UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF ENFORCEMENT
EPA-330/9-80-002
MICROBIAL BIOASSAY FOR TOXIC
AND HAZARDOUS MATERIALS
(AMES TEST FOR MUTAGENICITY)
August 1980
NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Denver, Colorado
&EPA
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UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF ENFORCEMENT
EPA-330/9-80-002
NEIC
MICROBIAL BIOASSAY FOR TOXIC
AND HAZARDOUS MATERIALS
(AMES TEST FOR MUTAGENICITY)
September 1980
William J. Stang
NATIONAL ENFORCEMENT INVESTIGATIONS CENTER
Denver, Colorado
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FOREWORD
Environmental measurements are required to determine the quality of
ambient waters and the character of waste effluents. Determination of the
toxicity of hazardous wastes has a high priority in the EPA water pollution
and hazardous waste control programs. This procedure is a modification of
the Ames test; it fills an urgent current need for a rapid screening meth-
od for determining mutagenicity and potential carcinogenicity. Federal
agencies, states, municipalities, universities, private laboratories and in-
dustry should find this manual of assistance in monitoring toxicity in the
environment.
Thomas P. Gallagher
Director, NEIC
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PREFACE
This manual on the Ames Mutagenicity Test was developed for use by the
NEIC. The document is provided as a uniform laboratory developmental meth-
od for rapid screening of complex environmental mixtures for mutagens and
potential carcinogens. Publication of this procedure acknowledges the po-
tential of the Ames test to determine some of the toxic effects of waste
materials.
Publication also culminates a series of historical events that make
use of the bioassay possible and refinement imperative:
• The use of epidemiological studies, long-term bioassays, and
short-term i_n vitro tests, such as the Ames test, were first
authorized in the Federal Water Pollution Control Act, Amend-
ments of 1972 and 1977. The Drinking Water Act of 1974, and
the Toxic Substances Control Act of 1976 to determine mutagen-
ic effects of toxic and hazardous materials.
The definition of toxic pollutants in the Clean Water Act of
1977 includes substances which cause mutations and cancer.
• In 1979 the Interagency Regulatory Liaison Group and the Regu-
latory Council encouraged the development of short-term screening
tests using microorganisims to detect carcinogenisis.
• In 1978 and 1979, the EPA Biological Advisory Committee held
discussions on the expanded use of bioassay tests on point
source and non-point source discharges and on ambient monitoring.
These short-term tests include the Ames test for mutagenicity.
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® An EPA Workshop "on the Ames Mutagenicity Test co-sponsored by
the National Enforcement Investigations Center and the. Environ-
mental Monitoring and Support Laboratory-Cincinnati was held
at the Federal Center, Denver, Colorado, December 5 and 6, 1979.
Attendees included representatives of the EPA Regional Surveil-
lance and Analyses Divisions, Air and Water Research Laborato-
ries, and interested program offices. The Workshop determined
the status of the Ames Mutagenicy Test in the Agency, explored
anticipated use of the test in response to program needs, re-
viewed a draft of the test procedure used by NEIC to analyze
complex waste mixtures, and considered standardization and
quality assurance of the methodology.
9 Following this workshop, the EPA Microbiological Methods Com-
mittee met in Denver and recommended that the NEIC draft pro-
cedure be modified based on attendees' suggestions and pub-
lished as a developmental method.
The manual is recommended for use in enforcement, monitoring, and
research. However, it is not intended to inhibit or prevent research
and development.
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CONTENTS
1 INTRODUCTION . . .
2 SUMMARY OF METHOD
3 SCOPE AND APPLICATION 2
ADVANTAGES 2
DISADVANTAGES 2
4 FACILITIES, EQUIPMENT AND SUPPLIES 3
GENERAL REQUIREMENTS 3
MAJOR EQUIPMENT 3
EXPENDABLE EQUIPMENT AND SUPPLIES 4
REAGENTS 5
PREPARED MEDIA 7
5 PREPARATION OF MEDIA AND REAGENTS 7
MINIMAL AGAR 7
VOGEL-BONNER MEDIUM "E" SOLUTION 8
NUTRIENT AGAR 8
NUTRIENT BROTH 8
TOP AGAR 9
L-HISTIDINE-HC1 (0.1 M) 9
D-BIOTIN (0.5 mM) 9
L-HISTIDINE-HC1 (0.5 mM)/D-BIOTIN (0.5 mM) SOLUTION. 10
SALT SOLUTION 10
SODIUM PHOSPHATE BUFFER 10
NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (0.1 M). 11
GLUCOSE-6-PHOSPHATE (1 M) 11
RAT LIVER ENZYME MIX 11
AMPICILLIN SOLUTION 11
6 SALMONELLA TEST STRAINS 12
TEST SYSTEM 12
TEST STRAINS 12
REGENERATION AND STORAGE OF TEST STRAINS 13
7 ANALYTICAL PROCEDURES 14
PREPARATION OF MASTER PLATES 14
TEST STRAIN FUNCTION 15
Histidine Requirement 15
Crystal-Violet/Ampicillin Sensitivity 16
UV Sensitivity 16
Spontaneous Reversion 19
COLLECTION AND PREPARATION OF SAMPLES 23
Sample Collection 23
Extraction and Concentration of Samples ...... 23
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CONTENTS (Cont.)
7 ANALYTICAL PROCEDURES (Cont.)
MUTAGENESIS ASSAYS 29
Plate Incorporation Procedure-Primary Screen ... 29
Plate Incorporation Procedure-Dose Response. ... 33
Spot Test 34
Preincubation Assay 35
8 QUALITY CONTROL PROCEDURES 38
VIABILITY COUNTS 38
AUTOMATED COLONY COUNTS 43
DIAGNOSTIC MUTAGENS 43
DISTILLED WATER BLANK 45
STERILITY CONTROLS 45
9 INTERPRETING AND REPORTING RESULTS 46
10 SAFETY CONSIDERATIONS 49
SAFETY PROCEDURES 51
WASTE DISPOSAL 53
REFERENCES 54
Tables
Genotype of Five Salmonella Strains Used for General
Screening in Mutagen Testing 13
Characteristic Reactions of Five Standard Salmonella
Test Strains to Diagnostic Mutagens 44
Data Summary, Mutagenic Activity of Textile
Manufacturing Company Effluent 48
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CONTENTS (Cont.)
Figures
1 Test Strain TA 98, Crystal-violet sensitivity (rfa-
character) and ampicillin resistance (R-factor) ... 17
2 Test Strain TA 1537, Crystal-violet sensitivity (rfa-
character) and ampicillin sensitivity (does not
contain R-factor) 17
3 Test Strain TA 98, Growth inhibition after exposure
to UV light (uvrB-deletion) 18
4 Filling 13 x 100 mm culture tubes with 2 ml top agar
(heating block set at 45° C) 18
5 Mixing top agar by rotating tube between palms .... 20
6 Pouring top agar onto minimal agar plate 20
7 Sealing agar plate in plastic bag : . 22
8 Example of a 1-gal, amber-glass sample bottle equipped
with teflon-lined cap 22
9 Sample Data Form 24
10 Extraction Procedure Data Form 27
11 Extraction Procedure for Environmental Water Samples . 28
12 Agar Plate Incorporation Procedure 30
13 Counting revertant colonies using digital, Quebec
Colony Counter 32
14 Dose-response effect of mutagenic material on Test
Strain TA 1537 32
15 Spot Test 36
16 Quality Control Data Form-Equipment 39
17 Quality Control Data Form-Media 40
18 Quality Control Data Form-Reagents 41
19 Quality Control Data Form-Spontaneous Revertants
and Diagnostic Mutagens 42
20 Dose Response Curve for Salmonella Test Strain TA 1537. 47
21 Ames Assay Data Form 50
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ACKNOWLEDGMENTS
The author gratefully acknowledges the efforts of the staff of NEIC,
EMSL-Cincinnati, HERL-RTP and certain EPA Regional offices for their sup-
port, editorial comments and reviews during the preparation of this docu-
ment.
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1. INTRODUCTION
There is increasing evidence that environmental mutagens are a major
cause of cancer and genetic birth defects, and contribute to aging and
heart disease. Humans are constantly exposed to mutagenic chemical contam-
inants in air, water, and food. Drinking water, municipal and industrial
discharges, hazardous waste disposal sites and receiving waters must be
monitored for mutagenic pollutants by systematic testing procedures and
corrective actions taken when such substances are found. However, monitor-
ing that uses complex biochemical or long-term animal tests is prohibitive-
ly expensive in time, manpower, and dollars.
Short-term tests to screen environmental wastes for mutagenicity are
needed. One rapid screening test, the Salmonel1 a/mammalian-microsome muta-
genicity assay (commonly known as the Ames test) has been investigated more
thoroughly than others and proven reliable for identification of mutagenic
and potentially carcinogenic substances.
