SIST EN ISO 11348-1:2009

Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:2007)

ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine bacterium Vibrio fischeri (NRRL B-11177). This part of ISO 11348 specifies a method using freshly prepared bacteria. The method is applicable to waste water, aqueous extracts and leachates, fresh water (surface and ground water), sea and brackish water, eluates of sediment (fresh water, brackish and sea water), pore water, singles substances, diluted in water.

Wasserbeschaffenheit - Bestimmung der Hemmwirkung von Wasserproben auf die Lichtemission von Vibrio fischeri (Leuchtbakterientest) - Teil 1: Verfahren mit frisch gezüchteten Bakterien (ISO 11348-1:2007)

Die Reihe ISO 11348 legt drei Verfahren zur Bestimmung der Hemmung der Lichtemission des marinen Bakteriums Vibrio fischeri (NRRL B 11177) fest. Dieser Teil von ISO 11348 beschreibt ein Verfahren mit frisch gezüchteten Bakterien.
Das Verfahren gilt für:
   Abwasser;
   wässrige Extrakte und Sickerwasser;
   Süßwasser (Oberflächenwasser und Grundwasser);
   Meerwasser und Brackwasser;
   Eluate von Sedimenten (Süßwasser, Brackwasser und Meerwasser);
   Porenwasser;
   Einzelstoffe, in Wasser gelöst.

Qualité de l'eau - Détermination de l'effet inhibiteur d'échantillons d'eau sur la luminescence de Vibrio fischeri (Essai de bactéries luminescentes) - Partie 1: Méthode utilisant des bactéries fraîchement préparées (ISO 11348-1:2007)

L'ISO 11348 décrit trois méthodes de détermination de l'inhibition de la luminescence émise par la bactérie marine Vibrio fischeri (NRRL B-11177). L'ISO 11348-1:2007 spécifie une méthode utilisant des bactéries fraîchement préparées.
Cette méthode est applicable aux eaux usées, aux extraits aqueux et aux lixiviats, aux eaux douces (eaux de surface ou souterraines), à l'eau de mer ou aux eaux saumâtres, aux éluats de sédiments (eau douce, eau saumâtre et eau de mer), aux eaux interstitielles et aux substances individuelles diluées dans l'eau.

Kakovost vode - Določevanje zaviralnega učinka vzorcev vode na oddajanje svetlobe Vibrio fischeri (preskus luminiscence bakterije) - 1. del: Metoda z uporabo sveže pripravljenih bakterij (ISO 11348-1:2007)

Standard ISO 11348 opisuje tri metode za določevanje zaviranja luminiscence, ki jo oddaja morska bakterija Vibrio fischeri (NRRL B-11177). Ta del standarda ISO 11348 določa metodo z uporabo sveže pripravljenih bakterij. Metoda se uporablja za odpadno vodo, vodne ekstrakte in izcedne vode, sladko vodo (površinsko in podtalno), morsko in slano vodo, izlužke sedimentov (sladka, slana in morska voda), vodo v porah, posamezne snovi, razredčene v vodi.

General Information

• Status: Published
• Public Enquiry End Date: 19-Sep-2008
• Publication Date: 14-Apr-2009
• Withdrawal Date: 04-Apr-2019
• ICS: 13.060.70 - Examination of biological properties of water
• Technical Committee: KAV - Water quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 24-Mar-2009
• Due Date: 29-May-2009
• Completion Date: 15-Apr-2009
• Ref Project: EN ISO 11348-1:2008 - Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:2007)

Relations

• Replaces: SIST EN ISO 11348-1:2000 - Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria (ISO 11348-1:1998)
• Effective Date: 01-May-2009
• Amended By: SIST EN ISO 11348-1:2009/A1:2019 - Water quality - Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly prepared bacteria - Amendment 1 (ISO 11348-1:2007/Amd 1:2018)
• Effective Date: 01-May-2019

