check
(Standard muta-
gens) Plate
test levels (in
appropriate sol-
vent) on nutrient
agar plates
Negative Control
(Spontaneous
Reversion Test)
Zero dose level.
non-activated
test conditions.
Solvent Controls
Zero dose level,
activated and
non-activated
test conditions
Positive Control
(Function Check)
Non-activated
test conditions,
direct-acting
standard
m uta gens.
Perform in duplicate on
day before each
experiment.
Perform in duplicate
when new S-9 batch is
received and for
cofactor solutions as
they are prepared.
Perform in duplicate
when new solutions of
mutagens are received
or prepared, or when
tester strains are
checked out
Perform in triplicate
for each bacterial
strain, when received
and with each assay.
Perform at least in
duplicate for each
bacterial strain and
condition with each
assay
Perform for each test
strain, when received
and with each assay.
No growth
(If growth,
filter sample
material through
0 8 /j glass-fiber
filter; use DMSO
as solvent
No growth
(If growth with
mix w/out S-9,
resterilize
stock solutions
and retest; if
growth with S-9,
filter sterilize
with 0 45 fjm
filter)
No growth (If
growth, for solu-
tions - filter
through 0 8 fjm
glass-fiber
filter; Suspen-
sions-leave in
DMSO overnight).
Background
growth and spon-
taneous revertant
colonies only
Background
growth and spon-
taneous revertant
colonies only
Background
growth and
revertant growth.
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March 1983
5-3
Section 5
Table 4. (Continued)
Test
Designation
Type
When Performed
Expected Results
PA
GC
VC
Positive Control
(Function Check}
Activated test
conditions.
standard muta-
gens requiring
S-9 activation
Colony Geno-
type Check-Rep-
lica plate colo-
nies onto Minimal
Agar with biotin.
Viability Check-
Serial dilutions
of overnight
culture plated on
nutrient agar
Perform for each test
strain, when received
and with each assay
When mutagenicity is
indicated, check plates
w/highest mutagenic
activity ratio.
When new tester
strains are received,
master plates are gen-
erated, or overnight
cultures to be used in
the assay are prepared.
Background
growth and
revertant growth.
Growth of
histidme revert-
ant colonies, no
growth of
phenocopy
colonies
Growth - compare
with instrumental
density measure-
ments of same
cultures to de-
termine cells/ml
or toxicity resulting from the solvent
New solvents, solvent batches, or
changes in distilled water supplies or
treatment warrant such a comparison,
even in laboratories where an adequate
historical data base for spontaneous
reversion of each tester strain has been
developed.
Water or DMSO solutions of standard
mutagens (see Table 3) known to yield
positive test results (induced revertants
>2 X spontaneous revertant rate, pos-
itive dose-response relationship and
background growth within normal
range') with specific strains under
known activation conditions are used in
tests PN and PA, as the test chemical in
the assay These positive controls
provide a means of confirming that the
test strains are responding predictably
and reproducibly Over time, a large
base of positive control data is devel-
oped within a testing laboratory These
data are useful m determining whether
subsequent tester strains have accept-
able mutagenic activity In orderto more
quickly build such an adequate data
base for each standard mutagen it may
be advantageous to limit the number of
such mutagens used to the minimum
required to show normal mutagenic
activity of the strains under the condi-
tions of the testing program. Afterward,
it may be useful to add selected
standard mutagens (e.g , known chemi-
cals of the same chemical class and
with physical/chemical characteristics
which approximate those of suspected
1That is, the "background lawn" or growth is not
absent or severely inhibited
components in test samples) to optimize
the test conditions for a specific assay A
data base for these additional selected
standard mutagens should be acquired
as soon as feasible.
General criteria for determining the
adequacy of Salmonella mutagenicity
testing data are detailed in Section 6.
To determine if colonies counted are,
in fact, true histidme-mdependent
revertant colonies rather than anoma-
lous growth of the histidme-dependent
background, colonies should be replated
on Minimal Agar medium supplemented
with biotin Histidme-independent cells
will form new colonies on the Minimal
Agar while the histidme-dependent
cells will not grow. Perhaps the most
effective way to accomplish this—
especially if the number of colonies on
the test plate is large—is to "replica
plate" the colonies from the test plate to
Minimal Agar. By this procedure surface
colonies are transferred on a piece of
clean, sterile, cotton velvet much as ink
is transferred from stamp pad to paper
on a rubber stamp. The pattern of
surface colonies is reproduced on the
minimal agar plate, and direct compari-
son of "donor" and "recipient" plates
confirms the histidine independence of
the colonies Additional confirmation of
the histidine independence of "back-
ground" colonies can be obtained by
replica plating onto biotin-supple-
mented, Minimal Agar plates with and
without added histidine and comparing
the recipient plates. For successful
transfer, the surface of the donor and
recipient plates should be dry, and the
velvet must be of good quality and
"wettable," i.e., free of sizings and other
additives (Larimer, pers comm ).'
Serial dilutions of overnight cultures
of test strains should be plated on
nutrient agar and incubated at 37°C
overnight to determine the viable cell
liter from each culture Spectrophoto-
metric, colorimetric, turbidimetric, or
particle-counter measurements used to
adjust culture densities do not estimate
viable cell count, but rather reflect the
density of bacterial material present. It
is necessary to develop and periodically
reconfirm standard curves within an
individual laboratory to relate the viable
cell count to instrumental measure-
ments. Changes in a laboratory's
standard curve over time may reflect
changes in incubation conditionsforthe
broth cultures (viable to total bacterial
count) changes in sensitivity or linearity
of the measurement procedure, or other
problems which must be addressed.
Salmeen and Durism (J981) suggest
that order-of-magnitude differences in
initial viable cell count can modify plate
counts, and the resulting slopes of
dose-response relationships. Figure 3
is an example of a form for recording
periodic measurements of instrument
values relative to plate counts of culture
dilutions
Results of all quality control tests
should be properly documented on QC
data forms (see Figures 4, 5 and 6) and
maintained as part of the permanent
data records for the testing program. In
addition, consistent with standard
'Meeting in May 1981 with Dr Frank Larimer,
Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tenn
-------
Section 5
5-4
March 1983
Date
Strain/
Culture ID
Initials
Date
Strain/
Culture ID
Initials
Dilution'1
Instrument
Reading
Plate
Count
Dilution'
Instrument
Reading
Plate
Count
10-'
10-'
70"
w-
Date
Dilution*
Strain/
Culture ID
Instrument
Reading
Initials
Plate
Count
Strain/
Date Culture ID
Instrument
Dilution' Reading
Initials
Plate
Count
w~
10-
7CT
10"
10-'
Date
Strain/
Culture ID
Initials
Date
Strain/
Culture ID
Initials
Dilution'
Instrument
Reading
Plate
Count
Dilution'
Instrument
Reading
Plate
Count
7cr
10-
1Q-
10"
jo-
1 Dilute overnight cultures (approximately 10 cells/ml) geometrically with sterile 0 9 percent NaCI
Figure 3. Quality Control Data Form - cell liter and viability measurements.
"good laboratory practices," the quality
of water sources and the performance
of laboratory equipment used in the
testing program (i.e. waterbaths, auto-
claves, incubators, refrigerators, freezers,
and cell- and colony-counting equip-
ment) should be periodically confirmed
and given regular preventive main-
tenance in accordance with manufac-
turers' recommendations. Equipment
and water quality QC data should be
recorded (Figure 7) and maintained as
part of the program's permanent data
record.