Modified versions of the Ames test have been developed and used suc-
cessfully with supporting chemical data by the EPA's NEIC in Denver, the
Health Effects Research Laboratory, Research Triangle Park, and several
Regions for air and wastewater characterization and health effects re-
search.
Other EPA Regional and research laboratories and program offices with
responsibility for toxic substances and hazardous materials have expressed
an immediate need for the application of the Ames test in their activities.
This manual was prepared to meet that need.
2. SUMMARY OF METHOD
The test system developed by Ames1 has been widely used as a rapid
screening procedure for the determination of mutagenic and potential car-
cinogenic hazards of complex environmental effluents and commercial prod-
ucts.
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The assay uses five standard strains of histidine-requiring Salmonella
mutants that revert after interaction with mutagens to the wild-type state
(histidine-independence). The rate of reversion indicates the degree of
mutagenicity. Relevancy of this i_n vitro bacterial test to carcinogenesis
is increased by adding rat-liver homogenate to the test system. The
rat-liver contains enzymes which perform several metabolic conversions
similar to those of mammalian organs. Test sensitivity is enhanced by
additional mutations in the test strains and by extrachromosomal resistance
transfer factors in two of the strains.
3. SCOPE AND APPLICATION
The Ames test has been used successfully as a screening procedure to
predict the mutagenic and potential carcinogenic hazards from pure compounds,
commercial products and complex discharges2'3'4'5.
3.1 ADVANTAGES
• The Ames test is simple, rapid, and relatively inexpensive.
• Positive test results reportedly show a high correlation
(85-93%) with substances that produce tumors in laboratory
animals1'5'6'7.
« The test system detects relatively low concentrations of
mutagens.
• The system has been tested and validated in several inde-
pendent studies5'6'7.
3.2 DISADVANTAGES
• Toxicity of complex mixtures may prevent measurement of muta-
genic effects.
9 Volatile organic compounds may be lost before measurement
can be determined.
• Some non-jnutagenic carcinogens, i.e. alkyl-halides, anti-
biotics, hormones, are not detectable in the test.
• The test does not consider pharmacokinetics involved in
mutagenesis and carcinogenesis.
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4. FACILITIES, EQUIPMENT, AND SUPPLIES
4.1 GENERAL REQUIREMENTS
Microbial assays for mutagenicity should be performed in a station-
ary laboratory. Generally, support equipment used in a typical water mi-
crobiology laboratory is suitable for use in the Ames test. This equip-
ment includes good quality autoclaves, colony counters, sterilization
ovens, incubators, waterbaths, water distillation systems, dishwashers,
refrigerators, freezers, balances (analytical and top loading) and the
usual pipettes and glassware for preparation of media and reagents.
These items are described in the EPA Microbiological Methods Manual2
and Standard Methods3.
A general list of equipment, supplies, media and reagents (re-
quirements for 20-30 samples) necessary for environmental mutagen-
esis testing is presented below:
4.2 MAJOR EQUIPMENT
ITEM
a. Membrane filtration system,
for sterilization of heat-
labile materials
b. Quebec Colony Counter,
darkfield, with elec-
tronic register
c. Automated Colony Counter,
(optional)
d. Petri dish Filler/Stacker,
(optional)
e. Laminar-Flow Biological
Safety and Fume Hood,
equipped with total
exhaust system
N0_.
2
1
1
1
SUGGESTED SOURCE"'
Millipore Corp.
Scientific Products, Inc.
New Brunswick Scientific
Co., Inc.
New Brunswick Scientific
Co., Inc.
Contamination Controls,
Inc.
* The mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
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f. "Shaker-in-the-Round", for 2
extraction procedure
g. Dri-block Heater, to hold 2-3
13 x 100 mm test tubes
with accessories
h. Evaporator, Rotovapor R/A, 2
with accessories
i. Shaker Waterbath, 1
for culturing test strains
j. Waterbath, for tempering 1
media
k. Ultra-freezer (-80°C), 1
12.5 cu ft
1. Explosion-proof Refrig- 1
erator, 3.5 cu ft, for
storage of standard diag-
nostic mutagens, equipped
with security lock
m. Mechanical Pipetting Device 2-3
n. Bag Sealing Device, for 1
sealing petri dishes in
plastic bags
Scientific Products,
Inc.
Scientific Products,
Inc.
Van Waters and Rogers
Scientific Co., Inc.
New Brunswick Scientific
Co., Inc.
Scientific Products,
Inc.
Revco Scientific Co.
Scientific Products,
Inc.
Bellco Glass, Inc.
Sears-Roebuck & Co.
4.3 EXPENDABLE EQUIPMENT AND SUPPLIES
ITEM
a. Micro-volume Pipettes
1 ul-1000 ul volume,
with sterile disposable
tips
b. Scalable Plastic Bags,
for sealing petri dishes
c. Surgical Gloves, latex,
disposable
d. Petri Dishes 15 x 100 mm,
gamma-i rradi ati on
sterilized, disposable
e. Test Tubes, 13 x 100 mm,
disposable
NO.
2-3
4-5 cases
of 500
1-2 cases
of 500
1-5 cases
of 500
4-5 cases
of 1000
SUGGESTED SOURCE
Cole-Parmer Instrument
Co.
Sears-Roebuck & Co.
Pharma Seal Laborato-
ries
Falcon Plastics,
Inc.
Bellco Glass, Inc.
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f. Caps, for 13 x 100 mm test 1 case of
tubes, color coded 1000 ea
g. Test Tubes, 20 x 125 mm, 1 case
screw-capped of 500
h. Reagent Bottles, screw- 1 doz. ea
capped for storage of
media and reagents, 100,
200, 300, 400, 500 ml volume
i. Pipettes, TD, disposable, 1 case of
sterile, glass, 1 ml, 2 ml, 1000 ea
5 ml and 10 ml volumes
j. Separatory funnels, 2000 ml 6-8
volume for extraction proce-
dure
k. Round-bottom flasks, 50, 100, 1 doz. ea
1000-ml volumes, to fit
evaporator rotovapor devices
1. Erlenmeyer Flasks, 250, 500, 1 doz. ea
1000, 2000 ml volumes
m. Sample Bottles, amber-glass, 3 doz.
1-gal volume
n. Kuderna-Danish Evaporative 5-10 ea
Concentrator, (flasks, 3-ball
Schneider columns, graduated
concentrator tubes)
Bellco Glass, Inc.
Corning Glass, Inc.
Bellco Glass Inc.
Scientific Products
Co.
Kimball Glass Co.
Kimball Glass Co.
Kimball Glass Co.
Kimball Glass Co.
Kontes Co.
4.4 REAGENTS
ITEM AMOUNT
a. Ampicillin, diagnostic 5g
reagent (special pre-
paration, high purity)
b. D-Biotin (M.W. 244.31) 5 g
c. Citric Acid 500 g
d. Crystal-violet 10 g
e. Dextrose 500 g
SUGGESTED SOURCE
Bristol Laboratories
Eastman Kodak Co.
(# 14635)
J. T. Baker Chemical Co.
Difco Laboratories, Inc.
Difco Laboratories, Inc.
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f. Methylene Chloride 5 gal
(Dichloromethane)
distilled in glass
g. Dimethyl Sulfoxide (DMSO) 1 gal
Spectrophotometric quality
h. Dipotassium Hydrogen 1 Ib
Phosphate (K2HP04)
i. Disodium Hydrogen 1 Ib
Phosphate (Na2HP04-7H20)
j. Glucose-6-Phosphate 10 g
(M.W. 282.1) anhydrous
k. Hydrochloric Acid (HC1) 9 Ib
1. L-Histidine (M.W. 192.7) 10 g
anhydrous
m. Magnesium Chloride (MgCl2-6H20) 1 Ib
n. Magnesium Sulfate (MgS04-7H20) 1 Ib
o. Nicotinamide Adenine 10 g
Dinucleotide Phosphate
(M.W. 765.4) anhydrous
p. Potassium Chloride (KC1) 1 Ib
q. Sodium Ammonium Phosphate 1 Ib
(NaNH4P04-H20)
r. Sodium Chloride (NaCl) 1 Ib
s. Sodium Dihydrogen Phosphate 1 Ib
(NaH2P04-H20)
t. Sodium Hydroxide (NaOH) 1 Ib
u. Sodium Sulfate (Na2S04) 5 Ib
anhydrous
Burdick & Jackson
Laboratories
Matheson, Coleman &
Bell (# MX1454)
Mallinckrodt Chemical
Works
Mallinckrodt Chemical
Works
Sigma Chemical Co.
(# G7879)
J. T. Baker Chemical Co.
Sigma Chemical Co.
(# H8125)
J. T. Baker Chemical Co.
J. T. Baker Chemical Co.
Sigma Chemical Co.