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 11348-1:2009
01-maj-2009
1DGRPHãþD
SIST EN ISO 11348-1:2000
.DNRYRVWYRGH'RORþHYDQMH]DYLUDOQHJDXþLQNDY]RUFHYYRGHQDRGGDMDQMH
VYHWOREH9LEULRILVFKHULSUHVNXVOXPLQLVFHQFHEDNWHULMHGHO0HWRGD]XSRUDER
VYHåHSULSUDYOMHQLKEDNWHULM,62
Water quality - Determination of the inhibitory effect of water samples on the light
emission of Vibrio fischeri (Luminescent bacteria test) - Part 1: Method using freshly
prepared bacteria (ISO 11348-1:2007)
Wasserbeschaffenheit - Bestimmung der Hemmwirkung von Wasserproben auf die
Lichtemission von Vibrio fischeri (Leuchtbakterientest) - Teil 1: Verfahren mit frisch
gezüchteten Bakterien (ISO 11348-1:2007)
Qualité de l'eau - Détermination de l'effet inhibiteur d'échantillons d'eau sur la
luminescence de Vibrio fischeri (Essai de bactéries luminescentes) - Partie 1: Méthode
utilisant des bactéries fraîchement préparées (ISO 11348-1:2007)
Ta slovenski standard je istoveten z: EN ISO 11348-1:2008
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
SIST EN ISO 11348-1:2009 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 11348-1:2009

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SIST EN ISO 11348-1:2009
EUROPEAN STANDARD
EN ISO 11348-1
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2008
ICS 13.060.70 Supersedes EN ISO 11348-1:1998
English Version
Water quality - Determination of the inhibitory effect of water
samples on the light emission of Vibrio fischeri (Luminescent
bacteria test) - Part 1: Method using freshly prepared bacteria
(ISO 11348-1:2007)
Qualité de l'eau - Détermination de l'effet inhibiteur Wasserbeschaffenheit - Bestimmung der Hemmwirkung
d'échantillons d'eau sur la luminescence de Vibrio fischeri von Wasserproben auf die Lichtemission von Vibrio fischeri
(Essai de bactéries luminescentes) - Partie 1: Méthode (Leuchtbakterientest) - Teil 1: Verfahren mit frisch
utilisant des bactéries fraîchement préparées (ISO 11348- gezüchteten Bakterien (ISO 11348-1:2007)
1:2007)
This European Standard was approved by CEN on 29 October 2008.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European
Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national
standards may be obtained on application to the CEN Management Centre or to any CEN member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by translation
under the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as the
official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,
Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2008 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11348-1:2008: E
worldwide for CEN national Members.

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SIST EN ISO 11348-1:2009
EN ISO 11348-1:2008 (E)
Contents Page
Foreword.3

2

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SIST EN ISO 11348-1:2009
EN ISO 11348-1:2008 (E)
Foreword
The text of ISO 11348-1:2007 has been prepared by Technical Committee ISO/TC 147 “Water quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 11348-1:2008 by
Technical Committee CEN/TC 230 “Water analysis” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an identical
text or by endorsement, at the latest by May 2009, and conflicting national standards shall be withdrawn at the
latest by May 2009.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
This document supersedes EN ISO 11348-1:1998.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech
Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia,
Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 11348-1:2007 has been approved by CEN as a EN ISO 11348-1:2008 without any
modification.

3

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SIST EN ISO 11348-1:2009

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SIST EN ISO 11348-1:2009

INTERNATIONAL ISO
STANDARD 11348-1
Second edition
2007-12-01

Water quality — Determination
of the inhibitory effect of water samples
on the light emission of Vibrio fischeri
(Luminescent bacteria test) —
Part 1:
Method using freshly prepared bacteria
Qualité de l'eau — Détermination de l'effet inhibiteur d'échantillons
d'eau sur la luminescence de Vibrio fischeri (Essai de bactéries
luminescentes) —
Partie 1: Méthode utilisant des bactéries fraîchement préparées





Reference number
ISO 11348-1:2007(E)
©
ISO 2007

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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
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©  ISO 2007
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
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ii © ISO 2007 – All rights reserved