-------
March 1983 5-5 Sections
STRAIN FUNCTION TESTS
Test Date/lnit TA1535 TA1537 TAJ 538 TA98 TAJ 00
1 Solvent/Negative Control'' 1
(specify)
2
3
Average
2 Ampilillm Sensitivity 2
3 Crystal Violet Sens/tivty2
4 U V. Sensitivity3 3 sec
5 Positive Controls^ 6 sec
Mutagen (Amt/p/ate) 9 sec
a I
b (
c (
d (
e (
Test Date/lnit. TAJ 535 TAJ 537 TAJ 538 TA98 TAJ 00
J Solvent/Negative Control 1.
(specify)
2
A verage
2 Ampilillm Sensitivity '
3. Crystal Violet Sens/tivty2
4. U V Sensitivity 3 sec
5. Positive Controls' 6 sec
Mutagen (Amt/p/ateJ 9 sec
a
b
c
d.
e.
^Plate counts
2 + = growth; - - no growth.
3+++ = 10° to 10~1 x control (not irradiated),
++ = 70~1 to W~2 x control,
+ = 10~2 to <0 x control, - = no growth.
Figure 4. Quality Control Data Form - strain function tests
-------
Section 5
5-6
March 1983
CUL JURE MEDIA RECORD
Media and
Lottt
Receive
Open
AMT
pH
Data
Int
Media and
Lot n
Receive
Open
AMT
pH
Data
Int.
Figure 5. Quality Control Data Form - culture media record
-------
March 1983
5-7
Section 5
REAGENTS, STANDARDS AND TEST MATERIALS
(Sterility, pH, etc )
Material
Test
Result
Date
/nit
Material
Test
Result
Date
Init
Figure 6. Quality Control Data Form - reagents, standards, and test materials.
-------
Section 5
5-8
March 1983
EQUIPMENT RECORD
Year. 19-
Month
Day:
STILL
Conductance
pH
Drained
WATERBATHS
1)
2)
3)
4)
5)
AUTOCLAVE
Pressure
Temp. (max)
OVEN
Temp (max)
INCUBATORS
1)
2)
3)
4)
5)
REFRIGERA TORS
1)
2)
FREEZERS
1)
2)
3)
COLONY COUNTERS
1)
2)
OTHER (Specify)
Figure 7. Quality Control Data Form - equipment record.
-------
March 1983
6-1
Section 6
Section 6
Data Analysis, Interpretation and Reports
Data Analysis
Steps in Evaluation
of Data
Chu et al. (1981) present a series of
steps that they have employed in eval-
uating large volumes of Ames test data
from collaborating laboratories-
1 Identification and removal of
spurious plate counts;
2 Determination of the adequacy of
the remaining data for making
decisions on the mutagenicity of
the test chemical,
3. Performance of statistical tests;
and
4 Interpretation of the results
This scheme should be followed in
evaluating Ames test data
Adequacy of Test Data
Five basic conditions outlined by
Dunkel and Chu (1980) for defining the
adequacy of test data and removing
spurious data have been adopted. These
criteria for data acceptance are'
1 Bacterial strain checks must be
satisfactory, i.e the crystal violet
and ampicillin checks for strain
characteristics should show all
strains to be sensitive to crystal
violet, and strains TA1535, TA1537
and TA1538 should be sensitive to
ampicillin. Strains TA98 and
TA100 should be ampicillin re-
sistant
2. Negative and solvent controls
(spontaneous reversion values)
must be acceptable, i.e. 2 of 2orat
least 2 of 3 (if in triplicate) negative
and solvent control plate counts
must fall within empirical 95
percent laboratory-control confi-
dence limits (determined within
each laboratory for each tester
strain)
3. Positive controls must be accept-
able, i.e 2 of 2 or at least 2 of 3 (if
in triplicate) standard mutagen
plate counts should exceed the
97.5th percentile of the historical
laboratory negative/solvent con-
trol single plate count (historical
average spontaneous revertant
rate for that strain).
4. Four acceptable dose levels must
be demonstrated in addition to the
solvent control. An acceptable
dose has to have at least two
acceptable plate counts and not
exhibit toxicity. Toxic dose level
was defined as any dose level
which was greater than that dose
eliciting the highest average
response (HAR) and in which
every plate count was less than
the lowest count in the HAR dose
level. Outlier plate counts were
identified by a Studentized range
procedure and eliminated Unless
at least two plate counts were
"within range" the dose level was
considered unacceptable and
eliminated.
5. The test could have no more than
one unacceptable dose level lower
than that dose giving the highest
average response
In addition to the above conditions
(acceptance criteria), replica plating
should confirm absence of "non-
revertant" (phenocopy) colonies, and all
sterility checks must be negative In
addition, all test data obtained from
plates with atypically sparse back-
ground lawn should be considered
questionable. Unfortunately, hard and
fast rules with respect to background
lawn have not been developed A recent
study (Salmeen and Durisin, 1981) was
conducted to quantify background lawn
using photomicrographs of plates
inoculated over a range of cell concen-
trations Tests which directly measure
toxicity (Waleh et al., in press) show
promise for being successfully coupled
to the Ames assay Additional studies of
this nature should be conducted so that
unambiguous guidance can be provided
as to what constitutes an "acceptable"
background growth (both in nature and
extent)
Graphical Approaches
A great deal of information about the
dose-response nature of an Amestest is
obtained by plotting the average plate
counts (Y axis) against the correspond-
ing dose level in milligrams (X axis) It
may be convenient to plot the data
points on log-log paper so that the nega-
tive control (spontaneous revertant rate,
zero-dose level)count isclearly resolved
from the zero line The spontaneous re-
version value of the appropriate strain
should be superimposed as a horizontal
straight line (see example, Figure 8) as
an aid in visually assessing the test-
dose responses The graph may include
plots of several "tests" (e.g , for all five
strains under one activation condition
or a single strain under multiple activa-
tion conditions). Appropriate spon-
taneous reversion values should be
included for each strain/condition
plotted
Graphs should be developed for both
screening and confirmation level test-
ing Inflections of dose-response curves
in screening tests are used as an aid in
selecting the dosing regimen for the
confirmatory tests The shape of result-
ing plots can also be used to select an
appropriate probability model for appli-
cation to the data (Sexton et al 1981).
All reports associated with graphical
representations should include the raw
data from which the average plate
counts for negative/solvent controls and
each test dose are calculated
Information on automated procedures
for graphical and statistical presenta-
tion/evaluation of mutagenicity data
using the In Vitro Information System
(MS) is presented in Lmhart, et al.
(1980)
Statistical Approaches
A generally accepted statistical test
for examining the results of the Salmo-
nella plate test has not been published
(de Serres and Shelby, 1979; Dunkel
and Chu, 1 980) However, a number of
statistical techniques can be applied to
the evaluation of Ames Test data Some
of these techniques are useful for deter-
mining the adequacy of data for further
analysis, e g , to detect changes in spon-
taneous revertant values over time,
determine the homogeneity of variances
among control and test data, determine
control limits for standard mutagen re-
sponse, and identify outlier data points
Other statistical tests are useful to de-
tect the presence and nature of dose ef-
fects, e g , tests of the homogeneity of
treatment means and tests for linear
trends (dose-response relationships)
The statistical tests generally assume
that the data are Gaussian distributions,
therefore, count and dose data usually
must be log-transformed before testing
Dose data should be "coded" (i e multi-
plied by a constant or added to 1 so all
values are greater than unity) before
transformation to avoid negative loga-
rithms. Table 5 presents a number of
useful statistical tests which should be
employed to determine (1) the adequacy
of the data and (2) the "positiveness" of
the test results.
-------
Section 6
6-2
March 1983
a.