(#N0505)
J. T. Baker Chemical Co.
J. T. Baker Chemical Co.
J. T. Baker Chemical Co.
Fisher Scientific Co.
J. T. Baker Chemical Co.
J. T. Baker Chemical Co.
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4.5 PREPARED MEDIA
ITEM AMOUNT SUGGESTED SOURCE
a. Ampicillin, "Dispens-o- 250 Difco Laboratories
Discs", 10 |jg (#6363)
b. Purified Agar (Oxoid, #L28) 5 Ib K. C. Biological Inc.
c. Nutrient Broth (Oxoid, #2) 5 Ib K. C. Biological Inc.
(# CM67)
d. Rat Liver Homogenate
Rat Liver Homogenate (RLH) is the supernatant from 9000 x g minced
rat-liver, using animals induced with Aroclor-1254 five days before sacri-
fice. It is recommended that laboratories purchase the prepared material
from commercial biological supply companies or from private laboratories
performing mutagen research.* Quick-freeze with dry ice immediately after
preparation, and store at -80°C in 2 ml, plastic vials.
5. PREPARATION OF MEDIA AND REAGENTS
5.1 MINIMAL AGAR
Composition
Solution A: Solution B:
Purified Agar (Oxoid, #L28) 5.0g Purified Agar 5.0 g
Distilled Water 500 ml Dextrose 20.0 g
Distilled Water 480 ml
Preparation
Dissolve the contents of each flask by heating in a boiling water
bath. Autoclave 15 minutes at 121°C (15 Ib pressure). Remove from the
autoclave and cool to 45°C in a water bath. Add 20 ml of Vogel-Bonner
Medium "E" (50X) solution (prewarmed to 45°C) to Solution B. Combine the
two solutions and dispense 30 ml/piate into 100 x 15 mm petri dishes.
* Rat Liver homogenate is available from the AMC Cancer Research Center and
Hospital c/o Dr. Elias Balbinder, 6401 W. Colfax Ave., Lakewood, CO 80214;
or from Litton Bionetics, 5516 Nicholson Lane, Kensington, MD 20795.
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5.2 VOGEL-BONNER MEDIUM "E" SOLUTION (SOX)
Composition
MgS04-7H20 10.0 g
Citric Acid 100.0 g
Dipotassium Hydrogen 500.0 g
Phosphate (K2HP04, anhydrous)
Sodium Ammonium Phosphate 175.0 g
(NaNH4HP04-4H20)
Distilled Water 670 ml
Preparation
Slowly dissolve the ingredients. Solution will require warming.
Final volume is 1 liter. Autoclave 15 minutes at 121°C (15 Ib pressure),
Store in refrigerator.
5.3 NUTRIENT AGAR
Composition
Purified Agar (Oxoid, #L28) 10.0 g
Lab-lemco powder 10.0 g
Peptone 10.0 g
Sodium Chloride (NaCl) 5.0 g
Distilled Water 1000 ml
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 30 ml/pi ate into 100 x 15 mm petri
dishes.
5.4 NUTRIENT BROTH (Oxoid #2)
Composition
Lab-lemco Powder 10.0 g
Peptone 10.0 g
Sodium Chloride (NaCl) 5.0 g
Distilled Water 1000 ml
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Preparation
Dissolve contents completely. Dispense into 20 x 125 mm screw-
capped tubes, 10 ml/tube. Autoclave 15 minutes at 121°C (15 Ib pres-
sure).
5.5 TOP AGAR
Composition
Purified Agar (Oxoid #L28) 1.2 g
Sodium Chloride (NaCl) 1.0 g
Distilled Water 200 ml
Preparation
Heat in a boiling water bath to dissolve completely. Dispense
into screw-capped bottles, 200 ml/bottle. Autoclave 15 minutes at 121°C.
Store in refrigerator. Before use, melt top agar in autoclave or micro-
wave oven, cool to 45°C and add 20 ml histidine/biotin solution/200 ml
(prewarmed to 45°C).
5.6 L-HISTIDINE-HC1 (0.1 M)
Composition
L-Histidine-HCl 1.927 g
Distilled Water 100 ml
Preparation
Sterilize by filtration through a 0.22 pro membrane filter. Store
in refrigerator.
5.7 D-BIOTIN (0.5 mM)
Composition
D-Biotin 0.0122 g
Distilled Water 100 ml
Preparation
Sterilize by filtration through a 0.22 pm membrane filter.
Store in refrigerator.
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10
5.8 L-HISTIDINE-HC1 (0.5 mM)/D-BIOTIN (0.5 mM) SOLUTION
Composition
L-Histidine-HCl 0.0240 g
D-Biotin 0.0305 g
Distilled Water 250 ml
Preparation
Sterilize by filtration through a 0.22 pro membrane filter.
Store in refrigerator.
5.9 SALT SOLUTION
Composition
1.65 M Potassium Chloride (KC1) 61.42 g
0.4 M Magnesium Chloride (MgCl2-6H20) 40.66 g
Distilled Water 500 ml
Preparation
Autoclave to sterilize. Store in refrigerator.
5.10 SODIUM PHOSPHATE BUFFER
Composition
Sodium Dihydrogen Phosphate 4.14 g
(NaH2P04'H20)
Disodium Hydrogen Phosphate 21.45 g
(Na2HP04 7H20)
Distilled'Water 550 ml
Preparation
Dissolve the dibasic salt in 400 ml H20, and the monobasic salt in
150 ml H20. Add dibasic solution to monobasic solution. Adjust pH to
7.4; autoclave to sterilize. Store in refrigerator.
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5.11 NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (0.1 M)
Composition
NADP 7.65 g
Distilled Water 100 ml
Preparation
Sterilize by filtration through a 0.22 urn membrane filter. Store
in 13 x 100 mm stoppered test tubes in 2 ml volumes in freezer at -20°C.
5.12 GLUCOSE-6-PHOSPHATE (1 M)
Composition
G-6-P 2.82 g
Distilled Water 10 ml
Preparation
Sterilize by filtration through a 0.22 pm membrane filter. Store
in 13 x 100 mm stoppered test tubes in 0.3 ml volumes in freezer at -20°C.
5.13 RAT LIVER ENZYME MIX
On the day of the test, using aseptic technique, the following in-
gredients are combined in a sterile graduate cylinder which has been
placed in an ice water bath. The rat liver enzyme mix must be chilled
in an ice bath during the test.
Rat Liver Homogenate 2.0 ml
Salt Solution 1.0 ml
G-6-P 0.25 ml
NADP 2.0 ml
Sodium Phosphate Buffer 25.0 ml
Sterile Distilled Water 19.75 ml
5.14 AMPICILLIN SOLUTION (8 mq/ml)
Composition
Ampicillin trihydrate 0.8 g
Sodium Hydroxide (0.02 N) 100 ml
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12
Preparation
Sterilize by filtration through a 0.22 urn membrane filter. Store
in refrigerator.
6. SALMONELLA TEST STRAINS
6.1 TEST SYSTEM
The Ames test is based on the use of five standard strains of
Salmonella typhimurium containing a specific mutation in the histidine
operon. These genetically altered strains cannot grow in the absence
of histidine; when they are placed in a histidine-free medium, only
those cells that revert spontaneously to the wild-type condition (his-
tidine- independence) are able to form colonies. The spontaneous muta-
tion rate of each strain is relatively constant, but if a chemical mut-
agen is added to the medium the mutation rate is increased significantly.
The sensitivity of the Salmonella test strains has been enhanced
by the introduction of two additional mutations, labeled uvrB and rfa.
The uvrB gene mutation results in an error-prone repair of DNA damage
caused by certain chemical or physical agents. The rfa (deep rough)
character allows increased cell permeability and greater penetration
of chemical mutagens into the bacterial cell, due to a defective lypo-
polysaccharide layer in the cell wall.
Certain chemicals will not display mutagenic activity unless they
are converted metabolically to an active form. The Ames test employs
rat-liver enzymes to simulate mammalian metabolism. Liver microsomes
are added to the test chemicals to detect the intermediate metabolites
that are often potent mutagens.
6.2 TEST STRAINS
The five standard Salmonella test strains used are described in
the table below. TA 1535 has a missense mutation in his G46 and is
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13
used to detect mutagens that cause base-pair substitutions (e.g., re-
placement of one nucleotide base pair in DMA by another). Strains
TA 1537 and TA 1538 are sensitive to mutagens that cause the frame
shift mutations (e.g., addition or removal of base pairs) his C3076
and his D3052, respectively.
Strains TA 100 and TA 98 are daughter strains of TA 1535 and
TA 1538, respectively; these contain the resistance transfer factor
plasmid (R-factor) pKM 101. The R-factor increases sensitivity to
certain mutagens, possibly through error-prone repair.
