Water quality - Determination of the genotoxicity of water and waste water - Salmonella/microsome fluctuation test (Ames fluctuation test)

This International Standard specifies a method for the determination of the genotoxic potential of water and waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chemicals that induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are involved in the biosynthesis of the amino acid, histidine. NOTE 1 ISO 13829[8] applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents. This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.
NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove necessary.

Qualité de l'eau - Évaluation de la génotoxicité des eaux résiduaires - Essai de Salmonella/microsome (essai d'Ames-fluctuation)

L'ISO 11350:2012 sp�cifie une m�thode d'�valuation de la g�notoxicit� des eaux r�siduaires en utilisant les souches bact�riennes Salmonella enterica subsp. enterica s�rotype Typhimurium TA 98 et TA 100 au cours d'un essai en fluctuation. Cette combinaison de souches permet d'�valuer la g�notoxicit� des substances chimiques qui produisent des mutations ponctuelles (substitutions de paires de base et mutations de changement de phase) dans les g�nes codant pour les enzymes impliqu�es dans la biosynth�se de l'acide amin� histidine.
Cette m�thode est applicable aux substances suivantes: a) eau douce; b) eaux r�siduaires; c) extraits aqueux et lixiviats; d) �luats de s�diments (eau douce); e) eau interstitielle; f) solutions aqueuses contenant des substances uniques ou des m�langes chimiques; g) eau potable.

Kakovost vode - Določevanje genotoksičnosti za vodo in odpadno vodo - Salmonella/mikrosomski fluktuacijski preskus (Amesov fluktuacijski preskus)

Ta mednarodni standard opisuje metodo za določevanje genotoksičnega potenciala vode in odpadne vode z uporabo bakterijskih sevov Salmonella enterica, podvrste enterica serotip Typhimurium TA 98 in TA 100 v nihajnem preskusu. Ta kombinacija sevov lahko izmeri genotoksičnost kemikalij, ki povzročajo točkovne mutacije (substitucijo baznih parov in mutacije okvirnega položaja) genov, ki kodirajo encime, vključene v biosintezo aminokisline histidin. OPOMBA 1 Standard ISO 13829[8] se uporablja za meritve genotoksičnosti vzorcev, ki vsebujejo agente za prečno povezovanje DNK. Ta metoda se uporablja za:
– sladko vodo,
– odpadno vodo,
– vodne ekstrakte in izcedne vode,
– izlužke sedimentov (sladka voda),
– porno vodo,
– vodne raztopine posameznih snovi ali kemičnih mešanic,
– pitno vodo.
OPOMBA 2 Pri preskušanju pitne vode je lahko potrebna ekstrakcija in predkoncentracija vodnih vzorcev.

General Information

Status
Published
Public Enquiry End Date
30-Jun-2012
Publication Date
26-Dec-2012
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
13-Dec-2012
Due Date
17-Feb-2013
Completion Date
27-Dec-2012

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INTERNATIONAL ISO
STANDARD 11350
First edition
2012-05-15
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome fluctuation test
(Ames fluctuation test)
Qualité de l’eau — Évaluation de la génotoxicité des eaux résiduaires —
Essai de Salmonella/microsome (essai d’Ames-fluctuation)
Reference number
ISO 11350:2012(E)
©
ISO 2012

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ISO 11350:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
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Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
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Published in Switzerland
ii © ISO 2012 – All rights reserved

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ISO 11350:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Interferences . 3
5 Principle . 4
6 Apparatus and materials . 4
7 Reagents, media and dilutions . 5
8 Sampling and samples . 9
9 Procedure . 9
9.1 Overnight culture . 9
9.2 Preparation of S9 mix .10
9.3 Testing of water samples .10
9.4 Measurement of revertant growth .13
9.5 Calculation of cytotoxicity .13
10 Validity criteria .14
11 Assessment criteria .14
12 Test report .14
Annex A (normative) Nutrient broth and agar .15
Annex B (normative) Preparation of ampicillin agar plates and stock cultures.16
Annex C (normative) Checking of genotype .17
Annex D (normative) S9 fraction .18
Annex E (informative) Example for application of samples on a 24 well plate .19
Annex F (informative) Example for reporting .21
Annex G (informative) Testing of chemicals .22
Annex H (informative) Precision data.25
Annex I (informative) Statistical assessment .27
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water —
A simplified evaluation for testing of waste water .33
Annex K (informative) Use of additional tester strains .35
Bibliography .36
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ISO 11350:2012(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11350 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2012 – All rights reserved