\
to
91
Q;
I
Qj
0>
(0
JO3
1C?
70'
Date
XXXXXX
Experiment/
Assay No.
XXX/XXX
Compound or
Sample ID
XXXXXXXX
Tester
Strain
XXXXXX
10"'
701
702
Figure 8.
70"1 70°
Concentration (mg/plate)
Key: • = non-activated; ® = Low S-9: * = High S-9
Spontaneous reversion rates'
= non-activated; — = low S-9; " " " " ^ high S-9
Example of graphical presentation of Salmonella mutagenicity test data
(simulated).
Table 5. Statistical Techniques Useful for Evaluating Mutagenicity Testing
Data
Test
Reference
Used to Determine
One-Way Analysis of
Variance
Linear Regression
Analysis
Bartlett's Test for
the Homogeneity of
Variances
Confidence Interval
Threshold
95% Confidence
Interval
Myers. 1979
Graybill, 1976
Draper and Smith,
1966
Sokal and Rohlf,
1969
Dunkel and Chu,
1980
Freund, 1979
Student/zed Range
Procedure
Myers. 1979
Differences among means
in control and test
data. Changes in
average control counts
over time.
Slope and significance
of linear data trends
Homogeneity of vari-
ances among control
and test data.
Responses greater than
a threshold value (a
function of concurrent
and historical negative
control statistics).
Acceptability of
positive control
response data;
acceptability of
concurrent negative
control data
(spontaneous revertant
value).
Outlier values in
test and control data.
Interpretation
Positive and Negative Results
Seven methods were tested with
Ames Test data for 1 7 pure compounds
in a major interlaboratory comparison
study (Dunkel and Chu, 1980) to deter-
mine their effectiveness in making
mutagenicity decisions. Rates of dis-
agreement with the consensus deter-
minations of the four laboratories were
calculated for each of the following
"decision rules"
Decision Rule
False False
Positive Negative
Rate Rate
Two-fold 7.1% 1.6%
[increase] rule
Modified 4.1% 1 8%
two-fold
[increase] rule
Positive 20.0% 0.4%
linear trend
Positive 180% 0.7%
homogeneity
Combined 11 6% 0 2%
statistical tests
(linear trend
and homogeneity)
97.5 th 1.8% 3.8%
percentile
Confidence 1 4% 4.1%
interval
(Dunkel and Chu, 1980)
The two-fold increase rule (Ames et
al., 1 975) is widely applied to Ames test
data as an indicator of positive muta-
genic test response. With the two-fold
increase rule a test on a single strain of
bacteria was considered positive if there
was a dose level with an average
response that was twice that of the
concurrent negative/solvent control
With the modified two-fold increase
rule, a test was considered positive if
two consecutive dose levels (or the
highest non-toxic dose level) produced
average responses at least twice that of
the negative/solvent control and at
least two of these consecutive doses
showed a dose-response relationship.
Tests for linear trends are based upon
regression analysis of log-log trans-
formed data (log counts and log (dose +
1 0)) in which the null hypothesis is that
the slope is equal to zero. The test is
considered positive if the linear trend
statistic is significant, i e , the probability
that the departure of the slope from zero
is a result of chance alone is less than 5
percent (P <0.05). The test for homo-
geneity compares the (log-transformed)
-------
March 1983
6-3
Section 6
responses for each dose using the one-
way analysis of variance The null
hypothesis is that the means of the
responses for all dose levels are equal
A test result is considered positive if the
homogeneity statistic is significant, i.e.
the probability that the differences
between average responses is a result
of chance alone is less than 5 percent (P
<005)
When the tests for linear trend and
homogeneity were combined, each had
to be significant (P <0.05 in each test)
for the results to be considered positive.
Note the very low (0.2%) rate—with the
combination of statistical tests—at
which tests indicated no mutagenicity
when laboratory consensus indicated
the compound to be mutagenic
The 97 5 th Percentile Rule compares
the responses for each dose to the
empirical 97.5 th percentile of the
laboratory negative/solvent control for
single plate counts to identify any dose
levels having two or more responses
(from triplicate plates) greater than the
97.5 th percentile of the laboratory
historical controls A test was consid-
ered positive if there were at least two
out of three consecutive dose levels
above the 97 5 th percentile of the
laboratory historical control, and the
consecutive doses showed a dose-
response relationship. In addition, a test
was also considered positive if the
highest non-toxic dose was above the
97 5 th percentile
The Confidence Interval Rule identi-
fies average dose level responses that
exceed a threshold level Y0 + K • SD(h),
where Y0 equals the average concurrent
control value, K is a constant which
takes into account the significance level
and the number of plates used, and
SD(h) is the standard deviation of the
log-transformed laboratory historical
negative/solvent controls for the strain
being used. If the threshold was
exceeded by two consecutive dose
levels or the last non-toxic dose, and at
least two consecutive dose levels
showed a dose-response relationship,
the test was considered positive
Several models based upon Poisson
(Stead et al. 1981) and negative
binomial distributions (Sexton et al.
1981; Margolin et al. 1981) have
recently been developed and tested and
show promise for increasing the object-
ivity of Ames test data interpretation.
The selection of test(s) to be appliedto
Ames test data should be based, in part,
on the purpose of the study and the
implications of the study findings. For
example, it isvitalthatthetestsselected
to evaluate data upon which the safety
of a consumer product (public health
concern) is determined yield the lowest
practicable rate of false negatives. That
is, we cannot afford to err in a direction
that would jeopardize public health by
incorrectly ascribing a negative finding
to a positive mutagen Among the tests
compared by Dunkel and coworkers, the
linear trend, homogeneity, and com-
bined tests yielded the lowest false-
negative rate
On the other hand results of tests
which yield an unacceptably high false-
positive rate can cause public alarm,
adverse economic impacts, and loss or
delayed development of useful chemi-
cals or products. The 97 5 th percentile
and confidence interval tests yielded the
lowest rates of false positives, that is,
branding as mutagenic those chemicals
that, by laboratory consensus, were not
in fact mutagenic by the Ames Test
As a single test, the modified two-fold
rule gave relatively low false-negative
and false-positive rates The authors
caution, however, that some modifica-
tion of the two-fold rule was probably
used in the decision-making process at
each collaborating laboratory and, as a
result, one would expect good agree-
ment between the two-fold tests and
the consensus determinations
It is recommended that at a minimum
all plate-incorporation assay data
should be tested with the modified two-
fold rule. Other tests should also be
applied as appropriate to the purpose of
the Ames testing activity so that
decisions can be made with a high
degree of confidence and objectivity
Ames test results must be reproduci-
ble (i.e. from screening test to confirma-
tory test or among repeated confirmatory
tests) before a final decision is made as
to whether a sample is mutagenic or not
by the Salmonella reverse-mutation
test system used Negative statements
should not be the natural offspring of
inconclusive data, but rather the result
of repeated testing which confirms the
hypothesis of no difference between
negative-control and test-dose counts
Even then, a negative result does not
necessarily mean the sample is non-
mutagenic, but rather that no mutagenic
effect was detectable under the condi-
tions of the test system used The Ames
test measures mutations at specific
base sequences in bacterial DNA; it
does not indicate overall mutagenic
potential. Sample materials should
show repeatable negative responses
when tested at levels of up to 5 - 10
mg/plate (toxicity and solubility permit-
ting) before discontinuing testing If the
confirmatory test that follows a "posi-
tive" screening test is negative, recheck
the suitability of the test conditions
relative to those of the screening test,
adjust as appropriate and rerun. Dis-
continue Ames testing if this retest fails
to meet criteria for a positive test and is
not confounded by toxic effects
The tests presented here, and others
used by investigators around the world,
are simply tools to aid the researcher in
making a correct decision based upon
the available data. They are not a substi-
tute for the judgement and expertise in
Ames test data interpretation developed
with years of experience. Data ex-
changes and independent confirmation
of data interpretation are strongly
recommended to minimize the chances
of making incorrect decisions.