GENOTYPE OF FIVE SALMONELLA TEST STRAINS
USED FOR GENERAL SCREENING IN MUTAGEN TESTING
Strain
TA 1535
TA 100
TA 1537
TA 1538
TA 98
Mutation
Missense Mutation his G46
Missense Mutation his G46
Frameshift Mutation his C3076
Frameshift Mutation his D3052
Frameshift Mutation his D3052
Repair
uvrB
uvrB
uvrB
uvrB
uvrB
LPS
rfa
rfa
rfa
rfa
rfa
R-Factor
pKM 101
pKM 101
The five Salmonella strains used in the Ames test may be obtained from
Dr. Bruce N. Ames, Biochemistry Department, University of California, Berkeley,
California 94720. The bacteria are impregnated onto small absorbent pads.
6.3 REGENERATION AND STORAGE OF TEST STRAINS
a. Upon receipt of the strains, streak each pad onto individual
nutrient agar plates. For strains TA 98 and TA 100, use
plates containing 25 ug/ml ampicillin solution*. Incubate
agar plates at 37°C ± 0.5° C for 24 hr.
* Ampicillin (0.1 ml of an 8 mg/ml solution) may be applied directly to
the agar plates using a sterile, glass spreader. Prepare the plates
one day in advance to allow the antibiotic solution to diffuse uni-
formly throughout the agar.
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14
b. After streaking, place each pad into a test tube containing
10 ml of nutrient broth. For TA 98 and TA 100 add 25 ug/ml
ampicillin solution.
c. Incubate plates at 37°C for 24 hr. Incubate broth cultures
at 37°C overnight, shaking constantly (16 hr maximum).
d. Frozen permanent stocks are prepared in duplicate as follows:
Pipette 0.8 ml of nutrient broth culture into a 2 ml, sterile,
glass, screw-capped vial with teflon liner. Add 0.07 ml di-
methyl sulfoxide (DMSO).
e. Quick-freeze vials in dry ice and place in freezer at -80°C.
f. Store agar plate cultures at 4°C until the broth cultures are
checked for proper function and determined to be satisfactory.
Use one culture of each strain to prepare a master plate (Sec-
tion 7). Open the remaining permanent culture only to regener-
ate the frozen stocks.
7. ANALYTICAL PROCEDURES
7.1 PREPARATION OF MASTER PLATES4
To avoid cell death by continued freezing and thawing of the per-
manent stocks, prepare master plates. These plates may be stored at
4°C for approximately six weeks if they are sealed to prevent dehydra-
tion.
a. Prepare fresh broth cultures by scraping the frozen broth
cultures with a sterile wooden applicator stick and inocu-
late nutrient broth tubes. For strains TA 98 and TA 100,
the broth contains 25 ug/ml ampicillin. Incubate, shaking
constantly (100 rpm), at 37°C overnight (16 hr maximum).
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15
b. Prepare minimal agar plates by spreading 0.1 ml of ster-
ile 0.1M L-histidine on the surface of each plate (for
TA 98 and TA 100 plates contain 25 |jg/ml ampicillin).
c. Allow the histidine solution to be absorbed into the agar.
d. Spread 0.1 ml of sterile 0.5 mM biotin on the surface of each
plate and allow the solution to be absorbed into the medium.
e. Using a sterile loop, streak each plate with the appropriate
test strain so that isolated colonies are obtained.
f. Incubate plates at 37°C for 48 hr.
g. Seal each plate by wrapping with tape or parafilm; store at
4°C.
h. Retain nutrient broth culture to observe test strain function.
7.2 TEST STRAIN FUNCTION
Routinely check test strains for reversion rates and proper gene-
tic markers.
7.2.1 Histidine Requirement
a. For each strain prepare two minimal agar plates by coat-
ing each plate with 0.1 ml of sterile 0.5 mM biotin.
Spread 0.1 ml of sterile 0.1 M histidine onto one of the
plates.
b. Apply a single cross-streak of broth culture to the
plates with each test strain. Incubate plates at 37°C
for 24 hr.
-------
16
Each strain should show growth on the plate containing
histidine; no growth should occur on the plate containing
biotin.
7.2.2 Crystal-violet (rfa character)/Ampicil1in Sensitivity
(R-factor)
a. Pipet 0.1 ml broth culture into 2 ml top agar (with his-
tidine/biotin solution added). Pour mixture onto a minimal
agar plate and allow to solidify. Using sterile tweezers,
place a filter paper disc (soaked with a 10% solution of crys-
tal-violet) off-center of the plate.
b. Place a 10 ug ampicillin impregnated disc (Difco #6363
"Dispens-o-Disc") off-center, opposite the crystal-violet
disc. Use a separate plate for each test strain. Incu-
bate 24 hours at 37°C.
All test strains should have a zone of growth inhibition (ap-
proximately 14 mm diameter) around the crystal-violet, indicating
the presence of the rfa mutation. Strains TA 1535, TA 1537 and
TA 1538 should show a zone of growth inhibition around the ampi-
cillin disc. Strains TA 98 and TA 100 (containing the R-factor)
are not inhibited by the ampicillin [Figures 1 and 2].
7.2.3 UV Sensitivity (uvrB-deletion)
Cross-streak each test strain on a nutrient agar plate. Ir-
radiate one-half of the plate under a 15 Watt germicidal lamp at a
distance of 33 cm (13 in) for 8 sec. Incubate plates at 37°C for
18 to 24 hr.
All strains should retain the u\/rB-deletion and demonstrate
growth on the unexposed portion of the plate only [Figure 3].
-------
17
Figure 1. Test Strain TA98, Crystal-violet sensitivity (rfa-
character) and ampicillin resistance (R-factor).
Figure 2. Test Strain TA1537, Crystal-violet sensitivity (rfa-
character) and ampicillin sensitivity (does not
contain R-factor).
-------
18
Figure 3. Test Strain TA98, Growth inhibition after ex-
posure to UV light (uvrB-deletion).
Figure 4. Filling 13 * 100 mm culture tubes with 2 ml top-
agar (heating block set at 45°C).
-------
19
7.2.4 Spontaneous Reversion
Spontaneous reversion control tests should be performed in
duplicate during each test. Results should be maintained as part
of the permanent quality control record for each study.
a. Prepare top agar (Section 5). Melt prepared, top agar
in autoclave for three minutes at 121°C, or melt the
agar in a microwave oven. Cool to 45°C in a waterbath.
b. Prepare histidine/biotin solution (Section 5). Warm
histidine/biotin solution in 45°C waterbath. Add 20 ml
histidine/biotin solution per 200 ml top agar.
c. Preset heating block containing sterile, capped, 13 x
100 mm culture tubes at 45°C. Pipet 2 ml molten top
agar into each tube [Figure 4].
The following steps should be performed under a laminar-
flow hood.
d. Add 0.1 ml broth culture of each test strain to each of
two duplicate tubes.
e. Add 0.5 ml rat liver enzyme mix (RLE), if RLE mix is be-
ing used in the mutagenesis assay.
f. Rotate each tube rapidly between the palms to mix the
material [Figure 5]. (A vortex-mixer may be used if
mixing is performed gently to prevent bubble formation
in the top agar.)
g. Pour the contents of each tube onto the surface of a min-
imal agar plate [Figure 6]. Gently tilt and rotate each
plate to spread the top agar uniformly over the surface
of the minimal agar.
-------
20
Figure 5. Mixing top-agar by rotating tube between palms.
Figure 6. Pouring top-agar onto minimal-agar plate.
-------
21
The organisms should remain at 45°C for no longer
than 2-min to avoid cell death. Also, RLE mix cannot be
subjected to 45°C for more than a few seconds. There-
fore, the above steps should be performed as quickly as
possible. If the top agar hardens in mid-operation, a
stippled agar surface will occur, which may cause diffi-
culties in counting the colonies.
h. Set each plate, with lid slightly ajar, on a flat sur-
face to dry for several minutes. Cover the plate, seal
in scalable plastic bag [Figure 7] and incubate at 37°C
for 48 hr in an inverted position.
i. Count the number of revertant colonies. Use a dissecting
microscope to note the presence of a light "lawn" of back-
ground growth which is due to the trace amount of histidine
in the top agar.
Expected Spontaneous
Strain
TA 1535
TA 1537
TA 1538
TA 100
TA 98
Revertants for Each Strain
No. Of Colonies
10-35
3-15
15-35
120-200
30-50
Revertant rates may be slightly higher on plates containing
RLE mix due to the histidine present in the homogenate. Each
laboratory should establish an acceptable range of spontaneous
revertants per plate to determine if the strains are respond-
ing properly.
-------
22
Figure 7. Sealing agar plate in plastic bag.
Figure 8. Example of a 1 -gallon amber-glass sample bottle
equipped with teflon-lined cap.