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INTERNATIONAL STANDARD ISO 11350:2012(E)
Water quality — Determination of the genotoxicity of water and
waste water — Salmonella/microsome fluctuation test (Ames
fluctuation test)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination of the genotoxic potential of water and
waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and
TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chemicals that
induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are
involved in the biosynthesis of the amino acid, histidine.
[8]
NOTE 1 ISO 13829 applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents.
This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.
NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove necessary.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable
for its application. For dated references, only the edition cited applies. For undated references, the latest edition
of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 7027, Water quality — Determination of turbidity
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ISO 11350:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
cofactor solution
aqueous solution of chemicals (e.g. NADP, glucose-6-phosphate, and inorganic salts) needed for the activity
of the enzymes in the S9 fraction
[10]
[Source: ISO 21427-2:2006, definition 3.2]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[6]
[Source: ISO 6107-6:2004, definition 24]
3.3
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water with dilution
water as integral number
NOTE 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. [In this International Standard,
the arrow indicates the transition from initial total volume to final total volume.] The corresponding and smallest possible
value of D is 1.
[6]
[Source: ISO 6107-6:2004, definition 28]
3.4
lowest ineffective dilution
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant increase in the
number of revertant wells compared with the negative control
NOTE 1 to entry: LID is determined for each incubation condition (strain, ±S9 mix). The highest LID value is decisive for
the overall assessment.
3.5
induction rate
difference between the mean value of wells with revertant growth counted on the plates treated with a dose
of the test sample or with a positive control, and the mean value of the corresponding wells treated with the
negative control using the same strain under identical conditions
[6]
[Source: ISO 6107-6:2004, definition 43, modified: “wells with revertant growth” replaces “mutant colonies”;
“corresponding wells” replaces “corresponding plates”]
3.6
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing preculture,
in fresh medium
[6]
[Source: ISO 6107-6:2004, definition 44]
3.7
negative control
dilution water without test sample
[6]
[Source: ISO 6107-6:2004, definition 51]
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ISO 11350:2012(E)
3.8
revertant growth
visible mutant colonies on the microplate at the end of the respective test
3.9
overnight culture
culture started late in the afternoon and incubated overnight (usually about 16 h) to be ready during the following
morning for purposes such as the inoculation of a preculture
[6]
[Source: ISO 6107-6:2004, definition 54]
NOTE 1 to entry For specification, see 9.1.
3.10
positive control
any well characterized material and/or substance, which, when evaluated by a specific test method, demonstrates
the suitability of the test system to yield a reproducible, appropriate positive or negative response in the test system
[7]
[Source: ISO 10993-12:—, definition 3.12]
NOTE 1 to entry The positive controls mentioned in this International Standard are dissolved in dimethyl sulfoxide (DMSO)
prior to use. For the purposes of this International Standard, the positive controls are known mutagens which are suitable
for the verification of the sensitivity of the method and/or the activity of the S9 mix.
3.11
S9 fraction
supernatant at 9 000g of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to
300 g) pretreated with a substance or substance combination appropriate for enzyme induction
[6]
[Source: ISO 6107-6:2004, definition 74]
3.12
S9 mix
mixture of S9 fraction and cofactor solution
[6]
[Source: ISO 6107-6:2004, definition 75]
3.13
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[6]
[Source: ISO 6107-6:2004, definition 87]
3.14
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all preparation
steps such as centrifugation, filtration, homogenization, pH adjustment and determination of ionic strength
[6]
[Source: ISO 6107-6:2004, definition 92]
4 Interferences
Bacteriotoxic effects of the test sample can lead to a reduction of viable bacteria and to a reduction of wells with
revertants due to a repression of revertant growth.
This method includes sterile filtration of water and waste water prior to the test. Due to this filtration, solid
particles are separated from the test sample. Thus, there is a possibility that genotoxic substances adsorbed
on particles are not detected.
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ISO 11350:2012(E)
5 Principle
The bacteria are exposed under defined conditions to various concentrations of the test sample and incubated
for 100 min at 37 °C ± 1 °C in 24 well plates. Due to this exposure, genotoxic agents enclosed in the test sample
can induce mutations in one or both marker genes of the bacterial strains used (hisG46 for TA 100 and hisD3052
for TA 98) in correlation with the applied concentrations. Induction of mutations causes a concentration-related
increase in the number of mutant colonies.
After exposure of the bacteria, reversion indicator medium (7.40), containing the pH indicator dye bromocresol
purple (7.7), is added to the wells. Subsequently, the batches are distributed to 384 well plates (48 wells for
each parallel) and incubated for 48 h to 72 h (9.3.2, 9.3.3).
Mutagenic activity of the test sample is determined by counting the number of purple to yellow shifted wells (per
48 wells of each parallel), treated with the undiluted or the diluted test sample, compared to the negative control.
The lowest dilution (1→N) of the test sample which induces no mutagenic effect under all experimental
conditions (if any mutagenic effect is induced by the test sample) is the criterion for evaluating the mutagenic
potential. Sample dilutions above this (1→A, A < N) shall induce a mutagenic effect according to the criteria of
this International Standard in at least one strain under at least one activation condition (with or without addition
of S9 mix). The respective LID value is N. If no mutagenic effect is observed under all experimental conditions,
this dilution is 1→1 and the respective LID value is 1.
6 Apparatus and materials
6.1 Temperature- and time-controlled incubator, 37 °C ± 1 °C.
6.2 pH meter.
6.3 Analytical balance.
6.4 Steam sterilizer.
6.5 Dry sterilizer.
6.6 Magnetic stirrer.
6.7 Rotary mixer.
6.8 Freezer, capable of being maintained at ≤ −18 °C and at ≤ −70 °C.
6.9 Pipettes, 0,1 ml, 0,5 ml, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, of glass or plastics.
6.10 Storage bottles, 250 ml and 1 000 ml.
6.11 Measuring cylinders, 100 ml and 200 ml.
6.12 Volumetric flasks, 20 ml, 200 ml and 500 ml.
6.13 Sterile filters, 0,2 µm and 0,45 µm.
6.14 Erlenmeyer flasks, 50 ml, 100 ml and 250 ml.
6.15 Inoculating loops.
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ISO 11350:2012(E)
6.16 Eight-channel multistepper pipette (repeater pipette).
6.17 Eight-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
6.18 Spectrophotometer.
6.19 Transparent sterile polystyrene 24 well and 384 well plates with flat bottom and lid.
6.20 Microplate photometer for 24 well plates and optionally for 384 well plates, filters: 420 nm ± 15 nm
and 595 nm ± 10 nm.
6.21 Clean bench.
6.22 Petri dishes with venting ribs, diameter approximately 94 mm, height approximately 16 mm.
6.23 Cryogenic vials, sterile, 1 ml, 10 ml.
7 Reagents, media and dilutions
7.1 General. As far as possible, use “reagent grade” chemicals. If hydrates of anhydrous compounds or hydrates
different from those specified are used, ensure that the appropriate mass of the main compound is employed.
When necessary, autoclave for 20 min at 121 °C ± 2 °C. Cover vessels loosely (e.g. with aluminium foil). Never
seal air-tight.
7.2 Water, grade 1, as defined in ISO 3696, or water with a conductivity of ≤5 µS/cm.
If sterile water is needed, sterilize by sterile filtration (0,2 µm) or autoclaving. Water as specified here is also
used for the stepwise dilution of the test sample.
7.3 Tester strains. Use mutant strains of Salmonella Typhimurium LT2, which enable detection of point
mutations, to determine the mutagenic potential of a test sample. Since point mutations can be subdivided into
two classes (frameshift mutations and base pair substitutions), the two tester strains TA 98 and TA 100 are used.
TA 98 contains as a marker the frameshift mutation (+2 type) hisD3052, whereas TA 100 bears the base pair
substitution hisG46.
In addition, both strains shall have the following genetic properties:
— they contain the plasmid pKM101, coding for ampicillin resistance;
— they are all deep rough, e.g. partly deficient in lipopolysaccharide side chains, enabling also larger
molecules to penetrate the bacterial cell wall and to cause mutations;
— due to a mutation in uvrB, the capability of the tester strains to repair DNA-damage is limited and the
likelihood that DNA-damage results in mutations is increased.
NOTE The use of additional tester strains is described in Annex K.
7.4 2-Aminoanthracene (2-AA), C H N, CAS No: 613-13-8.
14 11
7.5 Ampicillin sodium salt, C H N NaO S, CAS No: 69-52-3.
16 18 3 4
7.6 d-Biotin, C H N O S, CAS No: 58-85-5.
10 16 2 3
7.7 Bromocresol purple, sodium salt, CAS No: 62625-30-3.
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ISO 11350:2012(E)
7.8 Citric acid monohydrate, C H O •H O, CAS No: 5949-29-1.
6 8 7 2
7.9 Dimethylsulfoxide, DMSO, C H SO, CAS No: 67-68-5.
2 6
7.10 d-Glucose, anhydrous, C H O , CAS No: 50-99-7.
6 12 6
7.11 d-Glucose-6-phosphate disodium salt hydrate, G-6-P-Na , C H Na O P•2H O CAS No: 3671-99-6.
2 6 11 2 9 2
7.12 Hydrochloric acid solution, HCl, c(HCl) = 1 mol/l.
7.13 Magnesium chloride hexahydrate, MgCl •6H O, CAS No: 7791-18-6.
2 2
7.14 Magnesium sulfate heptahydrate, MgSO •7H O, CAS No: 10034-99-8.
4 2
7.15 Potassium chloride, KCl, CAS No: 7447-40-7.
7.16 Dipotassium hydrogenphosphate, K HPO , CAS No: 7758-11-4.
2 4
7.17 Sodium ammonium hydrogenphosphate tetrahydrate, NaNH HPO •4H O, CAS No: 7583-13-3.
4 4 2
7.18 Sodium chloride, NaCl, CAS No: 7647-14-5.
7.19 Sodium dihydrogenphosphate, anhydrous, NaH PO , CAS No: 7558-80-7.
2 4
7.20 Disodium hydrogenphosphate, anhydrous, Na HPO , CAS No: 7558-79-4.
2 4
7.21 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
7.22 β-Nicotinamide adenine dinucleotide phosphate sodium salt, NADP·H O, C H N NaO P ·H O,
2 21 27 7 17 3 2
CAS No: 698999-85-8.
7.23 Nitrofurantoin (NF), CAS No: 67-20-9.
7.24 4-Nitro-o-phenylenediamine (4-NOPD), CAS No: 99-56-9.
1)
7.25 Nutrient broth powder.
1)
7.26 S9 fraction (liver homogenate; induced by phenobarbital/β-naphthoflavone).
7.27 l-Histidine, C H N O , CAS No: 71-00-1.
6 9 3 2
7.28 Phosphate buffer.
7.28.1 Sodium dihydrogenphosphate buffer, c(NaH PO ) = 0,2 mol/l.
2 4
Dissolve 14,39 g NaH PO (or 16,55 g NaH PO •H O) in 600 ml of water (7.2).
2 4 2 4 2