Inconclusive Results
Occasionally a test will yield data that
suggest mutagenic activity but do not
meet one of the criteria for acceptance
as positive Although these cases can
often be resolved by modifying the test
conditions (e g , increasing the number
of closely-spaced test doses or opti-
mizing the amount or type of S-9
activation for the sample material),
some samples have such a limited
range between induction of mutagenic
response and cytotoxicity that they
cannot be readily characterized with
conventional plate-assay procedures
Several options are available to the
researcher in such cases:
• Retest with premcubation or
suspension assays
• Employ chemical fractionation
schemes to separate, if possible,
the mutagenic fraction from other
cytotoxic components, and rerun.
• Recommend testing with an alter-
nate mutagenicity test system,
e g , Saccharomyces forward/
reverse mutation tests
• Discontinue testing, and report
results as inconclusive
Once again, the option(s) selected may
be dictated, in part, by the specific
purposes of the testing program being
undertaken
A number of sample preparation/
chemical fractionation schemes have
been proposed and used by EPA's
National Enforcement Investigations
Center, Denver, Colorado; EPA's Health
Effects Laboratory, Research Triangle
Park, North Carolina; Oak Ridge National
Laboratory, Oak Ridge, Tennessee; and
others to identify those fractions of
complex industrial and environmental
samples that show mutagenicity. Sam-
ple preparative procedures and recom-
mendations for their use with Ames
testing will be reported in the near
future
-------
Section 6
6-4
March 1983
Reporting Test Results
Data Records
Test data should be entered directly
onto forms from which the data may
be keypunched and input to a data-
base system such as the Interim In
Vitro System developed by EPA A
description of the system, reporting
forms, and general instructions for
their use have been published (Sex-
ton et al 1981)
The reporting forms developed for the
HERL IN VITRO system (Figures 9, 10
and 11) are quite complete and are an
excellent record for the laboratory's
permanent data files The system is
undergoing some revision at this time
(Claxton, pers comm )1 to expand the
listings of coded elements and clarify
'Telephone conversation, July 1981 with Dr Larry
Claxton, Genetic Toxicology Division, Health Effects
Research Laboratory (EPA), Research Triangle
Park, N C
user instructions The updated listings
and instructions for the coded reporting
forms can be obtained from Mr Andrew
Stead, MD-57, EPA, Research Triangle
Park, NC 27711 Use of the reporting
forms is strongly recommended, wheth-
er or not the investigator chooses to
make use of the data base service at this
time or in the future
Data Presentation
While there are some distinct advan-
tages to standardizing the output format
for presenting Ames test data (e g , to
facilitate comparison of findings), it is
unlikely that such standardization will
be accomplished in the foreseeable
future However, for Ames test data to
be acceptable and to make independent
evaluation possible, minimum require-
ments must be met We agree with the
consensus findings reported by de
Serres and Shelby (1979) that the
following should be essential elements
in any Ames testing report:
1 Means and indications of variabil-
ity (e.g , standard deviation) of the
plate counts for the negative
control, the positive controls, and
each dose of the test compound,
2 The number of replicate plates in
each mean, and
3 When possible, individual plate
counts
If the volume of data is prohibitive to
report, complete data should be readily
available from the investigator upon
request.
In addition, the criteria used within
the reporting laboratory to determine a
positive test should be clearly stated.
HERL IN VITRO RESUL TS FORM
1 24
II '"
§_1 Systerr
5 -8
* IL
,10 Research
Lab ID
3-14
MO DA
Experiment
Animal
Organ
Inducer
Microorganism
©
Solvent
Positive
/Table 1 1}
43 4
SOL [J
Pos
E
YR
Datt
Remarks
Made'
Yes-1
15 78 79-20 27-24
LA
Test
J_r
B
Sam/
YR NUMBER
y/e Identification
Bss
Phenocopy
Check
Conclusion
Table 73]
Dose Level
f7) Units of
Concentration
Blank - mg ml
2 - ugrml
1 45
i a
: a
: n
] D
: a
: a
n
n
: a
© C
Stock con-
centration
46-50
\\
§
Amt Per
Plate f/j/J
51-54
i
636
Sterility
S-9 Mix
1 Not Contain
2-Contam
3 Not Checked
Plate A
©
Count
55 58
n
i
i
1!
B
G
59
U
n
c
n
_
D
D
25-30
Activation Batch
[3) 67-70
(
|
Activation
fixture
^er Plate ffj/J
Plate B
Count
60 63
rrrn
1
1
!
B
G
0
a
D
D
D
n
D
D
D
T
I
31-32
est Type
Table 10)
S71
Sample
Sterility
Check
1 -Not Contam 1
2-Conlam
3 -Not Checked
Plate C
© 1©
Count B
|G
itmn
p
p
p
p
p
b
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I
33 38
39-42
Strain Batch No
Microorganism
(7)72-73
U
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74 75
mm} Temp (Cent!
-Incubation
Plate D
© |(
Count
70-73 I
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r^
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n
k
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an
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75 78
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D
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n
HI
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Card
Code
Card
Code
[g]
[G]
Q
Remarks' Indicate Item Code and Card Code
SAMPLE
Figure 9. HERL IN VITRO System - results form
-------
March 1983
6-5
Section 6
HERL IN VITRO RESUL TS CONTINUATION FORM
if
I\V\R \ \ \
System Research
ID Lab ID
2) Solvent
Positive
(Table 1 1)
43-44
m
m
m
m
m
LD
m
m
\
\
m
m
m
m
m
(7) Units of
Concentration
Blank - mg/mi
2 - fjg/m/
45
0
0
0
D
0
D
D
0
D
D
D
D
n
D
0
\
1
1
1 1
\
MO DA YR
Experiment Date
CTTD-C
Lab Y
Test Sample
Dose Level
zJ Stock Con-
centration
46-50
x
II 1
\
HI
u
u
LUJLU
T
\
A
\\
\\
u
u
III
III
III
II 1
rr
III
r
III
\\
u
® Ami Per
Plate ffjl)
51
54
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am
n
i
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-------
Section 6
6-6
March 1983
System ID
2-4
HERL IN VITRO SYSTEM
SAMPLE IDE NT IF 1C A TION FORM (INTERIM)
SAMPLE
Sample ID
Lab
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Sample Description
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Forms Completion
Initials
Figure 11. HERL IN VITRO System - sample identification form (interim)
-------
March 1983
R-1
References
References
American Public Health Association.
1975. Standard Methods for the
Examination of Water and Waste-
water. 14th Edition. M.C Rand et
al, eds APHA, Washington, D C.
Ames, BN 1981. Supplement to the
Methods Paper [Ames etal 1975],
Revised February 1981. (Available
upon written request from Doro-
thy Maron, Ames Lab, Department
of Biochemistry, University of
California, Berkeley, CA 94720).
Ames, B IN., J. McCann and E Yamasaki
1975 Methods for detecting
carcinogens and mutagens with
the Sa/moA7e//a/mammalian-mi-
crosome mutagemcity test Mutat.