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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 2
4 Interferences . 2
5 Reagents and materials . 2
6 Apparatus . 4
7 Sampling and sample pretreatment. 5
8 Cultivation of luminescent bacteria. 5
9 Procedure . 7
10 Evaluation. 8
11 Expression of results . 10
12 Criteria of validity. 11
13 Precision. 12
14 Test report . 12
Annex A (informative) Colour-correction method. 13
Annex B (informative) Dilution level D – Preparation of the dilution series. 16
Annex C (informative) Precision data. 19
Annex D (informative) Testing salt water samples with the luminescent bacteria test with freshly
cultured bacteria. 20
Bibliography . 23

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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(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 11348-1 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
This second edition cancels and replaces the first edition (ISO 11348-1:1998), which has been technically
revised.
ISO 11348 consists of the following parts, under the general title Water quality — Determination of the
inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test):
⎯ Part 1: Method using freshly prepared bacteria
⎯ Part 2: Method using liquid-dried bacteria
⎯ Part 3: Method using freeze-dried bacteria
iv © ISO 2007 – All rights reserved

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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
Introduction
The measurements specified in ISO 11348 can be carried out using freshly prepared bacteria, as well as
freeze-dried or liquid-dried bacterial preparations.
Standardized work carried out by DIN Normenausschuss Wasserwesen and ISO/TC 147/SC 5/WG 1 has
shown that, in special cases, these different techniques may deliver different results, especially in the
presence of heavy metals.
Such varying sensitivity is caused by differences in media composition used in the preparation of freeze-dried
or liquid-dried bacteria. These protective media influence the bioavailability of toxicants and/or the light
emission of luminescent bacteria. This means that the origin and type of preparation need to be taken into
account when interpreting the results. This may be difficult sometimes, as freeze-dried and liquid-dried
bacteria may be obtained from different suppliers. This, in turn, can mean that the composition is not known in
detail and therefore cannot be interpreted by the user.
For this reason, in addition to toxicity measurements with liquid-dried bacteria (ISO 11348-2) and freeze-dried
bacteria (ISO 11348-3), a procedure with freshly prepared bacteria is described in this part of ISO 11348, the
performance of which can be interpreted by the user in every detail.
The laboratories responsible for the results have the opportunity to select the most suitable technique based
on expert judgement and information about the water sample to be tested.

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SIST EN ISO 11348-1:2009

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SIST EN ISO 11348-1:2009
INTERNATIONAL STANDARD ISO 11348-1:2007(E)