-------
23
7.3 COLLECTION AND PREPARATION OF SAMPLES
7.3.1 Sample Collection
a. Collect a gallon sample from each water and wastewater
source,* in a clean, amber-glass bottle with a teflon
lined cap [Figure 8]. To assure that wastewater sam-
ples are representative, collect them on a flow-compos-
ite basis over a 24-hr period. Collect time-propor-
tional composit samples where discharges are intermit-
tent. Grab samples are acceptable from wells, pools,
and lakes.
b. Ice the samples during collection and transport, and re-
frigerate at 0-4°C during storage. Samples should not
be held longer than 7 days between collection and extrac-
tion. DMSO extracts may be held for 30 days. Record
all sampling information as a permanent laboratory re-
cord [Figure 9].
7.3.2 Extraction and Concentration of Samples
Base-Neutral Extraction
Adjust the sample to pH 12 with 6N NaOH solution. Divide the
sample into four equal portions. Record the total volume and trans-
fer each portion into a 2-liter separatory funnel. Extract as fol-
lows:
a. Add 100 ml of methylene chloride (dichloromethane) to a
sample portion into a 2-liter separatory funnel.
Samples suspected of containing residual chlorine should be treated with
0.8 ml of a 10% solution sodium thiosulfate/liter of sample. This concen-
tration will neutralize approximately 15 mg/1 residual chlorine.
-------
24
Figure 9
SAMPLE DATA FORM
Station
No.
Stat ion
D escr ipt ion
Date
Time
Seq
No.
Grab
Comp
S am p lers
R em arks
(Preservative)
Signed
In By
-------
25
b. Shake the mixture for 2 min and place in a ring stand
until the phases separate. If an emulsion develops,
centrifuge at 2,000 rpm for 2 to 5 min and return the
mixture to the separatory funnel.
c. Remove the aqueous fraction and retain for further ex-
traction. Save the solvent fraction.
d. Extract the aqueous fraction two additional times using
60 ml of methylene chloride. Combine the solvent frac-
tion of the sample portion.
e. Retain the aqueous fraction for acid extraction.
f. Repeat Steps 'a' through 'e1 for the other three por-
tions of the sample.
g. Combine the solvent fractions from the entire sample
(approximately 880 ml) and pour through a drying column
containing 7 to 10 cm (3 to 4 in) of anhydrous sodium
sulfate, which has been pre-rinsed with 40 to 50 ml of
methylene chloride.
h. Add approximately 500 ml of the solvent extract to a
1000-ml Kuderna-Danish (K-D) flask equipped with a
3-ball Schneider-column and a 10 ml graduated concentra-
tor tube.
i. Place 1 or 2 boiling chips into the K-D flask and evap-
orate in a steam bath (adding the remaining solvent ex-
tract in stages) to a final volume of approximately 500
ml.
j. Add 150 ml of acetone to the concentrated solvent ex-
tract, and continue evaporation to a volume of 5 ml.
-------
26
k. Add acetone to bring volume to 10 ml and mix well. Re-
tain 2.5 ml for trace organic chemical analyses.
1. Add 10 ml of DMSO to the remaining 7.5 ml of sample
extract and mix well. Evaporate the extract to 10 ml
in a roto-evaporator at 65°C.
m. Take up the total residue in 25 ml of DMSO to a total
volume of 35 ml. Record all extraction information
[Figure 10].
A flow diagram of the extraction procedure is shown in Fig-
ure 11. Label the sample extract, and store at 4°C in a secured,
explosion-proof refrigerator until assayed by the Ames procedure.
Acid Extraction
n. Acidify the aqueous fraction from Step e to pH 2 with
6N HCL. Repeat the extraction procedure.
o. Discard the aqueous portion of the sample after the
final extraction.
p. Collect a gallon sample of mutagen-free, laboratory-pure
water in a clean, amber-glass bottle. Repeat Steps 'a1
through 'o1 on this water blank sample. (See Quality
Control Procedures, Section 8).
q. Record all extraction information [Figure 10].
-------
27
Figure 10
EXTRACTION PROCEDURE DATA FORM
Sta. No
Seq. No
Date
Sampled
OPERATION PERFORMED
pH
Adjust.
Int.
Solvent
Extract'n
Int.
Emuls'n.
Removec
Int.
Sample
Cone.
Int.
Signature/
Date
-------
28
GALLON SAMPLE
DIVIDE INTO 4 EQUAL PORTIONS
BASE-NEUTRAL EXTRACTION
ADJUST TO pH 12
EXTRACT EACH ALIQUOT WITH 100, 60, AND 60 ml MeCl
AQUEOUS PHASE
ACID EXTRACTION
ADJUST TO pH 2
SOLVENT PHASE
DRY WITH
Na?SO,
EXTRACT EACH ALIQUOT WITH TOO, 60, AND 60 ml MeCl2- CONC. IN K-D
AQUEOUS PHASE
DISCARD
SOLVENT PHASE
DRY WITH Na2S04
CONC. IN K-D
EXCHANGE INTO ACETONE
FINAL VOL= 10 mi
2.5 ml
7.5 ml
HOLD FOR • EXCHANGE ACETONE
GC/MS AND HPLC FOR 35 ml DMSO BY
EXCHANGE INTO ACETONE ROTOEVAPORATION
FINAL VOL= 10 ml |
AMES TEST
2.5 ml 7:5 ml
EXCHANGE ACETONE
FOR 35 ml DMSO BY
ROTOEVAPORATION
AMES TEST
HOLD FOR
GC/MS AND HPLC
Figure 11
Extraction Procedure for Environmental Water Samples
-------
29
7.4 MUTAGENESIS ASSAYS
7.4.1 Plate Incorporation Procedure - Primary Screen [Figure 12]
a. On the day prior to performance of the assay, pick single
isolated colonies from the master plates and inoculate
tubes (20 x 125 mm) containing 10 ml nutrient broth with
the five tester strains.*
b. Incubate tubes with constant agitation for 16 hr at 37°C.
c. Prepare top agar as outlined in Section 5. Melt prepared
top agar in autoclave for 3 min at 121°C, or melt the agar
in a microwave oven. Cool to 45°C in a waterbath.-
d. Prepare histidine/biotin solution as outlined in Section 5.
Place histidine/biotin solution in 45°C waterbath to warm.
Add 20 ml histidine/biotin solution per 200 ml top agar.
e. If possible, prewarm minimal agar plates to 37°C before
inoculation.
f. Preset heating block containing sterile 13 x 100 mm culture
tubes to 45°C. Pipet 2 ml molten top agar into each tube.
Perform the following steps under a laminar-flow hood;
wear fully fastened laboratory coat and surgical gloves.
g. Add 0.1 ml broth culture (from Step 'b1) [Figure 12] of
the desired test strain to each tube corresponding to
number of dilutions of extract (suspected mutagen) to be
tested. Broth cultures should be placed in an ice bath
* Do not add ampicillin to broth cultures for strains TA 98 and TA 100 during
the mutagenesis assays; the antibiotic can sometimes cause excessive bac-
terial killing if it is concentrated in test tube cultures.
-------
30
0.1 nil (10° CELLS)
109/ml HISTIDIIIE DEPENDENT
SALMONELLA TYPH1MUR1UM
(TA 93, TA 100, TA 1535
TA 1537, TA'1533)
TEST-COMPOUND
DISSOLVED IN DHSO
LIVER EXTRACT
(SUBCELLULAR FRACTION
WITH NADPH+ AMD G-fi-P
REGENERATION SYSTEM)
2 ml MOLTEN TOP ARAR
WITH TRACE AMOUNT OF
HISTIDINE AND BIOTIN
MINIMAL GLUCOSE AGAR
INCUBATE AT 37'C FOR 48 HOURS
COUNT NO. OF HISTIDINE
INDEPENDENT (REVERTANT) COLONIES
Figure 12
Agar Plate Incorporation Procedure
-------
31
for the duration of the test. Remember that upon inoc-
ulation the organisms must not remain at 45°C for more
than 2 min. Inoculate tubes singly to avoid prolonged
exposure to 45°C temperature
h. Add an appropriate concentration of test material to each
tube inoculated with culture. Suggested volumes of ex-
tract include 500 ul, 250 ul, 100 ul, 50 ul and 10 ul for
each test strain.
i. Rotate each tube rapidly between palms to thoroughly mix
the materials. Alternately, a Vortex-mixer may be used,
if mixing is performed gently to prevent bubble formation
in the top agar.
j. Pour the contents of each tube onto the surface of a min-
imal agar plate. Gently tilt and rotate each plate to
spread the top agar uniformly over the surface of the
minimal agar.
k. Set each plate, with lid slightly ajar, on a flat surface
to dry for several minutes. Cover the plates, seal in
scalable plastic bags, and incubate at 37°C for 48 hr in
an inverted position. Count the number of revertant col-
onies [Figure 13].