1) This reagent is commercially available. This information is given for the convenience of users of this International
Standard and does not constitute an endorsement by ISO of these products.
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ISO 11350:2012(E)
7.28.2 Disodium hydrogenphosphate buffer, c(Na HPO ) = 0,2 mol/l. Dissolve 28,39 g Na HPO in 1 000 ml
2 4 2 4
of water (7.2).
Add sodium dihydrogenphosphate buffer (7.28.1) to disodium hydrogenphosphate buffer (7.28.2) until a pH value
of 7,4 is reached and autoclave. Store at room temperature in the dark. The solution is stable for at least 1 year.
7.29 d-Biotin solution. Dissolve 12,2 mg d-biotin (7.6) in 100 ml of water (7.2) by boiling up. After cooling,
sterilize by filtration (0,2 µm filter). Store 10 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.30 l-Histidine solution. Dissolve 50 mg of l-histidine (7.27) in 50 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 1,5 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23). Aqueous solutions stored
as frozen aliquots are stable for at least 1 year.
7.31 Glucose-6-phosphate solution. Dissolve 0,68 g of d-glucose-6-phosphate (7.11) in 10 ml of water (7.2)
and sterilize by filtration (0,2 µm). Store aliquots (e.g. 200 µl) at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.32 NADP solution, c(NADP) = 0,04 mol/l. Dissolve the appropriate mass of NADP in 10 ml of water (7.2) to
obtain a final concentration of 0,04 mol/l and sterilize by filtration (0,2 µm). Store aliquots (e.g. 700 µl) at −18 °C
or below in sterile cryogenic vials (6.23). Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
Various hydrates of NADP are available. The actual molecular weight is specified in the product data sheet.
Calculate the amount of NADP required according to the molecular weight given.
7.33 Potassium chloride solution. Dissolve 74,56 g of KCl (7.15) in 1 000 ml of water (7.2) and autoclave.
Store at room temperature. The solution is stable for at least 1 year.
7.34 MgCl •6H O solution. Dissolve 50,83 g of MgCl •6H O (7.13) in 1 000 ml of water (7.2) and autoclave
2 2 2 2
the solution. Store at room temperature. The solution is stable for at least 1 year.
7.35 Bromocresol purple solution. Dissolve 51 mg of bromocresol purple sodium salt (7.7) in 30 ml of
water (7.2). Prepare this solution freshly before addition to the reversion indicator medium (7.40).
7.36 Ampicillin solution. Dissolve 500 mg of ampicillin (7.5) in 10 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 500 µl aliquots at −18 °C or below in sterile cryogenic vials (6.23). The solution is stable for
at least 6 months.
2)
7.37 Growth medium. Dissolve 4,7 g of nutrient broth powder and 0,31 g of sodium chloride (7.18) in 200 ml
of water (7.2). Adjust the pH to 7,5 ± 0,1. Add water (7.2) to 250 ml and autoclave the solution.
The following final concentrations in the growth medium shall result:
— 7,5 g/l meat extract;
— 7,5 g/l peptone;
— 5,0 g/l sodium chloride.
Solutions stored under sterile conditions as frozen aliquots are stable for at least 1 year.
2) Use nutrient broth powder containing 40 % meat extract, 40 % peptone, and 20 % sodium chloride.
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ISO 11350:2012(E)
7.38 Exposure medium. Prepare a medium for incubation of bacteria with the sample containing a low amount
of l-histidine to support a few cell divisions.
Dissolve consecutively the following ingredients in 900 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 1 000 ml, adjust the pH to 7,0 ± 0,2, if necessary, and sterilize by filtration (0,2 µm filter).
Store the medium at 2 °C to 8 °C.
Add, per 100 ml, 0,6 ml of d-biotin solution (7.29) and 0,1 ml of l-histidine solution (7.30) under sterile conditions.
Prepare only the amount of medium necessary for the next 2 weeks. Store the medium at 2 °C to 8 °C.
7.39 Exposure medium concentrate. Dissolve consecutively the following ingredients in 70 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 93 ml, adjust the pH, if necessary, and sterilize by filtration (0,2 µm filter). Store the medium
concentrate at 2 °C to 8 °C.
Add 6 ml of d-biotin solution (7.29) and 1 ml of l-histidine solution (7.30) under sterile conditions. Prepare only
the amount of medium necessary for the next 2 weeks. Store the medium concentrate at 2 °C to 8 °C.
7.40 Reversion indicator medium. Prepare a pH indicator medium without l-histidine.
7.40.1 Solution I. Dissolve the following ingredients in 950 ml water in the given order:
— 0,4 g magnesium sulfate heptahydrate (7.14);
— 4,0 g citric acid (7.8);
— 20,0 g dipotassium hydrogenphosphate (7.16);
— 7,0 g sodium ammonium hydrogenphosphate tetrahydrate (7.17).
Add water (7.2) to 1 000 ml and add 30,0 ml of bromocresol purple solution (7.35). Adjust the pH to 7,3 ± 0,1.
Transfer the solution one half each into two 1 000 ml flasks and autoclave.
7.40.2 Solution II. Dissolve 8,0 g of d-glucose (7.10) in 800 ml of water (7.2). Adjust the pH to 7,3 ± 0,1.
Transfer both halves of the solution into two 1 000 ml flasks and autoclave.
7.40.3 Mixing and storage. After cooling to ambient temperature, mix 515 ml of solution I (7.40.1) with 400 ml of
solution II (7.40.2) under sterile conditions. Add 20 ml of d-biotin solution (7.29) under sterile conditions to each flask.
Store the medium at room temperature in the dark. The medium is stable for at least 1 month.
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ISO 11350:2012(E)
7.41 Control solutions.
7.41.1 Negative controls. For preparation of the negative controls, always use the same solvent as for the samples
to be tested. This is usually water (7.2) when testing water samples and DMSO (7.9) when testing chemicals.
7.41.2 Positive controls. In general, dissolve 10 mg of each positive control substance in 10 ml of
DMSO (7.9). Prepare 50 µl aliquots as stock solutions in sterile cryogenic vials and store them at −18 °C or below.
Under these conditions stock solutions are stable for at least 1 year. On the day of the test, unfreeze one aliquot.
7.41.3 Strain TA 98 without S9 mix. Use 4-nitro-o-phenylenediamine (4-NOPD) (7.24) as positive control
substance for strain TA 98 without S9 mix.
Dilute the stock solution 1→2 with DMSO (7.9). This dilution is used in the test.
7.41.4 Strain TA 100 without S9 mix. Use nitrofurantoin (NF) (7.23) as a positive control substance for strain
TA 100 without S9 mix.
Dilute the stock solution 1→80 with DMSO. This dilution is used in the test.
7.41.5 Strain TA 98 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as a positive control substance for
strain TA 98 with S9 mix.
Dissolve the stock solution 1→200 with DMSO. This dilution is used in the test.
7.41.6 Strain TA 100 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as positive control substance for
strain TA 100 with S9 mix.
Dissolve the stock solution 1→50 with DMSO. This dilution is used in the test.
8 Sampling and samples
Test the samples immediately after sampling. If this is not possible, keep water samples at 0 °C to 5 °C (for
<48 h) or below −18 °C (for up to 2 months). For multiple testing divide larger samples in advance into appropriate
portions, since thawed samples can only be used on the same day.
Samples containing solids should be centrifuged to separate them. In this case, only the supernatant is
processed further.
Sterilize all samples using sterile filters (0,45 µm). Homogenize test samples by thoroughly shaking before use.
Adjust the sample to a pH of 7,2 ± 0,2 using either HCl (7.12) or NaOH solution (7.21). Select the acid or alkali
concentrations such that the added volumes are as small as possible. Avoid overtitration. Take into account the
[5]
change in the sample’s pH and resulting effects (see ISO 5667-16 ).
Perform dilutions of the test sample as specified in Tables 2 and 3 with sterilized water (7.2).
9 Procedure
9.1 Overnight culture
Under sterile conditions, pipette 20 ml of growth medium (7.37) supplemented with 20 µl of ampicillin
solution (7.36) into a 100 ml Erlenmeyer flask (6.14) hermetically sealed with caps or aluminium foil and mix by
gentle agitation.
Add 20 µl of the respective test strain (TA 98 or TA 100) immediately after thawing. Incubate the culture at
37 °C ± 1 °C for 10 h. If the required cell density (9.3.1, G.1.1) is not reached, extend incubation time to 12 h.
If the required cel
...

SLOVENSKI STANDARD
SIST ISO 11350:2013
01-januar-2013
.DNRYRVWYRGH'RORþHYDQMHJHQRWRNVLþQRVWL]DYRGRLQRGSDGQRYRGR
6DOPRQHOODPLNURVRPVNLIOXNWXDFLMVNLSUHVNXV $PHVRYIOXNWXDFLMVNLSUHVNXV
Water quality - Determination of the genotoxicity of water and waste water -
Salmonella/microsome fluctuation test (Ames fluctuation test)
Qualité de l'eau - Évaluation de la génotoxicité des eaux résiduaires - Essai de
Salmonella/microsome (essai d'Ames-fluctuation)
Ta slovenski standard je istoveten z: ISO 11350:2012
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST ISO 11350:2013 en,fr
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST ISO 11350:2013

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SIST ISO 11350:2013
INTERNATIONAL ISO
STANDARD 11350
First edition
2012-05-15
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome fluctuation test
(Ames fluctuation test)
Qualité de l’eau — Évaluation de la génotoxicité des eaux résiduaires —
Essai de Salmonella/microsome (essai d’Ames-fluctuation)
Reference number
ISO 11350:2012(E)
©
ISO 2012

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SIST ISO 11350:2013
ISO 11350:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s
member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved

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SIST ISO 11350:2013
ISO 11350:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Interferences . 3
5 Principle . 4
6 Apparatus and materials . 4
7 Reagents, media and dilutions . 5
8 Sampling and samples . 9
9 Procedure . 9
9.1 Overnight culture . 9
9.2 Preparation of S9 mix .10
9.3 Testing of water samples .10
9.4 Measurement of revertant growth .13
9.5 Calculation of cytotoxicity .13
10 Validity criteria .14
11 Assessment criteria .14
12 Test report .14
Annex A (normative) Nutrient broth and agar .15
Annex B (normative) Preparation of ampicillin agar plates and stock cultures.16
Annex C (normative) Checking of genotype .17
Annex D (normative) S9 fraction .18
Annex E (informative) Example for application of samples on a 24 well plate .19
Annex F (informative) Example for reporting .21
Annex G (informative) Testing of chemicals .22
Annex H (informative) Precision data.25
Annex I (informative) Statistical assessment .27
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water —
A simplified evaluation for testing of waste water .33
Annex K (informative) Use of additional tester strains .35
Bibliography .36
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International
Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 11350 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2012 – All rights reserved