Water quality — Determination of the inhibitory effect of water
samples on the light emission of Vibrio fischeri (Luminescent
bacteria test) —
Part 1:
Method using freshly prepared bacteria
WARNING — Persons using this part of ISO 11348 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 in accordance with this part of
ISO 11348 be carried out by suitably trained staff.
1 Scope
ISO 11348 describes three methods for determining the inhibition of the luminescence emitted by the marine
bacterium Vibrio fischeri (NRRL B-11177). This part of ISO 11348 specifies a method using freshly prepared
bacteria.
This method is applicable to:
⎯ waste water;
⎯ aqueous extracts and leachates;
⎯ fresh water (surface and ground water);
⎯ sea and brackish water;
⎯ eluates of sediment (fresh water, brackish and sea water);
⎯ pore water;
⎯ single substances, diluted in water.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 5667-16, Water quality — Sampling — Part 16: Guidance on biotesting of samples
ISO 5814, Water quality — Determination of dissolved oxygen — Electrochemical probe method
ISO 7027, Water quality — Determination of turbidity
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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
3 Principle
The inhibition of light emission by cultures of Vibrio fischeri is determined by means of a batch test. This is
accomplished by combining specified volumes of the test sample or the diluted sample with the luminescent
bacteria suspension in a test tube.
The test criterion is the luminescence, measured after a contact time of 15 min or 30 min and optionally 5 min,
taking into account a correction factor (f ), which is a measure of intensity changes of control samples during
kt
the exposure time. The inhibitory effect of the water sample can be determined as LID (see Annex B) or as
EC - and/or EC -values by means of a dilution series. (EC is the effective concentration).
20 50
4 Interferences
Insoluble, slightly soluble or volatile substances or substances which react with the dilution water or the
suspension, or alter their state during the test period, may affect the result or impair the reproducibility of the
test results.
Losses of luminescence caused by light absorption or light scattering may occur in the case of strongly
coloured or turbid waters. This interference can be compensated by a sample treatment for turbidity (7.2) or,
for example, by using a double-chambered absorption correction test tube (see Annex A).
[6]
Since oxygen is required for the bioluminescence , samples with a high oxygen demand (and/or a low
oxygen concentration) may cause a deficiency of oxygen and be inhibitory.
Readily biodegradable nutrients in the sample may cause a pollutant-independent reduction in
[1]
bioluminescence .
[6], [7]
Samples with a pH outside the range of pH = 6,0 and pH = 8,5 affect the luminescence of the bacteria .
An adjustment of the sample is required when the toxic effect of pH is not wanted.
As the test organism Vibrio fischeri is a marine bacterium, testing salt-water samples with the standard
procedure often leads to stimulation effects of bioluminescence, which may mask inhibition effects (see
Annex D).
Salt concentrations in the initial sample exceeding 30 g/l NaCl, or contents of other compounds giving equal
osmolarity may lead, together with the salt spiking required by the test, to hyperosmotic effects. The resulting
salt concentration in the test samples should not exceed the osmolarity of a 35 g/l NaCl solution in order to
avoid these effects.
5 Reagents and materials
Use chemicals of recognized analytical grade quality. Use distilled water or water of equivalent purity.
5.1 Test bacteria.
Use a strain of luminescence bacteria belonging to the species Vibrio fischeri NRRL B-11177. The bacteria
strain can be taken from commercially available freeze-dried or liquid-dried reagents or from culture
collections, e.g. Deutsche Sammlung für Mikrorganismen und Zellkulturen GmbH, Mascheroder Weg 10,
D-38124 Braunschweig, Germany, or NRRL, ARS Culture collection NCAUR, 1815 N, University Street,
Peoria, Illinois 61604, USA. The bacterial suspensions used for toxicity measurements shall be freshly
prepared from cultures.
5.2 Sodium chloride solution, as diluent.
Dissolve 20 g of sodium chloride (NaCl) in water and make up to 1 l with water.
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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
5.3 Sodium hydroxide solution, e.g. c(NaOH) = 1 mol/l.
5.4 Hydrochloric acid, e.g. c(HCl) = 1 mol/l.
For the adjustment of the pH, it may be necessary to use acids or bases of lower or higher concentration.