1. Initially, perform the assay without RLE mix. If muta-
genic activity is observed, determine a dose-response
with all active strains [Figure 14]. Then terminate the
test. However, if mutagenic activity is not observed,
repeat the above steps adding RLE mix after Step 'h'.
m. Add 0.5 ml of RLE mix to each tube. Immediately after
the RLE mix is added, quickly rotate the tube between
the palms and pour onto the surface of the minimal agar
plate (Steps 'i1 and 'j1). Allow only a few seconds for
the addition of RLE mix. Proceed with Step 'k1.
-------
32
Figure 13. Counting revertant colonies using digital,
Quebec Colony Counter.
Figure 14. Dose-response effect of mutagenic material on
Test Strain TA1537 ffop-50 n\, /eft-100/*!,
right-200 /aI of concentrated waste extract).
-------
33
7.4.2 Plate Incorporation Procedure - Dose Response
If increased numbers of revertants are noted, repeat all
steps of the primary screen in duplicate (preferably triplicate)
using additional doses of test material, with all active strains.
For example, if the 250 [jl concentration shows mutagenic activity,
volumes of choice might include 100 ul, 150 ul, 200 ul, 250 ul,
300 ul, 350 pi, 400 M"! » 450 ul, to establish a dose-response.
Proper test aliquots must be selected independently for each sam-
ple tested.
Perform control plates (at least duplicates) for each test
strain.
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 substance is toxic,
massive cell death will occur and the background lawn will be
sparse or absent compared to control plates. The sparse growth
is a result of more histidine being available to the surviving
bacteria. Survivors undergo more cell division and appear as
small colonies which can be mistaken for revertants; consequently,
the presence or absence of a normal lawn must be noted.
To check for histidine requirement, pick randomly selected
revertant colonies from plates showing mutagenicity and streak
onto a minimal agar plate.
All positive mutagenic responses should be verified by a
repeatable assay.
-------
34
7.4.3 Spot Test
The spot test is a simple method to determine if a com-
pound is mutagenic qualitatively. The test is particularly
useful to rapidly screen large numbers of compounds in a short
time. Also, the spot test may be used to determine an approx-
imate concentration range of suspected mutagen to employ in
the plate incorporation assay. Results of the spot test are
not conclusive; the test should be used only as an adjunct
to the agar plate incorporation procedure.
The spot test can only be used for analyses of water sol-
uble chemicals which are readily diffusible in agar. Also, it
is much less sensitive than the standard agar plate incorpora-
tion procedure, because fewer bacteria are exposed to the test
material at any dose level.
a. Prepare cultures, media, reagents and equipment (Steps
'a1 through 'j1 as in the Primary Screen assay, 7.4.1);
but omit the test chemical. Perform the test both with
and without 0.5 ml of rat liver enzyme mix added to the
top agar.
b. After the top agar has hardened, quickly add the sus-
pected mutagen to the agar surface in one of the fol-
lowing ways: (1) as crystals (for pure compounds), (2)
as a 10 (jl microdrop, or (3) as a 6 mm sterile filter
paper disc saturated with test chemical.
c. Seal the plates in plastic bags and incubate at 37°C
for 48 hr in an inverted position.
d. Perform all procedures at least in duplicate (prefer-
ably triplicate).
-------
35
e. Prepare a positive control by adding a known diagnostic
mutagen.
f. Prepare a negative control by adding a non-mutagenic
crystal, a 10 [jl microdrop of sterile distilled water,
or a blank 6 mm sensi disc saturated with sterile dis-
tilled water.
g. Read and record results.
A.positive result is usually indicated by a large increase
in colonies located in a circular position away from the spot
[Figure 15]. If the mutagen is non-toxic, colonies may develop
directly adjacent to the spot.
Always confirm definitive mutagenicity by demonstrating a
repeatable dose-response effect with the standard agar
plate incorporation procedure.
7.4.4 Preincubation Assay
The most widely used and successful modification to the stan-
dard Ames test is the preincubation assay described by Yahagi8.
This procedure is sometimes useful as an alternate test, if the
test material is toxic, or in cases where results of the standard
plate assay are inconclusive, e.g., detection of nitrosoamines.
Preincubation tests are performed at least in duplicate.
a. On the day before performing the assay, inoculate from
the master plates 20 x 125 mm tubes containing 10 ml
nutrient broth with the five tester strains.
b. Incubate tubes with constant agitation for 16 hr at 37°C.
c. Prepare top agar as outlined in Section 5. Melt prepared
top agar in autoclave for 3 min at 121°C, or melt the agar
in a microwave oven. Cool to 45°C in a waterbath.
-------
36
Figure 15. Spot test, Test Strain TA100 (/eft-sensi disc
contains 100/il of methyl methanesulfonate;
r/gf/?f-control showing spontaneous revertant
colonies).
-------
37
d. Prepare histidine/biotin solution as outlined in Sec-
tion 5. Place histidine/biotin solution in 45°C water-
bath to warm. Add 20 ml histidine/biotin solution/
200 ml top agar.
Perform the following steps under a laminar-flow hood;
wear fully fastened laboratory coat and surgical gloves.
e. Add appropriate concentrations of test material in dupli-
cate to sterile 13 x 100 ml test tubes which have been
placed in an ice bath (see Plate Incorporation Procedure-
Primary Screen, 7.4.1, Step 'h').
f. Inoculate each tube with 0.1 ml overnight broth culture
of the desired test strain.
g. Add 0.5 ml of RLE mix to each tube containing sample
extract and culture.
h. Quickly mix the contents of each tube and incubate with
constant agitation at 37°C for 15 min.
i. Add 2 ml of molten top agar (from Step 'd') to each tube,
and rotate each tube rapidly between the palms to thor-
oughly mix the materials. A Vortex-mixer may be used if
mixing is performed gently to prevent bubble formation
in the top agar.
j. Pour the contents of each tube onto the surface of a
minimal agar plate. Gently tilt and rotate each plate
to spread the top agar uniformly over the surface of
the minimal agar.
k. Set each plate, with lid slightly ajar, on a flat sur-
face to dry for several minutes. Cover the plates, seal
-------
38
in plastic bags, and incubate at 37°C for 48 hr in an in-
verted position. Count the number of revertant colonies.
1. If increased numbers of mutations are noted, repeat the
above steps in duplicate (preferably in triplicate) with
all active strains using additional concentrations of test
materials (see Plate Incorporation Procedure-Dose Response,
7.4.2).
m. Perform negative controls for each strain using phosphate
buffer instead of RLE mix.
n. Perform positive controls using an appropriate diagnos-
tic mutagen.
o. Perform control plates on solvent (DMSO) at similar con-
centrations.
8. QUALITY CONTROL PROCEDURES
Perform quality control tests and record results [Figures 16, 17, 18,
and 19] for all mutagenicity analyses. Include analyses of distilled water
extracts, analyses of test strain function and viability, sterility tests
of solvents (DMSO), extracts, reagents, culture media, and exposure of test
strains to diagnostic mutagens.
8.1 VIABILITY COUNTS
Bacteria density inoculated per plate is critical in determi-
nation of typical dose-responses and general reproducibility of results.
Bacteria densities of the 16 hr broth cultures should be approximately
109 viable organisms per ml for each test strain. Routine viability
counts (serial dilutions of nutrient agar pour plates) should be per-
formed prior to analyses until an acceptable and reproducible density
has been established. It may be necessary to reduce or increase the
-------
39
Figure 16
QUALITY CONTROL DATA FORM-EQUIPMENT
Year:
Mon lli
Day:
STILL
Conductance
pH
Drained
WATERBATHS
1)
2)
3)
4)
5)
AUTOCLAVE
Pressure
Temp. (max)
OVEN
Temp. (rn ax) :
INCUBATORS
1)
2)
3)
4)
5)
REFRIGERATORS
1)
2)
FREEZERS
1)
2)
3)
-
-
-------
40
Figure 17
QUALITY CONTROL DATA FORM-MEDIA
MEDIA AND
LOT tt .
RECEIVE
OPEI^
AMI
pH
DATE
INT
MEDIA AND
LOT tt.
RECEIVE
OPEN
AMI
pH
DATE
INT
-------
41
Figure 18
QUALITY CONTROL DATA FORM-REAGENTS
(Sterility, pH, etc.)
REAGENT
TEST
RESULT
DATE
INT.
REAGENT
TEST
RESULT
DATE
INT
-------
42
Figure 19
QUALITY CONTROL DATA FORM-SPONTANEOUS REVERTANTS AND DIAGNOSTIC MUTAGENS
DATE, INITIALS
MEDIA LOT tt
Spont. Revert.
Date:
Hist/8 iotin
B iotin
U.V. Sens.
Amp Sens.
C.V. Sens.
Spont. Revert.