Res. 31:347-364 (Also, as revised
April 1981)
Ashwood-Smith, M.J. 1979. Stability of
frozen microsome preparations for
use in the Ames' Salmonella
mutagemcity assay. Mutat. Res
69'1 99-200
Belser, W.L, S D. Shaffer, R.D. Bliss,
PM Hynds, L Yamamoto, J N.
Pitts and J.A. Winer. 1981. A
standardized procedure for quant-
ification of the Ames Salmonella/
mammalian-microsome muta-
gemcity test Env. Mutag. 3 123-
139.
Brusick, DJ, DW Matheson, D.R
Jaganath. 1980. Commercial
screening of environmental chem-
icals. In Chemical Mutagens, Vol
6. Eds. de Serres and Hollander.
pp. 81-108.
Chu, KC., KM. Patel, A.H. Lin, R.E.
Tarone, M S. Linhart and V.C.
Dunkel 1981. Evaluation of statis-
tical analyses and reproducibility
of microbial mutagenicity assay.
Mutat. Res 85-119-132.
Claxton, L.D. and H.M. Barnes 1981
The mutagemcity of diesel-ex-
haust particle extracts collected
under smog-chamber conditions
using the Salmonella typhimun-
um test system. Mutat. Res.
88(3)255-272.
de Serres, F.J. 1979. Problems associ-
ated with the application of short-
term tests for mutagemcity in
mass-screening programs Env.
Mutag 1:203-208.
de Serres, F.J. and M.D. Shelby. 1979.
Recommendations on data pro-
duction and analysis using the
Sa//r?o/7e//a/microsomal muta-
gemcity assay. Mutat. Res. 64:159-
165
Donnelly, K C. and K.W Brown. 1981
The development of laboratoryand
field studies to determine the fate
of mutagemc compounds from
land-applied hazardous wastes
In: Land Disposal Hazardous
Waste. EPA 600/9-81-002b pp
224-239.
Draper, N and H Smith 1966 Applied
Regression Analysis J Wiley and
Sons, Inc , New York, New York.
Dunkel, VC and K C. Chu. 1980.
Evaluation of methods for ana lysis
of microbial mutagemcity assays.
In. The Predictive Value of Short-
Term Screening Tests in Carcino-
gemcity G M. Williams, etal. eds.
Elsevier/North Holland Biomedi-
cal Press, pp 231-240.
Epler, J.L, B.R Clark, C-h. Ho, MR
Guerm and T K. Rao. 1978. Short-
term bioassay of complex mix-
tures Part II, Mutagenicitytestmg.
In: Application of Short-Term
Bioassays in the Fractionation and
Analysis of Complex Environmen-
tal Mixtures EPA-600/9-78-027.
Waters, et al. eds. pp. 269-289.
Federal Register. August 22, 1978. FR
37388
Freund, J.E. 1979. Modern Elementary
Statistics, Fifth Edition. Prentice-
Hall, Inc., Englewood Cliffs, New
Jersey
Graybill, F.A. 1976 Theory and Applica-
tion of the Linear Model. Duxbury
Press, Wadsworth Publ. Co., Inc.,
Belmont, California
King, L.L , M J. Kohan, A.C Austin, LD
Claxton and J. Lewtas. 1981.
Evaluation of the release of muta-
gens from diesel particles in the
presence of physiological fluids.
Env. Mutag. 3-109-122.
Linhart, M S., K Chu, U.N. Evans and
V.C Dunkel. 1980. In Vitro Inform-
ation System for collection and
analysis of experimental data. J.
Env Path. Tox. 4:1-21.
Loveday, K S. Standard Procedure:
Salmonella Microtoxicity Assay.
Unpublished protocol of Bioassay
Systems, Inc., Woburn, Massa-
chusetts 01801.
Margolin, B., N. Kaplan and E Zeiger.
1981. Statistical analysis of the
Ames Sa/mone//a/m\crosome test.
Proc Nat. Acad. Sci. 78'3779-
3783 p. 79
Maron, D., J. Katzenellenbogen and
B.N. Ames. 1981. Compatibility
of organic solvents with the
Salmonella/microsome test. Mu-
tat. Res. 88-343-350.
Myers, J.L 1979. Fundamentals of
Experimental Design. 3rd Edition.
Allyn and Bacon, Inc., Boston,
Massachusetts.
Nagao, M , T. Yahagi, Y. Semo, T
Sugimura and N. Ito 1977. Mu-
tagemcities of quinolme and its
derivatives Mutat. Res. 42.335-
342
Pellizan, E.D., LW Little, C Sparacmo,
TJ Hughes, L. Claxton and M.D
Waters. 1978. Integrating micro-
biological and chemical testing
into the screening of air samples
for potential mutagenicity In
Application of Short-Term Bioas-
says in the Fractionation and
Analysis of Complex Environmen-
tal Mixtures EPA-600/9-78-027.
Waters, et al., eds. pp. 331-351
Rosenkranz, H S., E C. McCoy, M.
Anders, W.T Speck and D. Bickers.
1978. The use of microbial assay
systems in the detection of envi-
ronmental mutagens in complex
mixtures In: Application of Short-
Term Bioassays in the Fractiona-
tion and Analysis of Complex En-
vironmental Mixtures EPA-600/
9-78-027. Waters, et al, eds pp
3-42
Salmeen, I and A.M Durism. 1981.
Some effects of bacterial popula-
tion on quantitation of Ames
5a//77OA7e//a-histidme reversion
mutagenesis assays. Mutat. Res
85-109-118
Sexton, N., L. Myers and T. Hughes.
1981 A plan to develop and
implement a quality assurance
program for the Ames/Salmonel-
la test. EPA-600/2-81 -054 pp 1-
27 and Appendices A-D. Available
through National Technical In-
formation Service, Springfield,
VA. 22161.
Sokal, R.R. and F.J. Rohlf. 1969.
Biometry. Freeman and Co., San
Francisco, California, pp. 370-
775.
Stead, A., V Hasselblad, J. Creason and
L Claxton. 1981. Modelling the
Ames test. Mutat. Res. 85:13-27.
Sugimura, T. and M. Nagao. 1980.
Modification of mutagenic activity.
/A7.-Chemical Mutagens, Vol. 6. F.J.
de Serres and A Hollaender, eds
Plenum Publ. Corp., New York,
New York. pp. 41-59.
-------
References R-2 March 1983
U S Environmental Protection Agency.
1978 Microbiological Methods for
Monitoring the Environment -
Water and Wastes EPA-600/8-
78-017 R BordnerandJ Winter,
eds EPA/EMSL, Cincinnati, Ohio
Waleh, N S , S J Rapport and K
Mortelmans Development of a
toxicity test to be coupled to the
Ames Salmonella assay and the
method of construction of the
required strains Mutat Res (In
press)
Yahagi, T., M Nagao, Y Semo, T
Matsushima, T Sugimura and M
Okada 1977 Mutagemcities of N-
nitrosammes on Salmonella Mu-
tat Res 48 121-130
Yamanaka, H , M Nagao, T Sugimura,
T. Furuya, S. Atsuko and T Mat-
sushima 1979 Mutagenicity of
pyrrolizidme alkaloids in the Sal-
mone//a/mammalian rmcrosome
test Mutat Res 68 211-216
Yoshikawa, K , T Nohmi, A Nagahara,
Y Inokawa, andM Ishidate 1980
Stability of S-9 during frozen
storage in Sa/mone/la/S-9 as-
says Mutat Res 74389-391
Zeiger, E , D A Pagano and I G C
Robertson 1981 A rapid and
simple scheme for confirmation of
Salmonella tester strain pheno-
type Envir Mutag 3 205-209
-------
March 1983
AI-1
Appendix I
Appendix I
Preparation of Media and Reagents
Stock Solutions
Vogel-Bonner Medium "E" (SOX)
(For Minimal Agar)
Ingredient
Magnesium Sulfate
(MgS04-7H20)
Citric acid
(monohydrate)
Dipotassium
hydrogen
phosphate
(K2HP04,
anhydrous)
Sodium ammonium
phosphate
(NaNH4HPO4-4H20)
Distilled water
Per Liter of
Solution
100g
1000g
500.0 g
175.0g
To final
volume of
1,000 ml
Preparation. Slowlydissolve, in order,
the first four ingredients in 600 ml of
warm (45°C) distilled water on a
magnetic stirring hot plate The K2HP04
dissolves slowly, therefore add no more
than 50 g at a time until completely
dissolved. Bring to 1,000 ml volume by
adding the distilled water. Pour 500 ml
of solution into each of two clean 1 -liter
glass bottles labelled "50 X VBME
Solution" and label with the date
prepared. Autoclave the two bottles,
loosely capped, for 15 minutes at
121°C. When bottles have cooled,
tighten caps and store in a cabinet at
room temperature Storage should not
exceed 2 months before use.