5.5 Solution for freshly prepared bacteria.
8,0 g D(+)-Glucose monohydrate (C H O ·H O)
6 12 6 2
20,0 g Sodium chloride (NaCl)
2,035 g Magnesium chloride hexahydrate (MgCl ·6 H O)
2 2
0,30 g Potassium chloride (KCl)
N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES)
11,9 g
Dissolve in water, stir for about 30 min and adjust the pH to 7,0 ± 0,2 with sodium hydroxide solution (5.3) or
hydrochloric acid (5.4). Make up to 1 l with water.
This solution may be stored in portions at −18 °C to −20 °C.
5.6 Reference substances.
Prepare the following reference-substance stock solutions with sodium chloride solution (5.2) as diluent
separately, without adjustment of the pH:
219,8 mg/l Zinc sulfate heptahydrate (ZnSO ·7 H O)
4 2
9 mg/l 3,5-Dichlorophenol (C H OCl ) (purity W 99 %)
6 4 2
22,6 mg/l Potassium dichromate (K Cr O )
2 2 7
These concentrations are approximately twice the expected EC -values for the respective reference
50
substances in this part of ISO 11348. The volumes required depend on the test set-up.
NOTE It is possible to use commercially available chemical preparations with defined concentrations of ZnSO and
4
K Cr O (titrisol) for the preparation of the stock solutions of the reference substances.
2 2 7
5.7 Liquid broth for pre- and main cultures.
30 g Sodium chloride (NaCl)
6,10 g Sodium dihydrogenphosphate monohydrate (NaH PO ·H O)
2 4 2
2,75 g Dipotassium hydrogenphosphate trihydrate (K HPO ·3 H O)
2 4 2
0,204 g Magnesium sulfate heptahydrate (MgSO ·7 H O)
4 2
0,500 g Diammonium hydrogenphosphate [(NH ) HPO ]
4 2 4
3 ml Glycerol
5,00 g Caso-peptone
0,50 g Yeast extract
Dissolve in water and adjust the pH to 7,0 ± 0,2 with sodium hydroxide solution (5.3) or hydrochloric acid (5.4).
Make up to 1 l with water. Transfer 50 ml each to Erlenmeyer flasks (e.g. 250 ml) and sterilize in an autoclave
at 121 °C for 20 min.
Caso-peptone and yeast extract offered by different suppliers can vary in quality. In case of problems (e.g.
growth inhibition), it is recommended to purchase the product from another manufacturer.
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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
5.8 Agar medium for stock cultures.
Adjust the liquid broth (5.7) to pH 7,0 ± 0,2.
Add 12 g of agar per litre and dissolve by gentle warming; sterilize and transfer to sterile Petri dishes.
5.9 Protective medium.
66 g D(+)-Glucose monohydrate (C H O ·H O)
6 12 6 2
4 g Sodium chloride (NaCl)
2 g L-Histidine
0,5 g Bovine serum albumin, BSA
Dissolve completely in water at about 37 °C and adjust the pH to 7,0 ± 0,2 at room temperature with sodium
hydroxide solution (5.3) or hydrochloric acid (5.4) as necessary. Make up to 100 ml with water.
Damage of bacterial cells during the freezing procedure is prevented by the use of the protective medium.
BSA offered by different suppliers can vary in quality. If problems occur, it is recommended to purchase the
product from another manufacturer.
Prepare protective medium freshly before use.
6 Apparatus
6.1 Thermostatically controlled thermo-block, to maintain the test samples at a temperature of
15 °C ± 1 °C. Within one test, the temperature deviation should be at most ± 0,3 °C.
6.2 Water bath or thermostatically controlled thermo-block, to maintain at least 12 ml volume (e.g.
reagent vessel) of the solution prepared in 5.5 at 15 °C ± 1 °C.
6.3 Luminometer, measuring cell maintained at 15 °C ± 1 °C, equipped with suitable test tubes.
6.4 Test tubes, made of a chemically inert material, appropriate for the selected luminometer, with a
capacity which facilitates the taking of a reading over the largest possible surface area and able to fit into the
thermo-block (6.1).
6.5 pH-meter.
6.6 Chronometer.
6.7 Piston pipettes or plastic syringes, 100 µl, 500 µl and 1 000 µl.
6.8 Piston pipettes, with variable volume, 10 ml to 200 ml and 200 µl to 5 000 µl.
6.9 Refrigerated centrifuge.
6.10 Magnetic stirrer and magnetic stirring bar.
6.11 Incubated shaker, for incubation of Erlenmeyer flasks.
6.12 Autoclave.
6.13 Incubator.
6.14 Spectral- or filter-photometer and test tubes, of optical path length 1 cm.
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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
6.15 Inoculating loop (or needle).
6.16 Conductometer.
6.17 Freezer, for the storage of solutions and suspensions.
6.18 Oxygen probe, in accordance with ISO 5814.
7 Sampling and sample pretreatment
7.1 Sampling
Collect samples in chemically inert, clean containers as specified in ISO 5667-16. Fill the containers
completely and seal them. Test the samples as soon as possible after collection. Where necessary, store