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SIST ISO 11350:2013
INTERNATIONAL STANDARD ISO 11350:2012(E)
Water quality — Determination of the genotoxicity of water and
waste water — Salmonella/microsome fluctuation test (Ames
fluctuation test)
WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.
IMPORTANT — It is absolutely essential that tests conducted according to this International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination of the genotoxic potential of water and
waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and
TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chemicals that
induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are
involved in the biosynthesis of the amino acid, histidine.
[8]
NOTE 1 ISO 13829 applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents.
This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.
NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove necessary.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable
for its application. For dated references, only the edition cited applies. For undated references, the latest edition
of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 7027, Water quality — Determination of turbidity
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3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
cofactor solution
aqueous solution of chemicals (e.g. NADP, glucose-6-phosphate, and inorganic salts) needed for the activity
of the enzymes in the S9 fraction
[10]
[Source: ISO 21427-2:2006, definition 3.2]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[6]
[Source: ISO 6107-6:2004, definition 24]
3.3
dilution level
D
denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water with dilution
water as integral number
NOTE 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. [In this International Standard,
the arrow indicates the transition from initial total volume to final total volume.] The corresponding and smallest possible
value of D is 1.
[6]
[Source: ISO 6107-6:2004, definition 28]
3.4
lowest ineffective dilution
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant increase in the
number of revertant wells compared with the negative control
NOTE 1 to entry: LID is determined for each incubation condition (strain, ±S9 mix). The highest LID value is decisive for
the overall assessment.
3.5
induction rate
difference between the mean value of wells with revertant growth counted on the plates treated with a dose
of the test sample or with a positive control, and the mean value of the corresponding wells treated with the
negative control using the same strain under identical conditions
[6]
[Source: ISO 6107-6:2004, definition 43, modified: “wells with revertant growth” replaces “mutant colonies”;
“corresponding wells” replaces “corresponding plates”]
3.6
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing preculture,
in fresh medium
[6]
[Source: ISO 6107-6:2004, definition 44]
3.7
negative control
dilution water without test sample
[6]
[Source: ISO 6107-6:2004, definition 51]
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3.8
revertant growth
visible mutant colonies on the microplate at the end of the respective test
3.9
overnight culture
culture started late in the afternoon and incubated overnight (usually about 16 h) to be ready during the following
morning for purposes such as the inoculation of a preculture
[6]
[Source: ISO 6107-6:2004, definition 54]
NOTE 1 to entry For specification, see 9.1.
3.10
positive control
any well characterized material and/or substance, which, when evaluated by a specific test method, demonstrates
the suitability of the test system to yield a reproducible, appropriate positive or negative response in the test system
[7]
[Source: ISO 10993-12:—, definition 3.12]
NOTE 1 to entry The positive controls mentioned in this International Standard are dissolved in dimethyl sulfoxide (DMSO)
prior to use. For the purposes of this International Standard, the positive controls are known mutagens which are suitable
for the verification of the sensitivity of the method and/or the activity of the S9 mix.
3.11
S9 fraction
supernatant at 9 000g of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to
300 g) pretreated with a substance or substance combination appropriate for enzyme induction
[6]
[Source: ISO 6107-6:2004, definition 74]
3.12
S9 mix
mixture of S9 fraction and cofactor solution
[6]
[Source: ISO 6107-6:2004, definition 75]
3.13
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[6]
[Source: ISO 6107-6:2004, definition 87]
3.14
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all preparation
steps such as centrifugation, filtration, homogenization, pH adjustment and determination of ionic strength
[6]
[Source: ISO 6107-6:2004, definition 92]
4 Interferences
Bacteriotoxic effects of the test sample can lead to a reduction of viable bacteria and to a reduction of wells with
revertants due to a repression of revertant growth.
This method includes sterile filtration of water and waste water prior to the test. Due to this filtration, solid
particles are separated from the test sample. Thus, there is a possibility that genotoxic substances adsorbed
on particles are not detected.
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5 Principle
The bacteria are exposed under defined conditions to various concentrations of the test sample and incubated
for 100 min at 37 °C ± 1 °C in 24 well plates. Due to this exposure, genotoxic agents enclosed in the test sample
can induce mutations in one or both marker genes of the bacterial strains used (hisG46 for TA 100 and hisD3052
for TA 98) in correlation with the applied concentrations. Induction of mutations causes a concentration-related
increase in the number of mutant colonies.
After exposure of the bacteria, reversion indicator medium (7.40), containing the pH indicator dye bromocresol
purple (7.7), is added to the wells. Subsequently, the batches are distributed to 384 well plates (48 wells for
each parallel) and incubated for 48 h to 72 h (9.3.2, 9.3.3).
Mutagenic activity of the test sample is determined by counting the number of purple to yellow shifted wells (per
48 wells of each parallel), treated with the undiluted or the diluted test sample, compared to the negative control.
The lowest dilution (1→N) of the test sample which induces no mutagenic effect under all experimental
conditions (if any mutagenic effect is induced by the test sample) is the criterion for evaluating the mutagenic
potential. Sample dilutions above this (1→A, A < N) shall induce a mutagenic effect according to the criteria of
this International Standard in at least one strain under at least one activation condition (with or without addition
of S9 mix). The respective LID value is N. If no mutagenic effect is observed under all experimental conditions,
this dilution is 1→1 and the respective LID value is 1.
6 Apparatus and materials
6.1 Temperature- and time-controlled incubator, 37 °C ± 1 °C.
6.2 pH meter.
6.3 Analytical balance.
6.4 Steam sterilizer.
6.5 Dry sterilizer.
6.6 Magnetic stirrer.
6.7 Rotary mixer.
6.8 Freezer, capable of being maintained at ≤ −18 °C and at ≤ −70 °C.
6.9 Pipettes, 0,1 ml, 0,5 ml, 1 ml, 2 ml, 5 ml, 10 ml and 25 ml, of glass or plastics.
6.10 Storage bottles, 250 ml and 1 000 ml.
6.11 Measuring cylinders, 100 ml and 200 ml.
6.12 Volumetric flasks, 20 ml, 200 ml and 500 ml.
6.13 Sterile filters, 0,2 µm and 0,45 µm.
6.14 Erlenmeyer flasks, 50 ml, 100 ml and 250 ml.
6.15 Inoculating loops.
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6.16 Eight-channel multistepper pipette (repeater pipette).
6.17 Eight-channel pipettes, 5 µl to 50 µl and 50 µl to 300 µl.
6.18 Spectrophotometer.
6.19 Transparent sterile polystyrene 24 well and 384 well plates with flat bottom and lid.
6.20 Microplate photometer for 24 well plates and optionally for 384 well plates, filters: 420 nm ± 15 nm
and 595 nm ± 10 nm.
6.21 Clean bench.
6.22 Petri dishes with venting ribs, diameter approximately 94 mm, height approximately 16 mm.
6.23 Cryogenic vials, sterile, 1 ml, 10 ml.
7 Reagents, media and dilutions
7.1 General. As far as possible, use “reagent grade” chemicals. If hydrates of anhydrous compounds or hydrates
different from those specified are used, ensure that the appropriate mass of the main compound is employed.
When necessary, autoclave for 20 min at 121 °C ± 2 °C. Cover vessels loosely (e.g. with aluminium foil). Never
seal air-tight.
7.2 Water, grade 1, as defined in ISO 3696, or water with a conductivity of ≤5 µS/cm.
If sterile water is needed, sterilize by sterile filtration (0,2 µm) or autoclaving. Water as specified here is also
used for the stepwise dilution of the test sample.
7.3 Tester strains. Use mutant strains of Salmonella Typhimurium LT2, which enable detection of point
mutations, to determine the mutagenic potential of a test sample. Since point mutations can be subdivided into
two classes (frameshift mutations and base pair substitutions), the two tester strains TA 98 and TA 100 are used.
TA 98 contains as a marker the frameshift mutation (+2 type) hisD3052, whereas TA 100 bears the base pair
substitution hisG46.
In addition, both strains shall have the following genetic properties:
— they contain the plasmid pKM101, coding for ampicillin resistance;
— they are all deep rough, e.g. partly deficient in lipopolysaccharide side chains, enabling also larger
molecules to penetrate the bacterial cell wall and to cause mutations;
— due to a mutation in uvrB, the capability of the tester strains to repair DNA-damage is limited and the
likelihood that DNA-damage results in mutations is increased.
NOTE The use of additional tester strains is described in Annex K.
7.4 2-Aminoanthracene (2-AA), C H N, CAS No: 613-13-8.
14 11
7.5 Ampicillin sodium salt, C H N NaO S, CAS No: 69-52-3.
16 18 3 4
7.6 d-Biotin, C H N O S, CAS No: 58-85-5.
10 16 2 3
7.7 Bromocresol purple, sodium salt, CAS No: 62625-30-3.
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7.8 Citric acid monohydrate, C H O •H O, CAS No: 5949-29-1.
6 8 7 2
7.9 Dimethylsulfoxide, DMSO, C H SO, CAS No: 67-68-5.
2 6
7.10 d-Glucose, anhydrous, C H O , CAS No: 50-99-7.
6 12 6
7.11 d-Glucose-6-phosphate disodium salt hydrate, G-6-P-Na , C H Na O P•2H O CAS No: 3671-99-6.
2 6 11 2 9 2
7.12 Hydrochloric acid solution, HCl, c(HCl) = 1 mol/l.
7.13 Magnesium chloride hexahydrate, MgCl •6H O, CAS No: 7791-18-6.
2 2
7.14 Magnesium sulfate heptahydrate, MgSO •7H O, CAS No: 10034-99-8.
4 2
7.15 Potassium chloride, KCl, CAS No: 7447-40-7.
7.16 Dipotassium hydrogenphosphate, K HPO , CAS No: 7758-11-4.
2 4
7.17 Sodium ammonium hydrogenphosphate tetrahydrate, NaNH HPO •4H O, CAS No: 7583-13-3.
4 4 2
7.18 Sodium chloride, NaCl, CAS No: 7647-14-5.
7.19 Sodium dihydrogenphosphate, anhydrous, NaH PO , CAS No: 7558-80-7.
2 4
7.20 Disodium hydrogenphosphate, anhydrous, Na HPO , CAS No: 7558-79-4.
2 4
7.21 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
7.22 β-Nicotinamide adenine dinucleotide phosphate sodium salt, NADP·H O, C H N NaO P ·H O,
2 21 27 7 17 3 2
CAS No: 698999-85-8.
7.23 Nitrofurantoin (NF), CAS No: 67-20-9.
7.24 4-Nitro-o-phenylenediamine (4-NOPD), CAS No: 99-56-9.
1)
7.25 Nutrient broth powder.
1)
7.26 S9 fraction (liver homogenate; induced by phenobarbital/β-naphthoflavone).
7.27 l-Histidine, C H N O , CAS No: 71-00-1.
6 9 3 2
7.28 Phosphate buffer.
7.28.1 Sodium dihydrogenphosphate buffer, c(NaH PO ) = 0,2 mol/l.
2 4
Dissolve 14,39 g NaH PO (or 16,55 g NaH PO •H O) in 600 ml of water (7.2).
2 4 2 4 2