Calcium Chloride Solution (10 mM)
(For Minimal Agar)
Ingredient
Calcium chloride
(anhydrous)
Distilled water
Per Liter of
Solution
0 115 g
To final
volume of
1,000 ml
Preparation. Dissolve 0 115 g CaCI2 in
1,000 ml of distilled water. Transfer 500
ml to each of two clean 1 -liter glass bot-
tles labelled "CaCI2 Solution, 10mM"
Autoclave, cool and store at room tem-
perature. Stable as long as solution is
sterile.
Glucose Solution (20%)
(For Minimal Agar)
Ingredient
D-glucose
Distilled water
Per Liter of
Solution
200 g
To final
volume of
1,000 ml
Preparation Dissolve 200 g D-
glucose in about 600 ml of distilled
water in a 1-liter volumetric flask Add
distilled water to make up the total
volume to 1 liter Sterilize by autoclav-
mg or filtering through a 0 22 fjm
membrane filter Store in sterile glass
bottle(s) at room temperature Discard
if, on visual inspection, solution appears
turbid or a surface film has formed
Solution is stable on storage, as long as
sterility is maintained
L-Histidme HCI (0.1 M)
(For Master Plates)
Ingredient
L-Histidme-HCI
(M W. = 191 56)
Distilled water
Per 100 ml
of Solution
1 916g
To final
volume of
100 ml
Preparation. Prepare in a volumetric
flask, shake vigorously to dissolve
Sterilize by autoclaving or filtering
through a 0 22 /urn membrane filter
Store in properly labelled sterile glass
bottle, at 4°C for up to 1 month, wrap
bottle in aluminum foil to protect from
light during storage Discard solution if
it has yellowed
D-Biotin (0.5 mM)
(For Master Plates)
Ingredient
D-Biotm
Distilled water
Per 100 ml
of Solution
0.01 2 g
To final
volume of
100 ml
Preparation. Prepare in a volumetric
flask, shake vigorously, and warm if
necessary, to dissolve. Sterilize by
autoclaving or filtering through a 0.22
/um membrane filter. Store up to 3
months at room temperature m properly
labelled glass bottle(s). Heat gently
before use if the solution has precipi-
tated on storage.
L-Histidme HCI (0.5 mM)/D-Biotm
(0.5 mM) Solution
(For addition to top agar before use)
Per 250 ml
of Solution
0024g
Ingredient
L-Histidme-HCI
(M W = 191 56)
D-Biotm
(M W = 24431)
Distilled water
0030g
To final
volume of
250 ml
Preparation Sterilize by filtration
through a 0.22 pm membrane filter.
Store at 4°C in properly labelled glass
bottle(s) for up to 1 month. Wrap
bottle(s) m aluminum foil to protect from
light Discard solution if it hasyellowed.
Salt Solution
(For S-9 Mix)
Ingredient
1.65 M Potassium
chloride (KCI)
0 4 Magnesium
chloride
(MgCI2-6H20)
Distilled water
Per 500 ml
of Solution
61.42 g
4066 g
To final
volume of
500 ml
Preparation Autoclave to sterilize
Store at room temperature m properly
labelled glass bottle(s) Stable for long
periods if sterility is maintained
Sodium Phosphate Buffer
(0.2 M, pH 7.4)
(For S-9 Mix)
Ingredient
Sodium dihydrogen
phosphate
(NaH2P04-H20
Disodium hydrogen
phosphate
(Na2HP04-7H20)
Distilled water
Per 500 ml
of Solution
3.77 g
1951 g
To final
volume of
500 ml
Preparation. Dissolve the dibasic salt
in 300 ml H20, andthe monobasic salt in
-------
Appendix I
AI-2
March 1983
150 ml HaO. Add dibasic solution to
monobasic solution and adjust volume
to 500 ml. Adjust pH to 7.4 with HCI;
autoclave to sterilize. Store at room
temperature in properly labelled glass
bottle(s). Stable for long periods if
sterility is maintained.
Nicotinamide Adenine Dinucleotide
Phosphate (NADP) (0.1 M)
(For S-9 Mix)
Per 100 ml
Ingredient of Solution
Nutrient Agar
Composition:
Ingredient
Purified agar
(Oxoid # L28 or
equivalent)
Nutrient broth
(prepared as above)
Per Liter
of Medium
10g
To final
volume of
1,000 ml
Top Agar
Composition:
Ingredient
Purified agar
(Oxoid #L28
or equivalent)
Sodium
chloride (NaCI)
Distilled water
Per Liter
of Medium
6.0 g
50 g
To final
NADP (MW - 765.4)
Distilled water
7.65 g
To final
volume of
100ml
Preparation. Sterilize by filtration
through a 0.22 /jm membrane filter.
Store in labelled, 13 x 100 mm,
stoppered glass test tubes in 2 ml
volumes in freezer at -20°C. Note:
Check the molecular weight of each
lot, as it varies according to impurities
present.
Glucose-6-Phosphate (G-6-P) (1 M)
Preparation Heat in a boiling water
bath to dissolve completely. Autoclave
15 minutes at 121°C (15 Ib pressure).
Remove from autoclave and cool to
45°C in water bath. Dispense 25
ml/plate into 100 x15 mm petri dishes
using an automatic dish filler which has
been adjusted to level. Place plates in a
sealed container and store up to 1
month at room temperature. In general,
do not prepare more plates than will be
used during a 2-week period.
(For S-9 Mix)
Ingredient
G-6-P (MW = 282.2)
Distilled water
Per 10 ml
of Solution
2.82'g
To final
volume of
10ml
Minimal Agar
Composition:
Ingredient
Oxoid #L28 agar
(or equivalent)
Preparation. Sterilize by filtration
through a 0.22 /urn membrane filter.
Store in labelled, 13 x 100 mm,
Stoppered glass test tubes in 0.3 ml
volumes in freezer at -20°C.
Nutrient Broth
Composition:
50 X VBME
stock solution
20% glucose
stock solution
(10 mM CaCI2
stock solution1
Distilled water
Ingredient
Oxoid Media #2
Nutrient
Broth Powder
Distilled Water
Per Liter
of Medium
25 g
To final
volume of
1,000 ml
Per Liter
of Medium
15g
20ml
100 ml
1 ml)
To final
volume of
1,000 ml
Preparation. Dissolve nutrient broth
powder completely and dispense 10ml
aliquots of the broth into sterile 50 ml
screw-capped culture or centrifuge
tubes. Label appropriately (broth, vol-
ume, date of preparation). Autoclave for
15 minutes at 121°C (15 Ib pressure).