Date:
Hist/B iotin
Biotin
U.V. Sens.
Amp Sens.
C.V. Sens.
Spont. Revert.
Date:
Hist/B iotin
Biotin
U.V. Sens.
Amp Sens.
C.V. Sens.
Hist/B iotin
Biotin
U.V. Sens.
Amp Sens.
C.V. Sens.
Hist/B iotin
B iotin
U.V. Sens.
Amp Sens.
C.V. Sens.
MNNG
MMS
9AA
.4NNO
D nu n.
2AF
NOTES
Average
Average
Average
Average
Average
TA 1535
I
I
I
I
I
TA 1537
I
I
I
I
I
TA 1538
I
I
I
I
I
TA 98
I
I
I
I
I
TA 1OO
1
1
- 1
1
1
-------
43
incubation period accordingly to obtain 109 cells per ml. After a
proper incubation period has been determined, viability counts
should be performed at routine intervals. As an alternate approach,
correlate viability counts with spectrophotometric data so that bac-
teria densities can be determined quickly.
8.2 AUTOMATED COLONY COUNTS
Observed differences often occur between automated and manual
counting procedures for a specific group of plates. Differences are
due to several factors including: (1) masking of colonies around the
periphery of the agar plates, (2) overlapping or clustered colonies
which are not fully counted, and (3) bubble formation in the agar me-
dia.
Automated colony counter manufacturers supply calibration proce-
dures which are unique to each instrument. Establish a calibration
technique for each counting application (different batches of agar me-
dia, test strains, and each counting run) to verify automated with man-
ual counts. Analysts should be thoroughly familiar with instrument oper-
ation and calibration. Examine all agar plates before use and discard
those containing excessive bubbles.
8.3 DIAGNOSTIC MUTAGENS
Positive diagnostic mutagens are included with each test to con-
firm proper test strain function. Because it is undesirable to handle
and store the concentrated mutagenic materials in a laboratory that is
not equipped with elaborate safety controls (high hazard area), it is
recommended that diagnostic mutagens be purchased in diluted quantities
from a commercial laboratory*. Some mutagenic materials are extremely
* Diluted diagnostic mutagen standards may be obtained from Nanogens
International, P. 0. Box 1025, Watsonville, California 95076.
-------
44
unstable. However, those dissolved in DMSO seem to retain their poten-
cy for longer periods. Routinely, check for potency of diagnostic
mutagens; many are usable for 6 months or more. Dose response curves
should be established for each diagnostic mutagen. This curve is used
to monitor and possibly adjust the mutagen concentration over time.
The following table contains the characteristic reversion pat-
terns of the five standard test strains to several diagnostic muta-
gens. Additional mutagenic compounds may be incorporated for quality
control. These include sodium azide, benzo (a) pyrene and 7,12-di-
methylbenzanthracene (DMBA). Each laboratory should use positive con-
trols at concentrations yielding reproducible counts to confirm the
reversion properties of each strain. Often the choice of controls may
be influenced by the suspected chemical characteristics of the sample.
CHARACTERISTIC REACTIONS OF FIVE STANDARD
SALMONELLA TEST STRAINS TO DIAGNOSTIC MUTAGENS1
Mutagen
Methyl methane-
sulfonate
4-Nitroqui no-
li ne-N-oxide
MNNGd
9-Aminoacridine
Daunomycin Hydro-
chloride
2-Aminof luorene
Aflatoxin Bl
Amount S-9
Used (pi) Mix
10
2.5
5
100
100
50 0.5 ml
50 0.5 ml
a Plate incorporation assay.
b All chemicals are dissolved (1 r
Test Strains
TA 1535 TA 1537 TA 1538 TA 98 TA 100
Mutagenic
±c -c
+
+ ±
+
ng/ml) in DMSO exce
Response
± +c
± + +
± - +
+ -
+ + +
± + +
pt for Daunomycin,
which is dissolved (1 mg/ml) in distilled water.
'+' = at least 2.5 times spontaneous reversion rate; '-' = < spontan-
eous reversion rate; '±' = slightly elevated reversion rate, but less
than 2.5 times spontaneous reversion rate.
N-methyl-N1-nitro-N-nitrosoguanidine.
-------
45
8.4 DISTILLED WATER BLANK
Fill a clean, gallon, amber-glass bottle with sterile distilled
water and treat as a sample. This provides background information on
the quality of the sample bottles, distilled water, extract solvents,
emulsion removal and the concentration process.
Test a DMSO blank at concentrations similar to those used in the
assay to ensure that this solvent is not toxic and does not interfere
with test results.
8.5 STERILITY CONTROLS
Perform sterility controls on solvents, extracts, liver homogen-
ate, RLE mix, and culture media. Examine test reagents for sterility
by a "spread plate" technique. Inoculate 1 ml aliquots of test re-
agents onto sterile nutrient agar plates, spreading the test materials
uniformly by gently tilting and rotating the plates, or spread the
material with a small, sterile, glass "hockey stick".
Incubate plates at 37°C for 24 hr. Discard contaminated reagents
or, if appropriate, re-sterilize. Pre-incubate all culture media at 37°C
for 24 hr.
DMSO extracts are usually sterile as a result of the bactericidal
action of the solvents in the extraction procedure. Occasionally, a
waste concentrate may consist of viscous materials, and the extracts
remain contaminated. Contaminated DMSO extracts may be filter-steril-
ized through a fritted-glass filter or a 0.45 |jm teflon filter prior
to assay.
For a more complete discussion of quality control procedures, see
Microbiological Methods for Monitoring the Environment Water and Wastes2.
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46
9. INTERPRETING AND REPORTING RESULTS
A typical dose-response curve showing mutagenicty of a textile waste
sample is presented in Figure 20. Generally, dose-response curves for pure
mutagenic compounds are linear. However, non-linear curves are often ob-
served in the case of complex materials. Misinterpretation of the mutagen-
ic activity can result if too high concentrations of mutagenic materials
are tested. High concentrations of bactericidal materials can cause exces-
sive mortality, which results in a decrease in the number of revertants.
Initial screening of suspected mutagens should include a wide range of con-
centrations. All positive results must be confirmed using a narrower
range of concentrations. At least 500 pi of material should be tested be-
fore a negative result is reported. An example of a data summary report is
shown on page 48.
Most investigators consider a chemical to be mutagenic if the number
of induced revertants is two or more times greater than the number of spon-
taneous revertants1. A two-fold increase in induced revertants coincident
with a typical dose-response correlates closely (>90% probability) with
inducement of tumors by organic compounds in laboratory animals i'5'6'7.
The NEIC uses a mutagenic activity ratio* (MAR) of 2.5 or greater as
one of the two criteria for determination of a positive mutagenic response.
Commoner and co-workers9"11 have provided a basis for selection of this ra-
tio. In tests of approximately 100 compounds (50 carcinogens and 50 non-
carcinogens), these investigators concluded that approximately 83% of the
presumptive carcinogens yield ratios above 2.0, and the same percentage of
noncarcinogens yield ratios below that value. However, an MAR of 2.5 or
greater increases the probability to 95%, that a substance would produce
tumors if administered to laboratory animals, i.e, that it is a presumptive
carcinogen.
* This is defined as E-C/c where E is the number of mutant colonies/plate
when the compound is present; C is the corresponding value for the con-
trol, with the compound absent, obtained on the same day; and c is the
average historical control value determined for each laboratory.
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300
oo
h^
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DATA SUMMARY, MUTAGENIC ACTIVITY OF TEXTILE MANUFACTURING COMPANY EFFLUENT
October-December 1978
Salmonella
Station No. Sample Date/Time Extract Test
& Description Type Collected pH Strain
01 Composite 10/16/78 Basic TA 1537
0600
Textile Manufacturing Acidic TA 1537
Co. Treatment Pond
Effluent
,
Volume of
Concentrate
Tested (ul)
100
95
85
75
60
50
35
25
20
10
5
2.5
1.0
100
95
85
75
60
50
35
25
20
10
5
2.5
1.0
Equivalent
Volume of
Sample (ml)
(Calculated)
19.4
18.5
16.5
14.6
11.7
9.7
6.8
4.9
3.9
1.9
1.0
. 0.5
0.2
19.4
18.5
16.5
14.6
11.7
9.7
6.8
4.9
3.9
1.9
1.0
0.5
0.2
No. of Revertant
Colonies/Plate
Control Experimental
8 264
249
225
214
181
147
125
68
69
61
46
25
9
8 307
277
258
166
138
113
72
48
55
54
24
28
11
Mutagenic
Activity
Ratio0
36.5
34.4
31.0
29.4
24.7
19.8
16.7
8.6
8.7
7.6
5.4
2.4
1.0
42.7
38.4
35.7
22.6
18.6
15.0
9.1
5.7
6.7
6.6
2.3
2.8
0.5
a Composite sample - Compositing was hourly over a 24-hr period; date and time listed is date and time period ended.
b Value based on average of two control values.
c Average of two plates
d Mutagenic Activity Ratio = E-C/c where: E is the number of colonies/experimental plate
C is the Average Control Value based on two plates
c is the historical control value averaged over 100 tests (TA 1537 = 7)
CO
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49
A substance Is determined mutagenic if: (1) an MAR of 2.5 is obtained,
and (2) a typical and reproducible dose-response is observed.