1) This reagent is commercially available. This information is given for the convenience of users of this International
Standard and does not constitute an endorsement by ISO of these products.
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7.28.2 Disodium hydrogenphosphate buffer, c(Na HPO ) = 0,2 mol/l. Dissolve 28,39 g Na HPO in 1 000 ml
2 4 2 4
of water (7.2).
Add sodium dihydrogenphosphate buffer (7.28.1) to disodium hydrogenphosphate buffer (7.28.2) until a pH value
of 7,4 is reached and autoclave. Store at room temperature in the dark. The solution is stable for at least 1 year.
7.29 d-Biotin solution. Dissolve 12,2 mg d-biotin (7.6) in 100 ml of water (7.2) by boiling up. After cooling,
sterilize by filtration (0,2 µm filter). Store 10 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.30 l-Histidine solution. Dissolve 50 mg of l-histidine (7.27) in 50 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 1,5 ml aliquots at −18 °C or below in sterile cryogenic vials (6.23). Aqueous solutions stored
as frozen aliquots are stable for at least 1 year.
7.31 Glucose-6-phosphate solution. Dissolve 0,68 g of d-glucose-6-phosphate (7.11) in 10 ml of water (7.2)
and sterilize by filtration (0,2 µm). Store aliquots (e.g. 200 µl) at −18 °C or below in sterile cryogenic vials (6.23).
Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
7.32 NADP solution, c(NADP) = 0,04 mol/l. Dissolve the appropriate mass of NADP in 10 ml of water (7.2) to
obtain a final concentration of 0,04 mol/l and sterilize by filtration (0,2 µm). Store aliquots (e.g. 700 µl) at −18 °C
or below in sterile cryogenic vials (6.23). Aqueous solutions stored as frozen aliquots are stable for at least 1 year.
Various hydrates of NADP are available. The actual molecular weight is specified in the product data sheet.
Calculate the amount of NADP required according to the molecular weight given.
7.33 Potassium chloride solution. Dissolve 74,56 g of KCl (7.15) in 1 000 ml of water (7.2) and autoclave.
Store at room temperature. The solution is stable for at least 1 year.
7.34 MgCl •6H O solution. Dissolve 50,83 g of MgCl •6H O (7.13) in 1 000 ml of water (7.2) and autoclave
2 2 2 2
the solution. Store at room temperature. The solution is stable for at least 1 year.
7.35 Bromocresol purple solution. Dissolve 51 mg of bromocresol purple sodium salt (7.7) in 30 ml of
water (7.2). Prepare this solution freshly before addition to the reversion indicator medium (7.40).
7.36 Ampicillin solution. Dissolve 500 mg of ampicillin (7.5) in 10 ml of water (7.2) and sterilize by filtration
(0,2 µm filter). Store 500 µl aliquots at −18 °C or below in sterile cryogenic vials (6.23). The solution is stable for
at least 6 months.
2)
7.37 Growth medium. Dissolve 4,7 g of nutrient broth powder and 0,31 g of sodium chloride (7.18) in 200 ml
of water (7.2). Adjust the pH to 7,5 ± 0,1. Add water (7.2) to 250 ml and autoclave the solution.
The following final concentrations in the growth medium shall result:
— 7,5 g/l meat extract;
— 7,5 g/l peptone;
— 5,0 g/l sodium chloride.
Solutions stored under sterile conditions as frozen aliquots are stable for at least 1 year.
2) Use nutrient broth powder containing 40 % meat extract, 40 % peptone, and 20 % sodium chloride.
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7.38 Exposure medium. Prepare a medium for incubation of bacteria with the sample containing a low amount
of l-histidine to support a few cell divisions.
Dissolve consecutively the following ingredients in 900 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 1 000 ml, adjust the pH to 7,0 ± 0,2, if necessary, and sterilize by filtration (0,2 µm filter).
Store the medium at 2 °C to 8 °C.
Add, per 100 ml, 0,6 ml of d-biotin solution (7.29) and 0,1 ml of l-histidine solution (7.30) under sterile conditions.
Prepare only the amount of medium necessary for the next 2 weeks. Store the medium at 2 °C to 8 °C.
7.39 Exposure medium concentrate. Dissolve consecutively the following ingredients in 70 ml water:
— 0,2 g magnesium sulfate heptahydrate (7.14);
— 2,0 g citric acid (7.8);
— 10,0 g dipotassium hydrogenphosphate (7.16);
— 3,5 g sodium ammonium hydrogenphosphate tetrahydrate (7.17);
— 4,0 g d-glucose (7.10).
Add water (7.2) to 93 ml, adjust the pH, if necessary, and sterilize by filtration (0,2 µm filter). Store the medium
concentrate at 2 °C to 8 °C.
Add 6 ml of d-biotin solution (7.29) and 1 ml of l-histidine solution (7.30) under sterile conditions. Prepare only
the amount of medium necessary for the next 2 weeks. Store the medium concentrate at 2 °C to 8 °C.
7.40 Reversion indicator medium. Prepare a pH indicator medium without l-histidine.
7.40.1 Solution I. Dissolve the following ingredients in 950 ml water in the given order:
— 0,4 g magnesium sulfate heptahydrate (7.14);
— 4,0 g citric acid (7.8);
— 20,0 g dipotassium hydrogenphosphate (7.16);
— 7,0 g sodium ammonium hydrogenphosphate tetrahydrate (7.17).
Add water (7.2) to 1 000 ml and add 30,0 ml of bromocresol purple solution (7.35). Adjust the pH to 7,3 ± 0,1.
Transfer the solution one half each into two 1 000 ml flasks and autoclave.
7.40.2 Solution II. Dissolve 8,0 g of d-glucose (7.10) in 800 ml of water (7.2). Adjust the pH to 7,3 ± 0,1.
Transfer both halves of the solution into two 1 000 ml flasks and autoclave.
7.40.3 Mixing and storage. After cooling to ambient temperature, mix 515 ml of solution I (7.40.1) with 400 ml of
solution II (7.40.2) under sterile conditions. Add 20 ml of d-biotin solution (7.29) under sterile conditions to each flask.
Store the medium at room temperature in the dark. The medium is stable for at least 1 month.
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7.41 Control solutions.
7.41.1 Negative controls. For preparation of the negative controls, always use the same solvent as for the samples
to be tested. This is usually water (7.2) when testing water samples and DMSO (7.9) when testing chemicals.
7.41.2 Positive controls. In general, dissolve 10 mg of each positive control substance in 10 ml of
DMSO (7.9). Prepare 50 µl aliquots as stock solutions in sterile cryogenic vials and store them at −18 °C or below.
Under these conditions stock solutions are stable for at least 1 year. On the day of the test, unfreeze one aliquot.
7.41.3 Strain TA 98 without S9 mix. Use 4-nitro-o-phenylenediamine (4-NOPD) (7.24) as positive control
substance for strain TA 98 without S9 mix.
Dilute the stock solution 1→2 with DMSO (7.9). This dilution is used in the test.
7.41.4 Strain TA 100 without S9 mix. Use nitrofurantoin (NF) (7.23) as a positive control substance for strain
TA 100 without S9 mix.
Dilute the stock solution 1→80 with DMSO. This dilution is used in the test.
7.41.5 Strain TA 98 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as a positive control substance for
strain TA 98 with S9 mix.
Dissolve the stock solution 1→200 with DMSO. This dilution is used in the test.
7.41.6 Strain TA 100 with S9 mix. Use 2-aminoanthracene (2-AA) (7.4) as positive control substance for
strain TA 100 with S9 mix.
Dissolve the stock solution 1→50 with DMSO. This dilution is used in the test.
8 Sampling and samples
Test the samples immediately after sampling. If this is not possible, keep water samples at 0 °C to 5 °C (for
<48 h) or below −18 °C (for up to 2 months). For multiple testing divide larger samples in advance into appropriate
portions, since thawed samples can
...