Store in dark up to 2 months at room
temperature.
Preparation of Minimal Agar Medium
(For 1 liter - approximately 40 plates).
Add 15gofOxoid#L28agarto880mlof
distilled water in a 2-liter flask. Adjustto
1 liter volume with distilled water, if
necessary. Autoclave for 35 minutes
using slow exhaust. When solution has
cooled slightly, add 20 ml of "VBME 50
X" salt solution, 100 ml of 20 percent
glucose, and 1 ml 10 mM CaCb
solution. Mix and place in 45°C water-
bath.
volume of
1 liter
Histidine/biotin solution (20 ml
added to each 200 ml portion of medium
before use).
Preparation. Heat in an autoclave
with flowing steam or in a boiling water
bath to completely dissolve the agar.
Dispense into screw-capped glass
bottles, 200 ml/bottle. Autoclave 15
minutes at 121°C. Store in refrigerator.
Before use, melt top agar in autoclave or
microwave oven, cool and maintain at
45°C in a waterbath. Add 20 ml of
sterile histidme/biotin solution (pre-
warmed to 45°C) to each 200 ml portion
of top agar, and swirl contents thorough-
ly to obtain a uniform mixture. Dispense
the top agar into 100 mm disposable,
sterile test tubes (2 ml/tube); cap the
tubes and place in a 45°C heating block
to equilibrate (about 5 -10 minutes). Top
agar should only be melted once, as
repeated meltings may cause crystalsto
form when top agar is poured on plates
and make colony counting more difficult.
Strain Function Test Materials
Ampicillin Solution (7 mg/ml)
Ingredient
Ampicillin
trihydrate
Sodium hydroxide
(0.02 N)
Per 100ml
of Solution
0.7 g
To final
volume of
100ml
Preparation. Sterilize by filtration
through a 0.22 ,um membrane filter.
Store up to 1 month in glass bottle at
4°C or up to 6 months frozen.
Crystal Violet Solution (0.1 %)
'Optional; use if needed to control filamentous
growth of Salmonella cells
Ingredient
Crystal violet
(Gentian violet,
methyl-rosaniline
chloride)
Ethanol
solution (70%)
Per 100 ml
of Solution
0.1 g
100 ml
-------
March 1983
AI-3
Appendix I
Preparation. Bring up to 100 ml in a
volumetric flask. Store in glass bottle at
room temperature Stable for prolonged
periods if stored out of direct light.
Preparation of Crystal Violet discs (10
/j\ of 01% Crystal Violet per disc)
Prepare 100 ml of crystal violet in 70%
alcohol. Using a sharp (new), clean
paper punch and Whatman filter paper
#1 or #2, punch out individual discs (or
multiple discs and separate the layered
discs) Arrange discs on a large petri
dish (single discs, no overlapping). Add
10 /ul of the crystal violet solution to
each disc, allow to dry and autoclave at
121°C for 15 minutes
Rat Liver Enzyme Mix (S-9
Mix)1
Composition.
Ingredient
Rat Liver S-9
Arochlor-1254-
induced)
Salt Solution
Glucose-6-
Phosphate
NADP
Sodium
Phosphate
Per 50
Low S-9 Mix
20 ml
1 0 ml
025 ml
20 ml
250 ml
ml of Mix
High S-9 Mix
100 ml
1 0 ml
025 ml
20 ml
250 ml
Buffer
Sterile
Distilled
Water
19 75 ml
11 75 ml
Preparation of S-9 Mix. On the day of
the test, combine the ingredients
indicated above (under Composition),
using aseptic technique, in a sterile
graduated cylinder that has been placed
in an icewater bath. Freshly prepared S-
9 Mix can be kept on ice several hours
before running the test. Alternatively, it
is convenient, and acceptable, to mix all
ingredients of the S-9 Mix (except the
rat liver homogenate) in large batches,
dispense into convenient aliquots, and
store at -20°C so that only the microso-
mal preparation (S-9) need be added on
the day of the test.
It is recommended that laboratories
purchase the microsomal preparation
(S-9) from a commercial biological
supply company or private laboratory
performing mutagen research. This
material is available from Litton Bionet-
ics, 5516 Nicholson Lane, Kensington,
MD 20795; or from the Meloy Labora-
tories, c/o Dr. Carol Richardson, 6715
'Other rat tissues and tissues of other mammals
may be used as a source of S-9 preparation Also
other chemicals (e g , phenobarbital) may be used
to induce the mammal
Electronic Drive, Springfield, VA 22151.
Quick-freeze with dry ice immediately
after preparation, and store at -80°C in
2-ml plastic vials. The frozen S-9 has
been shown to retain full activity for at
least 1 month at -80°C (Yoshikawa et
al., 1980) and up to several years for
selected enzymes, if maintained at
temperatures below-130°C(Ashwood-
Smith, 1979).
S-9 preparations vary in protein
content It is recommended that new S-
9 batches be tested with standard
mutagens and strains of known activity
-------
March 1983
AIM
Appendix II
Appendix II
Facilities, Equipment and Supplies
General Requirements
Microbial assays for mutagenicity
should be performed in a stationary
laboratory. Generally, support equip-
ment used in a typical water micro-
biology laboratory is suitable for use in
the Ames test. This equipment includes
good quality autoclaves, colony counters,
sterilization ovens, incubators, water-
baths, water distillation systems, dish-
washers, refrigerators, freezers, balances
(analytical and top loading) and the
usual pipettes and glassware for pre-
paration of media and reagents. These
items are described in the EPA's
microbiological methods manual (EPA
1978) and Standard Methods (APHA
1975). Facility requirements are also
detailed in Brusick et al., (1980).
Autoclaves
Unit(s) selected must maintain stand-
ard autoclave conditions and be capable
of accepting large volume reagent
bottles and flasks. It is recommended
that an exhaust hood be installed over
the autoclave to evacuate volatile
chemicals, heat, and moisture from the
laboratory area.
Incubators
Unit(s) should employ forced-air (or a
mechanism of equivalent effectiveness)
to minimize temperature differences
throughout the chamber and must be
able to maintain 37° + 0.5°C. Incuba-
tor(s) should be directly or indirectly
(e g., through an exhaust hood with
appropriate filtration) exhaustible; the
use of a microswitch on doors to
activate an exhaust fan is recommended.
It is recommended that a temperature
recorder be attached to each incubator
to verify temperature stability over
prolonged periods of operation. Units
which provide positive humidity control
are useful to minimize dessication of, or
condensation on the plates. Incubators
should not be overloaded in use;
overloading can result in the establish-
ment of temperature gradients (Belser,
et al 1981) which affect growth rates
Refrigerators and Freezers
Units should be lockable If used for
solvent or organic chemical storage,
they must be explosion proof Tempera-
ture recorders should be provided to
monitor tr*e performance of the re-
frigerator and freezer units A visual or
audible alarm should be provided to
indicate power outages and significant
temperature deviations. An auxiliary
power supply for freezer units can
protect deep-frozen culture materials
from the damaging effects of power
interruption, and is strongly recom-
mended.