A continuing average of all spontaneous revertant controls for each
test strain should be recorded. Use of this average in calculating the MAR
serves to "smooth out" bias that can occur with various daily control values.
With complex environmental mixtures, it is important to record the total vol-
ume of sample extracted. This measurement can be used to calculate the equi-
valent volume of orginal sample that resulted in mutagenic activity. A sug-
gested data reporting form is presented in Figure 21.
Because of the limitations of the sample concentration procedure and
the inherent limitations of the Salmonella mutagenicity test, a negative
result from any sample is inconclusive. A negative result does not neces-
sarily mean the sample is non-mutagenic, but that the mutagenic effect is
not detectable under the conditions of the test system used. The Salmonella
mutagenicty test measures mutations at specific base sequences in bacterial
DNA; it does not indicate overall mutagenic potential.
The method described will yield a low estimate of mutagenic activity
from complex materials for the following reasons: (1) the volatile alkyl
halides are lost in the dichloromethane/acetone/DMSO exchange, and (2) some
of the important chlorinated carcinogens such as carbon tetrachloride, chlo-
roform, and hexachlorobenzene are not detected. Therefore, an analysis by
gas chromatography/mass spectrometry should always be used to complement the
Salmonella mutagenesis assay. Volatile organic chemical analyses should be
performed, also.
10. SAFETY CONSIDERATIONS
Any laboratory equipped to do general microbiological analyses can be
adapted to provide the necessary safety equipment for mutagen analyses.
Simple application and adherence to "common sense" rules when handling mu-
tagenic and carcinogenic material are extremely important.
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Figure 21
AMES ASSAY DATA FORM
50
DATE
STATION NO.
Extract
,PH
..
Quant
(ul)
S-9
Mix
SEQUENCE NO.
Number Of Revertants
1535
1537
1538
98
100
COMPOUND NAME
Extract
.PH
Qu ant
(ul)
S-9
Mix
ANALYST
Number Of Revertants
1535
1537
1538
98
"
1OO
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51
10.1 SAFETY PROCEDURES
Listed below are safety procedures and adaptations followed by
the NEIC laboratory during performance of microbial mutagen analyses.
The reader is referred to the EPA Safe Practices Handbook on Carcino-
gens12 for additional information.
a. Perform all organic solvent extraction procedures in a prop-
erly ventilated explosion-proof hood.
b. Wear fully fastened laboratory coats and latex surgical
gloves when handling mutagens or potentially mutagenic ma-
terial.
c. Mouth pipetting is strictly prohibited. Perform all pipet-
ting operations with a mechanical device so that no possible
contact can be made between the laboratory workers and test
materials.
d. The Federal Occupational Safety and Health Act of 1972 re-
quires biological hazard signs and tags to identify presence
of potential health hazard. Place signs properly to identify
equipment, containers, rooms, materials, experimental animals,
or combinations of each of these items. Where chemical car-
cinogens are present, post work and storage areas with signs
bearing the legend: DANGER-CHEMICAL CARCINOGEN - AUTHORIZED
PERSONNEL ONLY.
e. Add test materials (suspected mutagens) inside a total-exhaust
laminar-flow hood to prevent worker exposure and contamination
of culture materials. Each laboratory-type hood or device used
for containment of mutagenic or carcinogenic materials must bear
the legend: DANGER - CHEMICAL CARCINOGEN.
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52
f. Prevent exposure of potentially toxic, volatile materials to
the laboratory environment or between individual inoculated
plates by sealing plates with scalable plastic bags immedi-
ately after inoculation. Open plates and plastic bags only
under a properly vented laboratory hood.
g. Permit only authorized personnel to enter work and storage
areas where chemical carcinogens are present.
h. Cover all work surfaces (bench tops, hood floors, etc.) on
which chemical mutagens and carcinogens are used with stain-
less steel or plastic trays, dry absorbent plastic-backed
paper, or other impervious material.
i. Keep only minimum working quantities of chemical carcinogens
and mutagens in the work area.
j. Properly label storage vessels containing chemical mutagens
or carcinogens. Catalog and store stock quantities of chemi-
cal mutagens and carcinogens in a specific storage area that
is secured at all times.
k. Use unbreakable outer containers, such as metal desiccators
to transport chemical carcinogens and mutagens.
1. Place materials contaminated with chemical carcinogens in sep-
arate sealed plastic bags or other suitable impermeable con-
tainers before transferring from work areas to disposal areas.
Label both with the name of the carcinogen and a sign DANGER -
CHEMICAL CARCINOGEN, before transporting.
m. Use housekeeping procedures which suppress the formation of
aerosols; dry sweeping and dry mopping are prohibited.
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53
n. Use special precautions when handling DMSO. This chemical
is readily absorbed through the skin, along with any mater-
ials contained within the solvent. DMSO must be stored in
a locked cabinet.
o. Autoclaving any mutagenic test materials or cultures is strict-
ly prohibited.
p. A thorough safety procedure for handling hazardous materials,
including a health monitoring program for employees, shall be
established for each Laboratory.
10.2 WASTE DISPOSAL
The NEIC laboratory performs all mutagen tests using disposable
test tubes, pipettes and petri dishes. All potentially toxic materials
are put into plastic bags, labeled, sealed and disposed of in 55-gal
plastic-lined drums. Ultimate disposal of all materials is by a pri-
vate contract firm that uses an approved hazardous waste disposal site.
High temperature incineration is the preferable means of disposal if
this service is available.
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54
REFERENCES
1. Ames, B. N., McCann, J., and Yamansaki, E., Methods for Detecting Car-
cinogens and Mutagens with the Salmonella/Mammalian-Microsome Mutagen-
icity Test. Mutation Research, 31 (1975) 347-364; (Supplemental Revis-
ion, June 1979).
2. Bordner, R. and Winter, J., eds. Microbiological Methods for Monitoring
the Environment, Water and Wastes, Environmental Monitoring and Support
Laboratory, Office of Research and Development, U. S. Environmental Pro-
tection Agency, EPA-600/8-78-017, Cincinnati, Ohio (December 1978).
3. Rand, M. et a!, 1975. Standard Methods for the Examination of Water
and Wastewater. 14th Ed.APHA - AWWA - WPCF, 1193 pp.
4. Dutka, B. J., ed., Canada Department of Environment "Methods for Micro-
biological Analysis of Waters, Wastewaters and Sediments" Manual. Mi-
crobiology Laboratories, Applied Research Division, Canada Centre for
Inland Waters, Burlington, Ontario, 1978 revision.
5. McCann, J., Ames, B. N., Detection of Carcinogens as Mutagens, in the
Salmonel1 a/Microsome Test: Assay of 300 Chemical, Proc. Nat. Acad. Sci.,
73 (1976) 950-954.
6. Sugimura, T., et. al., Overlapping of Carcinogens and Mutagens, In Magee
P. N., S. Takayama, T. Sugimura, and T. Matsushima, eds. Fundamentals
in Cancer Prevention, Univ. Park Press, Baltimore, MD, pp 191-215, 1976.
7. Purchase, I. F. H., et. al., An Evaluation of 6 Short-term Tests for De-
tecting Organic Chemical Carcinogens. British Journal of Cancer, 37,
(1978) 873-902.
8. Yahagi, T., et. al., Mutagenicity of Carcinogenic Azo Dyes and Their
Derivatives, Cancer Letters, 1, (1975) 91-96.
9. Commoner, B., Chemical Carcinogens in the Environment; Presentation at
the First Chemical Congress of the North American Continent, Mexico
City, Mexico, Dec. 1975.
10. Commoner, B., Development of Methodology, Based on Bacterial Mutagene-
sis and Hyperfine Labelling, For the Rapid Detection and Identification
of Synthetic Organic Carcinogens in Environmental Samples, Research
Proposal Submitted to National Science Fdn., Feb., 1976.
11. Commoner, B., Henry, J. I., Gold, J. C., Reading, J. J., Vithayathil,
A. J., "Reliability of Bacterial Mutagenesis to Distinguish Carcino-
genic and Non-carcinogenic Chemicals", EPA-600/1-76-011, Government
Printing Office, Washington, D. C. (April 1976).
12. Safe Practices Handbook on Carcinogens, Safety Standards for Research,
U.S. Environmental Protection Agency, Washington, D. C. (In Preparation).
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