NORME ISO
INTERNATIONALE 11350
Première édition
2012-05-15
Qualité de l’eau — Évaluation de la
génotoxicité des eaux résiduaires —
Essai de Salmonella/microsome (essai
d’Ames-fluctuation)
Water quality — Determination of the genotoxicity of water and waste
water — Salmonella/microsome fluctuation test (Ames fluctuation test)
Numéro de référence
ISO 11350:2012(F)
©
ISO 2012

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ISO 11350:2012(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2012
Droits de reproduction réservés. Sauf prescription différente, aucune partie de cette publication ne peut être reproduite ni utilisée sous
quelque forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie et les microfilms, sans l’accord écrit
de l’ISO à l’adresse ci-après ou du comité membre de l’ISO dans le pays du demandeur.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Publié en Suisse
ii © ISO 2012 – Tous droits réservés

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ISO 11350:2012(F)
Sommaire Page
Avant-propos .iv
1 Domaine d’application . 1
2 Références normatives . 1
3 Termes et définitions . 2
4 Interférences . 3
5 Principe . 4
6 Appareillage et matériaux . 4
7 Réactifs, milieux et dilutions . 5
8 Échantillonnage et échantillons .10
9 Mode opératoire .10
9.1 Culture d’une nuit .10
9.2 Préparation du mélange S9 .10
9.3 Essais des échantillons d’eau . 11
9.4 Mesure de la croissance des révertants .13
9.5 Calcul de la cytotoxicité .14
10 Critères de validité .14
11 Critères d’évaluation .14
12 Rapport d’essai .14
Annexe A (normative) Bouillon nutritif et gélose .16
Annexe B (normative) Préparation des plaques de gélose à l’ampicilline et des cultures mères .17
Annexe C (normative) Vérification du génotype .18
Annexe D (normative) Fraction S9 .19
Annexe E (informative) Exemple d’application d’échantillons sur une plaque à 24 puits.20
Annexe F (informative) Exemple de rapport .22
Annexe G (informative) Essai des substances chimiques .23
Annexe H (informative) Données de fidélité .26
Annexe I (informative) Évaluation statistique .28
Annexe J (informative) Mesure de la plus faible dilution sans effet (LID) d’une eau résiduaire —
Évaluation simplifiée des essais portant sur les eaux résiduaires .34
Annexe K (informative) Utilisation de souches de contrôle supplémentaires .36
Bibliographie .37
© ISO 2012 – Tous droits réservés iii

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ISO 11350:2012(F)
Avant-propos
L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes nationaux de
normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est en général confiée aux
comités techniques de l’ISO. Chaque comité membre intéressé par une étude a le droit de faire partie du comité
technique créé à cet effet. Les organisations internationales, gouvernementales et non gouvernementales,
en liaison avec l’ISO participent également aux travaux. L’ISO collabore étroitement avec la Commission
électrotechnique internationale (CEI) en ce qui concerne la normalisation électrotechnique.
Les Normes internationales sont rédigées conformément aux règles données dans les Directives ISO/CEI, Partie 2.
La tâche principale des comités techniques est d’élaborer les Normes internationales. Les projets de Normes
internationales adoptés par les comités techniques sont soumis aux comités membres pour vote. Leur publication
comme Normes internationales requiert l’approbation de 75 % au moins des comités membres votants.
L’attention est appelée sur le fait que certains des éléments du présent document peuvent faire l’objet de droits
de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable de ne pas avoir
identifié de tels droits de propriété et averti de leur existence.
L’ISO 11350 a été élaborée par le comité technique ISO/TC 147, Qualité de l’eau, sous-comité SC 5,
Méthodes biologiques.
iv © ISO 2012 – Tous droits réservés

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NORME INTERNATIONALE ISO 11350:2012(F)
Qualité de l’eau — Évaluation de la génotoxicité des eaux
résiduaires — Essai de Salmonella/microsome (essai
d’Ames-fluctuation)
AVERTISSEMENT — Il convient que l’utilisateur de la présente Norme internationale connaisse bien
les pratiques courantes de laboratoire. La présente Norme internationale n’a pas pour but de traiter
tous les problèmes de sécurité qui sont, le cas échéant, liés à son utilisation. Il incombe à l’utilisateur
d’établir des pratiques appropriées en matière d’hygiène et de sécurité, et de s’assurer de la conformité
à la réglementation nationale en vigueur.
IMPORTANT — Il est absolument essentiel que les essais réalisés conformément à la présente Norme
internationale soient exécutés par un personnel ayant reçu une formation adéquate.
1 Domaine d’application
La présente Norme internationale spécifie une méthode d’évaluation de la génotoxicité des eaux résiduaires
en utilisant les souches bactériennes Salmonella enterica subsp. enterica sérotype Typhimurium TA 98 et
TA 100 au cours d’un essai en fluctuation. Cette combinaison de souches permet d’évaluer la génotoxicité des
substances chimiques qui produisent des mutations ponctuelles (substitutions de paires de base et mutations
de changement de phase) dans les gènes codant pour les enzymes impliquées dans la biosynthèse de l’acide
aminé histidine.
[8]
NOTE 1 L’ISO 13829 est applicable pour la détermination de la génotoxicité des échantillons contenant des agents
de réticulation de l’ADN.
Cette méthode est applicable aux substances suivantes:
— eau douce;
— eaux résiduaires;
— extraits aqueux et lixiviats;
— éluats de sédiments (eau douce);
— eau interstitielle;
— solutions aqueuses contenant des substances uniques ou des mélanges chimiques;
— eau potable.
NOTE 2 Lors de l’analyse de l’eau potable, il peut être nécessaire d’extraire et de préconcentrer les échantillons d’eau.
2 Références normatives
Les documents suivants, en tout ou partie, sont référencés de manière normative dans le présent document
et sont indispensables pour son application. Pour les références datées, seule l’édition citée s’applique. Pour
les références non datées, la dernière édition du document de référence s’applique (y compris les éventuels
amendements).
ISO 3696, Eau pour laboratoire à usage analytique — Spécification et méthodes d’essai
ISO 7027, Qualité de l’eau — Détermination de la turbidité
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ISO 11350:2012(F)
3 Termes et définitions
Pour les besoins du présent document, les termes et définitions suivants s’appliquent.
3.1
solution de cofacteurs
solution aqueuse de produits chimiques (par exemple NADP, glucose-6-phosphate et sels inorganiques)
nécessaires à l’activité des enzymes de la fraction S9
[10]
[Source: ISO 21427-2:2006 , définition 3.2]
3.2
milieu de culture
substances nutritives se présentant sous une forme et une phase (liquide ou solidifiée) favorisant la croissance
microbiologique
[6]
[Source: ISO 6107-6:2004 , définition 24]
3.3
niveau de dilution
D
dénominateur du coefficient de dilution (le numérateur étant 1) d’un mélange d’eau ou d’eaux résiduaires et
d’eau de dilution en tant que nombre entier
NOTE 1 à l’article: Pour de l’eau ou pour des eaux résiduaires non diluées, ce coefficient est, par définition, égal à
1→1. [Dans la présente Norme internationale, la flèche indique la transition de volume total initial à volume total final.] La
valeur D correspondante la plus petite possible est 1.
[6]
[Source: ISO 6107-6:2004 , définition 28]
3.4
plus faible dilution sans effet
LID
plus faible dilution dans un lot d’essai qui ne présente aucun effet, c’est-à-dire aucune augmentation
statistiquement significative du nombre de puits de révertants par rapport au témoin négatif
NOTE 1 à l’article: La LID est déterminée pour chaque condition d’incubation (souche, ± mélange S9). La valeur
LID maximale est décisive pour l’évaluation globale.
3.5
taux d’induction
différence entre la valeur moyenne des puits avec croissance de révertants dénombrés sur les plaques et
traités avec une dose de l’échantillon pour essai ou avec un témoin positif, ainsi que des puits correspondants
traités avec le témoin négatif en utilisant la même souche, dans les mêmes conditions
[6]
[Source: ISO 6107-6:2004 , définition 43, modifiée: “puits avec croissance de révertants dénombrés” remplace
“colonies mutantes dénombrées”; “puits correspondants” remplace “plaques correspondantes”]
3.6
inoculum
fraction d’une culture de micro-organismes utilisée pour démarrer une nouvelle culture ou une préculture à
croissance exponentielle dans un nouveau milieu de culture
[6]
[Source: ISO 6107-6:2004 , définition 44]
3.7
témoin négatif
eau de dilution sans l’échantillon pour essai
[6]
[Source: ISO 6107-6:2004 , définition 51]
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ISO 11350:2012(F)
3.8
croissance de révertants
colonies mutantes visibles sur la microplaque à la fin de l’essai respectif
3.9
culture d’une nuit
culture commencée en fin d’après-midi et dont l’incubation dure toute une nuit (généralement environ 16 h), afin
qu’elle puisse être utilisée le matin suivant à des fins, par exemple, d’inoculation d’une préculture
[6]
[Source: ISO 6107-6:2004 , définition 54]
NOTE 1 à l’article: Pour les spécifications, voir 9.1.
3.10
témoin positif
matériau et/ou substance bien caractérisés qui, lors d’essais réalisés selon un mode opératoire spécifique,
démontrent l’aptitude du système à fournir une réponse positive ou négative reproductible lors de
l’expérimentation
[7]
[Source: ISO 10993-12:— , définition 3.12]
NOTE 1 à l’article: Les témoins positifs mentionnés dans la présente Norme internationale sont dissous dans du
diméthylsulfoxyde (DMSO) avant utilisation. Pour les besoins de la présente Norme internationale, les témoins positifs
sont des mutagènes connus pour vérifier la sensibilité de la méthode et/ou l’activité du mélange S9.
3.11
fraction S9
surnageant à 9 000g d’un homogénat de tissus dans du KCl à 0,15 mol/l, obtenu à partir de foies de rats mâles (200 g
à 300 g), prétraités avec une substance ou une combinaison de substances appropriée pour l’induction d’enzymes
[6]
[Source: ISO 6107-6:2004 , définition 74]
3.12
mélange S9
mélange de la fraction S9 et de la solution de cofacteurs
[6]
[Source: ISO 6107-6:2004 , définition 75]
3.13
culture mère
culture d’une souche d’organismes maintenue dans certaines conditions afin de conserver ses caractéristiques
d’origine, telles que les séquences des nucléotides
[6]
[Source: ISO 6107-6:2004 , définition 87]
3.14
échantillon pour essai
portion non diluée, diluée ou préparée autrement d’un échantillon soumis à essai, après exécution de toutes
les étapes de préparation, telles que la centrifugation, la filtration, l’homogénéisation, l’ajustement du pH et la
détermination de la force ionique
[6]
[Source: ISO 6107-6:2004 , définition 92]
4 Interférences
Les effets bactériotoxiques de l’échantillon pour essai peuvent entraîner une réduction du nombre de bactéries
viables et des puits contenant des révertants, en raison d’une répression de la croissance des révertants.
Cette méthode inclut la filtration stérile de l’eau et des eaux résiduaires avant essai. Cette filtration permet de
séparer les particules solides de l’échantillon pour essai. Ainsi, il est possible que les substances génotoxiques
adsorbées sur les particules ne soient pas détectées.
© ISO 2012 – Tous droits réservés 3