samples at 2 °C to 5 °C in the dark in the containers for not longer than 48 h. For periods up to two months,
store at u −18 °C. Do not use chemicals to preserve the samples. Perform the necessary pH-adjustment and
salt addition immediately before testing.
7.2 Sample preparation
Measure the oxygen concentration in all samples. An oxygen concentration > 3 mg/l is required for the test. If
the oxygen concentration of the undiluted sample is less than 3 mg/l, use adequate methods to oxygenate the
sample, e.g. aeration or stirring.
Measure the pH of all samples. If the pH is between 6,0 and 8,5, an adjustment is usually not necessary.
Adjustment of the pH-value, however, may alter the nature of the sample. On the other hand, the pH of the
sample and the pH of the test batch may differ because of the buffer capacity of the test medium. It may be
necessary to carry out tests on both the pH-adjusted and the non-pH-adjusted samples.
If necessary, adjust the pH of the sample by adding either hydrochloric acid (5.4) or sodium hydroxide solution
(5.3). Depending on the purpose of the test, the pH may be adjusted to 7,0 ± 0,2 or to the upper (8,5 ± 0,2)
and lower limits (6,0 ± 0,2). Choose the concentration of the hydrochloric acid or the sodium hydroxide
solution to restrict the volume added to not more than 5 % of total volume.
Add 20 g of sodium chloride per litre to the water sample or to the neutralized water sample.
For samples with high salt concentrations, measure the salinity and add the amount of salt which is necessary
to adjust the osmolarity to 20 g/l NaCl.
If the sample contains between 20 g/l and 50 g/l NaCl-equivalents, add no salt. The resulting salt
concentration in the test samples shall not exceed the osmolarity of a 35 g/l sodium chloride solution.
For salt water samples, Annex D gives further information.
Strongly turbid samples should be allowed to settle for 1 h or centrifuged, for example for 10 min at 5 000g, or
should be filtered. Use the supernatant or filtrate for the test.
8 Cultivation of luminescent bacteria
8.1 Stock culturing
Transfer luminescent bacteria of strain Vibrio fischeri NRRL B-11177 (5.1) under sterile conditions to Petri
dishes containing the agar medium for stock cultures (5.8).
Incubate in an incubator for 2 d to 3 d at 20 °C ± 1 °C.
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SIST EN ISO 11348-1:2009
ISO 11348-1:2007(E)
Mark luminescent single colonies using visual observations in the dark, and store dishes in the refrigerator
afterwards.
Transfer marked colonies under sterile conditions to fresh dishes after a storage period of one week to a
maximum of two weeks.
Commercially available vials of preserved bacteria usually are not dispensed under sterile conditions. For
cultivation of pure cultures, several single-colony passages are recommended. To prevent genetic alterations,
a new vial of preserved bacteria can be opened and used approximately every 6 months.
NOTE The luminescence of luminescent bacterial colonies can decrease during storage.
8.2 Preparation of pre-cultures
Inoculate 50 ml of pre-culture broth (5.7) in Erlenmeyer flasks (e.g. 250 ml) under sterile conditions with one
luminescent single colony of a stock culture aged 2 d to 3 d.
–1
Shake for 21 h ± 1 h at 20 °C ± 1 °C with a periodicity of at least 180 r·min .
Determine the turbidity of a 1 in 10 dilution of the culture in sodium chloride solution (5.2), e.g. in formazine
absorption units (FAU) at 578 nm, as specified in ISO 7027.
8.3 Preparation of main culture
Inoculate 50 ml of the main culture broth (5.7) in 250 ml Erlenmeyer flasks with an appropriate volume of
pre-culture (8.2) to result in an estimated initial turbidity of 10 FAU.
–1
Shake for 20 h ± 1 h at 20 °C ± 1 °C with a periodicity of at least 180 r·min .
Determine turbidity, in FAU, of a 1 in 10 dilution in sodium chloride solution (5.2) photometrically at 578 nm.
NOTE Following the above conditions, the undiluted main culture usually exhibits a turbidity of 700 FAU to
1 800 FAU.
8.4 Preparation of stock suspension
Pre-cool sodium chloride solution (5.2) and protective medium (5.9) on ice.
Centrifuge bacterial suspension from the main culture (8.3) at 4 °C ± 2 °C in a pre-cooled refrigerated
centrifuge, for 15 min to 20 min at 6 000g ± 2 000g.
Decant supernatants and resuspend pellets in 5 ml to 10 ml, per 50 ml of main culture, of ice-cold sodium
chloride solution (5.2).
Repeat centrifugation under the same conditions.
Decant supernatant and resuspend pellets in 0,5 ml, per 50 ml of main culture, of ice-cold sodium chloride
solution (5.2).
Transfer the bacterial suspension to a precooled beaker (e.g. 100 ml) and place on ice.
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