Laminar-Flow Safety Cabinets
Must be an OSHA-approved type,
designed to protect both the personnel
and work area Intake air must be
filtered with a high efficiency particu-
late air (HEPA) filter with not greater
than 30 percent recirculation and 100
percent of intake air must be exhausted
Exhaust air should flow through a HEPA
filter and an appropriate trap (e g ,
activated charcoal) for organic chemicals
A general list of equipment, supplies,
media and reagents (requirements for
20-30 samples) necessary for environ-
mental mutagenesis testing is pre-
sented below For many items, equi-
valent products are available from other
sources Listing does not constitute a
specific endorsement
Major Equipment
Essential Items
Item
• Membrane filtration system,
for sterilization of heat-labile
materials
• Bacterial colony counter,
darkfield, with electronic
register
• Automated Colony Counter
Laminar-flow safety cabinet
Dri-block heater, w/accessories,
to hold 13x100 mm test tubes
Incubator shaker (e.g..
Controlled Environment Incubator
Shaker)
or
Shaker waterbath, for culturmg
test strains (37°C)
Waterbath, for tempering media
No Needed Suggested Source
2 Nuclepore Corp.;
Millipore Corp
1 Scientific Products, Inc.
1 New Brunswick Scientific
Co., Inc
1 Contamination Controls,
Inc
3 Scientific Products, Inc
1 New Brunswick Scientific
Co , Inc.
1 New Brunswick Scientific
Co., Inc.
1 Scientific Products, Inc.
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Appendix II
AII-2
March 1983
Item
• Utra-freezer (-80°C) (e.g.,
So-Low PR120E, 5 cu. ft.
capacity)
• Incubator (e.g., Forma
Scientific model 3028 C02
incubator)
• Refrigerator-compact, explosion-
proof, lockable, for storage of
standard mutagens
• Spectrophotometer, Turbidimeter,
or Particle Counter (Coulter-
type). Determination/adjustment
for bacterial culture density.*
• Micro-Volume Pipettes (1 /ul -
1000/1/1 volumes)
Optional Items (recommended)
• Petri dish filler/stacker
• Mechanical Pipetting Device
• Bag Sealing Device (for sealing
petri dishes in plastic bags)
Expendable Equipment and Supplies
Item
Sterile Disposable Tips for
Micro-Volume Pipettes
• Scalable Plastic Bags, for
sealing petri dishes
• Surgical Gloves, latex,
disposable
• Petri Dishes 15x100 mm, gamma-
irradiation sterilized,
disposable; or #1028 Muta-assay®
cold-sterilized plates
• Test Tubes, 13x100 mm,
disposable
• Volumetric Flasks, 10, 25, 50,
100, 500 and 1,000 ml
• Membrane Filters (pore size
<022/um)
• Glass-fiber Filters (pore size
<08//m)
• Laboratory Tape, white, and
heavy-base dispenser
• Caps, for 13x100 mm test tubes,
color coded
No Needed Suggested Source
1 So-Low Environmental
Equipment Co.
1 Forma Scientific
Scientific Products, Inc.
1 (Choice) Beckman Co.;
Coulter Electronics;
Perkm-Elmer Co
Cole-Parmer Instrument Co.
1 New Brunswick Scientific
Co , Inc
2-3 Bellco Glass, Inc.
1 Sears-Roebuck and Co.
No. Needed Suggested Source
1 Box of
1,000 for
each Volume
Pipette
4-5 cases
of 500
1 -2 cases
of 500
1-5 cases
of 500
4-5 cases
of 1,000
6 each
Cole-Parmer Instrument Co.
Sears-Roebuck and Co.
Pharma Seal Laboratories
Falcon Plastics, Inc.
Bellco Glass, Inc.
Cole-Parmer Instrument Co
Nuclepore Corp.,
Milhpore Corp
Whatman Corp.
Dispenser and
1 doz. rolls
1 case of
1,000ea.
Bellco Glass, Inc.
"NOTE Whichever method is selected, standard curves must be prepared (and
periodically reconfirmed) with viable cell counts determined by the
dilution and plate method.
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March 1983
AII-3
Appendix II
Item
• Test Tubes, 20x125 mm, screw-
capped
• Reagent Bottles, screwcapped
for storage of media and
reagents, 1 00, 200, 300, 400,
500 ml volume
• Pipettes, TD, disposable,
sterile, glass, 1 ml, 2 ml, 5 ml
and 10 ml volumes
• Erlenmeyer Flasks, 50, 125, 250,
500, 1,000, 2,000 ml volumes
with Morten culture tube
closures or equivalent
No. Needed Suggested Source
1 case of Corning Glass, Inc.
500
1 doz. ea Bellco Glass, Inc.
1 case of Scientific Products Co.
each volume
1 doz ea Kimball Glass Co.
Reagents (Reagent grade unless otherwise indicated)
Item
• Ampicillm, diagnostic reagent
(special preparation, high purity)
• D-Biotm (M.W 24431)
• Calcium Chloride (CaCb)
• Citric Acid
• Crystal-violet
• Glucose
• Methylene Chloride (Dichloromethane)
distilled in glass
• Dimethyl Sulfoxide (DMSO)
spectroptiotometric quality
• Dipotassium Hydrogen Phosphate
(K2HP04)
• Disodium Hydrogen Phosphate
(Na2HPO4-7H20)
• Glucose-6-Phosphate (M W. 282.1)
anhydrous
• Hydrochloric Acid (HCI)
• L-Histidme (M.W. 1927)
anhydrous
• Magnesium Chloride
(MgCI2-6H20)
• Magnesium Sulfate
(MgS04-7H20)
• Nicotmamide Adenme Dmucleotide
Phosphate (M W. 765.4) anhydrous
• Potassium Chloride (KCI)
• Sodium Ammonium Phosphate
(NaNH4P04'4H2O)
• Sodium Chloride (NaCI)
• Sodium Dihydrogen Phosphate
(NaH2P04-H2O)
Amount Suggested Source
5 g Bristol Laboratories
5g Eastman Kodak Co (# 14635)
1 Ib. J.T. Baker Chemical Co.
500 g J.T. Baker Chemical Co.
10 g Difco Laboratories, Inc.
500 g Difco Laboratories, Inc.
5 gal Burdick and Jackson
Laboratories
1 gal Mathesoh, Coleman and
Bell (# MX1454)
1 Ib Mallmckrodt Chemical
Works
1 Ib. Mallmckrodt Chemical
Works
10 g Sigma Chemical Co
(# G7879)
9 Ib J.T Baker Chemical Co.
10 g Sigma Chemical Co.
(# H8125)
1 Ib. J.T. Baker Chemical Co.
1 Ib. J.T. Baker Chemical Go.
10 g Sigma Chemical Co.
(# N0505)
1 Ib. J.T Baker Chemical Co.
1 Ib. J.T. Baker Chemical Co.
1 Ib. J.T. Baker Chemical Co.
1 Ib Fisher Scientific Co.
-------
Appendix II
AII-4
March 1983
Item
• Sodium Hydroxide (NaOH)
• Sodium Sulfate
(Na2S04) anhydrous
Prepared Media
Item
• Ampicillm, "Dispens-o-Discs,"
lO/jg
• Purified Agar (Oxoid # L28
or equivalent)
• Nutrient Broth Powder (Oxoid #2
or equivalent)
• Rat Liver Enzymes (Induced
with Aroclor 1254) (S-9 preparation)
Amount
1 Ib
5 Ib.
Amount
250
5 Ib.
5 Ib
Suggested Source
J.T Baker Chemical Co
J T Baker Chemical Co.
Suggested Source
Difco Laboratories
(# 6363)
K C Biological Inc
K.C. Biological Inc.
(# CM67)
Litton Bionetics; AMC Cancer
Research Center, Meloy
Laboratories, Inc
Additional sources of equipment and materials for Sa/mo«e//a/microsomal mutagemcity tests are suggested in Ames
(1981)
U S GOVERNMENT PRINTING OFFICE 1983 - 659-095/1918
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