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ISO 11350:2012(F)
5 Principe
Les bactéries sont exposées, dans des conditions définies, à différentes concentrations de l’échantillon pour
essai et incubées pendant 100 min à 37 °C ± 1 °C sur des plaques à 24 puits. En raison de cette exposition,
les agents génotoxiques contenus dans l’échantillon pour essai peuvent induire des mutations dans un ou
deux gènes marqueurs des souches bactériennes utilisées (hisG46 pour TA 100 et hisD3052 pour TA 98) en
corrélation avec les concentrations appliquées. L’induction de mutations provoque une augmentation liée à la
concentration du nombre de colonies mutantes.
Après exposition des bactéries, le milieu indicateur de réversion (7.40) contenant le colorant indicateur de pH,
le pourpre de bromocrésol (7.7), est ajouté dans les puits. Ensuite, les lots sont répartis sur des plaques à
384 puits (48 puits pour chaque parallèle) et incubés pendant 48 h à 72 h (9.3.2, 9.3.3).
L’activité mutagène de l’échantillon pour essai est déterminée en comptant le nombre de puits passant de la
couleur pourpre à jaune (pour 48 puits de chaque parallèle), traités avec l’échantillon pour essai dilué ou non,
et comparée au témoin négatif.
La plus faible dilution (1→N) de l’échantillon pour essai qui n’induit aucun effet mutagène sous toutes les
conditions expérimentales (si un effet mutagène est induit par l’échantillon pour essai) est le critère d’évaluation
du potentiel mutagène. Des dilutions d’échantillons supérieures à celle-ci (1→A, A < N) doivent induire un effet
mutagène conforme aux critères de la présente Norme internationale dans au moins une souche, sous au
moins une condition d’activation (avec ou sans ajout de mélange S9). La valeur LID respective est N. Si aucun
effet mutagène n’est observé sous toutes les conditions expérimentales, cette dilution est 1→1 et la valeur LID
respective est égale à 1.
6 Appareillage et matériaux
6.1 Incubateur à température et à durée contrôlées, 37 °C ± 1 °C.
6.2 pH-mètre.
6.3 Balance analytique.
6.4 Stérilisateur à vapeur.
6.5 Stérilisateur à sec.
6.6 Agitateur magnétique.
6.7 Mélangeur rotatif.
6.8 Congélateur, pouvant être maintenu à ≤ −18 °C et à ≤ −70 °C.
6.9 Pipettes, 0,1 ml, 0,5 ml, 1 ml, 2 ml, 5 ml, 10 ml et 25 ml, en verre ou en plastique.
6.10 Flacons de stockage, 250 ml et 1 000 ml.
6.11 Éprouvettes graduées, 100 ml et 200 ml.
6.12 Fioles jaugées, 20 ml, 200 ml et 500 ml.
6.13 Filtres stériles, 0,2 µm et 0,45 µm.
6.14 Fioles Erlenmeyer, 50 ml, 100 ml et 250 ml.
4 © ISO 2012 – Tous droits réservés

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ISO 11350:2012(F)
6.15 Boucles d’ensemencement.
6.16 Pipette multistepper à huit canaux (pipette à répétition).
6.17 Pipettes à huit canaux, 5 µl à 50 µl et 50 µl à 300 µl.
6.18 Spectrophotomètre.
6.19 Plaques à 24 puits et 384 puits en polystyrène stérile transparent à fond et à couvercle plats.
6.20 Photomètre pour microplaques pour plaques à 24 puits et facultativement pour plaques à
384 puits, filtres: 420 nm ± 15 nm et 595 nm ± 10 nm.
6.21 Banc stérile.
6.22 Boîtes de Petri avec nervures d’évacuation, diamètre d’environ 94 mm, hauteur d’environ 16 mm.
6.23 Tubes cryogéniques, stériles, 1 ml, 10 ml.
7 Réactifs, milieux et dilutions
7.1 Généralités. Dans la mesure du possible, utiliser des produits chimiques de «qualité réactif». Si des
hydrates de composés anhydriques ou des hydrates différents de ceux spécifiés sont utilisés, s’assurer que la
masse du composé principal est appropriée.
Si nécessaire, effectuer un autoclavage pendant 20 min à 121 °C ± 2 °C. Couvrir les récipients de façon non
hermétique (par exemple avec une feuille d’aluminium). Ne jamais fermer hermétiquement.
7.2 Eau, de qualité 1, conformément à l’ISO 3696, ou eau ayant une conductivité ≤ 5 µS/cm.
S’il est nécessaire d’utiliser de l’eau stérile, stériliser par filtration stérile (0,2 µm) ou par autoclavage. L’eau
spécifiée ici est également utilisée pour la dilution fractionnée de l’échantillon pour essai.
7.3 Souches de contrôle. Utiliser des souches mutantes de Salmonella Typhimurium LT2, qui permettent
de détecter les mutations ponctuelles, pour déterminer le potentiel mutagène d’un échantillon pour essai. Étant
donné que les mutations ponctuelles peuvent être subdivisées en deux classes (mutations de changement
de phase et substitutions de paires de base), les deux souches de contrôle TA 98 et TA 100 sont utilisées.
TA 98 contient, comme marqueur, la mutation de changement de phase (type +2) hisD3052, tandis que TA 100
contient la substitution de paires de base hisG46.
De plus, les deux souches doivent avoir les propriétés génétiques suivantes:
— elles contiennent le plasmide pKM101, codant pour la résistance à l’ampicilline;
— elles sont toutes «deep rough», c’est-à-dire partiellement déficientes en chaînes latérales
lipopolysaccharidiques, ce qui permet également à de plus grosses molécules de pénétrer la paroi
cellulaire bactérienne et de provoquer des mutations;
— en raison d’une mutation dans uvrB, la capacité des souches de contrôle à réparer les dommages de l’ADN
est limitée et la probabilité pour que les dommages de l’ADN provoquent des mutations est augmentée.
NOTE L’utilisation d’autres souches de contrôle est décrite dans l’Annexe K.
© ISO 2012 – Tous droits réservés 5

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ISO 11350:2012(F)
7.4 2-Aminoanthracène (2-AA), C H N, numéro CAS: 613-13-8.
14 11
7.5 Sel sodique d’ampicilline, C H N NaO S, numéro CAS: 69-52-3.
16 18 3 4
7.6 d-Biotine, C H N O S, numéro CAS: 58-85-5.
10 16 2 3
7.7 Pourpre de bromocrésol, sel sodique, numéro CAS: 62625-30-3.
7.8 Acide citrique monohydraté, C H O , H O, numéro CAS: 5949-29-1.
6 8 7 2
7.9 Diméthylsulfoxyde, DMSO, C H SO, numéro CAS: 67-68-5.
2 6
7.10 d-Glucose, anhydre, C H O , numéro CAS: 50-99-7.
6 12 6
7.11 Sel disodique de d-Glucose-6-phosphate hydraté, G-6-P-Na , C H Na O P, 2H O, numéro
2 6 11 2 9 2
CAS: 3671-99-6.
7.12 Solution d’acide chlorhydrique, HCl, c(HCl) = 1 mol/l.
7.13 Chlorure de magnésium hexahydraté, MgCl , 6H O, numéro CAS: 7791-18-6.
2 2
7.14 Sulfate de magnésium heptahydraté, MgSO , 7H O, numéro CAS: 10034-99-8.
4 2
7.15 Chlorure de potassium, KCl, numéro CAS: 7447-40-7.
7.16 Hydrogénophosphate de dipotassium, K HPO , numéro CAS: 7758-11-4.
2 4
7.17 Hydrogénophosphate de sodium et d’ammonium tétrahydraté, NaNH HPO , 4H O, numéro
4 4 2
CAS: 7583-13-3.
7.18 Chlorure de sodium, NaCl, numéro CAS: 7647-14-5.
7.19 Dihydrogénophosphate de sodium, anhydre, NaH PO , numéro CAS: 7558-80-7.
2 4
7.20 Hydrogénophosphate de disodium, anhydre, Na HPO , numéro CAS: 7558-79-4.
2 4
7.21 Solution d’hydroxyde de sodium, c(NaOH) = 1 mol/l.
7.22 Sel sodique de β-nicotinamide adénine dinucléotide phosphate, NADP, H O, C H N NaO P , H O,
2 21 27 7 17 3 2
numéro CAS: 698999-85-8.
7.23 Nitrofurantoïne (NF), numéro CAS: 67-20-9.
7.24 4-Nitro-o-phénylènediamine (4-NOPD), numéro CAS: 99-56-9.
1)
7.25 Poudre pour bouillon nutritif .
1)
7.26 Fraction S9 (broyat de foie; induit par le phénobarbital/la β-naphtoflavone) .
1) Ce réactif est disponible dans le commerce. Cette information est donnée à l’intention des utilisateurs du présent
document et ne signifie nullement que l’ISO approuve ou recommande l’emploi exclusif du produit ainsi désigné.
6 © ISO 2012 – Tous droits réservés

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ISO 11350:2012(F)
7.27 l-Histidine, C H N O , numéro CAS: 71-00-1.
6 9 3 2
7.28 Tampon phosphate.
7.28.1 Tampon dihydrogénophosphate de sodium, c(NaH PO ) = 0,2 mol/l.
2 4
Dissoudre 14,39 g de NaH PO (ou 16,55 g de NaH PO , H O) dans 600 ml d’eau (7.2).
2 4 2 4 2
7.28.2 Tampon hydrogénophosphate de disodium, c(Na HPO ) = 0,2 mol/l. Dissoudre 28,39 g de Na HPO
2 4 2 4
dans 1 000 ml d’eau (7.2).
Ajouter le tampon dihydrogénophosphate de sodium (7.28.1) au tampon hydrogénophosphate de
disodium (7.28.2) jusqu’à ce qu’une valeur pH de 7,4 soit atteinte et passer à l’autoclave. Conserver à
température ambiante et à l’abri de la lumière. La solution est stable pendant au moins un an.
7.29 Solution de d-biotine. Dissoudre 12,2 mg de d-biotine (7.6) dans 100 ml d’eau (7.2) par ébullition. Après
refroidissement, stériliser par filtration (filtre de 0,2 µm). Conserver des aliquotes de 10 ml à une température
inférieure ou égale à −18 °C dans des tubes cryogéniques stériles (6.23). Les solutions aqueuses conservées
sous forme d’aliquotes congelées sont stables pendant au moins un an.
7.30 Solution de l-histidine. Dissoudre 50 mg de l-histidine (7.27) dans 50 ml d’eau (7.2) et stériliser par
filtration (filtre de 0,2 µm). Conserver des aliquotes de 1,5 ml à une température inférieure ou égale à −18 °C
dans des tubes cryogéniques stériles (6.23). Les solutions aqueuses conservées sous forme d’aliquotes
congelées sont stables pendant au moins un an.
7.31 Solution de glucose-6-phosphate. Dissoudre 0,68 g de d-glucose-6-phosphate (7.11) dans 10 ml
d’eau (7.2) et stériliser par filtration (filtre de 0,2 µm). Conserver des aliquotes (par exemple 200 µl) à une
température inférieure ou égale à −18 °C dans des tubes cryogéniques stériles (6.23). Les solutions aqueuses
conservées sous forme d’aliquotes congelées sont stables pendant au moins un an.
7.32 Solution de NADP, c(NADP) = 0,04 mol/l. Dissoudre la masse appropriée de NADP dans 10 ml
d’eau (7.2) de façon à obtenir une concentration finale de 0,04 mol/l et stériliser par filtration (filtre de 0,2 µm).
Conserver des aliquotes (par exemple 700 µl) à une température inférieure ou égale à −18 °C dans des tubes
cryogéniques stériles (6.23). Les solutions aqueuses conservées sous forme d’aliquotes congelées sont stables
pendant au moins un an.
Différents hydrates de NADP sont disponibles. La masse moléculaire réelle est spécifiée dans la fiche technique
du produit. Calculer la quantité de NADP nécessaire en fonction de la masse moléculaire indiquée.
7.33 Solution de chlorure de potassium. Dissoudre 74,56 g de KCl (7.15) dans 1 000 ml d’eau (7.2) et
passer à l’autoclave. Conserver à température ambiante. La solution est stable pendant au moins un an.
7.34 Solution de MgCl , 6H O. Dissoudre 50,83 g de MgCl , 6H O (7.13) dans 1 000 ml d’eau (7.2) et passer
2 2 2 2
la solution à l’autoclave. Conserver à température ambiante. La solution est stable pendant au moins un an.
7.35 Solution de pourpre de bromocrésol. Dissoudre 51 mg de sel sodique de pourpre de bromocrésol (7.7)
dans 30 ml d’eau (7.2). Préparer cette solution immédiatement avant d’ajouter le milieu indicateur de réversion (7.40).
7.36 Solution d’ampicilline. Dissoudre 500 mg d’ampicilline (7.5) dans 10 ml d’eau (7.2) et stériliser par
filtration (filtre de 0,2 µm). Conserver des aliquotes de 500 µl à une température inférieure à −18 °C dans des
tubes cryogéniques stériles (6.23). La solution est stable pendant au moins six mois.
© ISO 2012 – Tous droits réservés 7

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ISO 11350:2012(F)
2)
7.37 Milieu de croissance. Dissoudre 4,7 g de poudre pour bouillon nutritif et 0,31 g de chlorure de
sodium (7.18) dans 200 ml d’eau (7.2). Ajuster le pH à 7,5 ± 0,1. Compléter à 250 ml avec de l’eau (7.2) et
passer cette solution à l’autoclave.
On doit obtenir les concentrations finales suivantes dans le milieu de croissance:
— 7,5 g/l d’extrait de viande;
— 7,5 g /l de peptone;
— 5,0 g/l de chlorure de sodium.
Les solutions conservées dans des conditions stériles sous forme d’aliquotes congelées sont stables pendant
au moins un an.
7.38 Milieu d’exposition. Préparer un milieu d’incubation des bactéries avec l’échantillon contenant une
faible quantité de l-histidine pour supporter quelques divisions cellulaires.
Dissoudre consécutivement les ingrédients suivants dans 900 ml d’eau:
— 0,2 g de sulfate de magnésium heptahydraté (7.14);
— 2,0 g d’acide citrique (7.8);
— 10,0 g d’hydrogénophosphate de dipotassium (7.16);
— 3,5 g d’hydrogénophosphate de sodium et d’ammonium tétrahydraté (7.17);
— 4,0 g de d-glucose (7.10).
Compléter à 1 000 ml avec de l’eau (7.2), ajuster le pH à 7,0 ± 0,2, si nécessaire, et stériliser par filtration (filtre
de 0,2 µm). Conserver le milieu entre 2 °C et 8 °C.
Pour 100 ml, ajouter 0,6 ml de solution de d-biotine (7.29) et 0,1 ml de solution de l-histidine (7.30) dans des
conditions stériles. Ne préparer que la quantité de milieu nécessaire pour les deux prochaines semaines.
Conserver le milieu entre 2 °C et 8 °C.
7.39 Milieu d’exposition concentré. Dissoudre consécutivement les ingrédients suivants dans 70 ml d’eau:
— 0,2 g de sulfate de magnésium heptahydraté (7.14);
— 2,0 g d’acide citrique (7.8);
— 10,0 g d’hydrogénophosphate de dipotassium (7.16);
— 3,5 g d’hydrogénophosphate de sodium et d’ammonium tétrahydraté (7.17);
— 4,0 g de d-glucose (7.10).
Compléter à 93 ml avec de l’eau (7.2), ajuster le pH, si nécessaire, et stériliser par filtration (filtre de 0,2 µm).
Conserver le milieu concentré entre 2 °C et 8 °C.
Ajouter 6 ml de solution de d-biotine (7.29) et 1 ml de solution de l-histidine (7.30) dans des conditions stériles.
Ne préparer que la quantité de milieu nécessaire pour les deux prochaines semaines. Conserver le milieu
concentré entre 2 °C et 8 °C.
7.40 Milieu indicateur de réversion. Préparer un milieu d’indication du pH sans l-histidine.
2) Utiliser une poudre pour bouillon nutritif contenant 40 % d’extrait de viande, 40 % de peptone et 20 % de chlorure de
sodium.
8 © ISO 2012 – Tous droits réservés

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ISO 11350:2012(F)
7.40.1 Solution I. Dissoudre les ingrédients suivants dans 950 ml d’eau, dans l’ordre ci-dessous:
— 0,4 g de sulfate de magnésium heptahydraté (7.14);
— 4,0 g d’acide citrique (7.8);
— 20,0 g d’hydrogénophosphate de dipotassium (7.16);
— 7,0 g d’hydrogénophosphate de sodium et d’ammonium tétrahydraté (7.17).
Compléter à 1 000 ml avec de l’eau (7.2) et ajouter 30,0 ml de solution de pourpre de bromocrésol (7.35).
Ajuster le pH à 7,3 ± 0,1. Transférer deux volumes identiques de solution dans deux flacons de 1 000 ml et
passer à l’autoclave.
7.40.2 Solution II. Dissoudre 8,0 g de d-glucose (7.10) dans 800 ml d’eau (7.2). Ajuster le pH à 7,3 ± 0,1.
Transférer les deux moitiés de la solution dans deux flacons de 1 000 ml et passer à l’autoclave.
7.40.3 Mélange et conservation. Après refroidissement à température ambiante, mélanger 515 ml de
solution I (7.40.1) avec 400 ml de solution II (7.40.2) dans des conditions stériles. Ajouter 20 ml de solution de
d-biotine (7.29) dans chaque flacon, dans des conditions stériles.
Conserver le milieu à température ambiante et à l’abri de la lumière. L
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