SIST EN ISO 10705-2:2001
(Main)Water quality - Detection and enumeration of bacteriophages - Part 2: Enumeration of somatic coliphages (ISO 10705-2:2000)
Water quality - Detection and enumeration of bacteriophages - Part 2: Enumeration of somatic coliphages (ISO 10705-2:2000)
Wasserbeschaffenheit - Nachweis und Zählung von Bakteriophagen - Teil 2: Zählung von somatischen Coliphagen (ISO 10705-2:2000)
Dieser Teil von ISO 10705 legt ein Verfahren zum Nachweis und zur Zählung
von somatischen Coliphagen durch Bebrütung der Probe mit einem geeigneten
Wirtsstamm fest. Das Verfahren kann für alle Arten von Wasser, Sedimenten und
Schlämmen angewandt werden, wenn notwendig nach Verdünnung. Das Ver-fahren
kann auch für Schalentierextrakte angewandt werden.
Bei geringer Phagenanzahl kann eine Aufkonzentration notwendig sein, für die
eine gesonderte Internationale Norm entwickelt werden wird.
ANMERKUNG Es ist wünschenswert, dass so weit wie möglich nach Internationalen Normen
verfahren wird. Dieser Teil von ISO 10705 beinhaltet einen Verweis auf alternative Verfahren, die
keine teuren Materialien oder Zubehör erfordern, welche in Entwicklungsländern möglicherweise
nicht ohne weiteres verfügbar sind. Die Anwendung dieser Alternativen hat keinen Einfluss auf
die Durchführung dieses Verfahrens.
Qualité de l'eau - Détection et dénombrement des bactériophages - Partie 2: Dénombrement des coliphages somatiques (ISO 10705-2:2000)
La présente partie de l'ISO 10705 spécifie une méthode de détection et de dénombrement des coliphages somatiques par incubation de l'échantillon avec une souche-hôte appropriée. La méthode est applicable à tous les types d'eaux, aux extraits de sédiments et de boues, si nécessaire après dilution. La méthode est également applicable aux extraits de coquillages. Dans le cas d'une faible population de phages, une étape de préconcentration peut s'avérer nécessaire pour laquelle une Norme internationale distincte sera élaborée ultérieurement.
NOTE Il est souhaitable que les Normes Internationales soient adoptées le plus largement possible. La présente partie de l'ISO 10705 comporte des références à des procédures alternatives qui parent à la nécessité d'utiliser du matériel ou des équipements coûteux qui peuvent ne pas être aisément disponibles dans les pays en développement. L'utilisation de ces procédures alternatives n'affecte pas les performances de la présente méthode.
Kakovost vode - Ugotavljanje prisotnosti in števila bakteriofagov - 2. del - Ugotavljanje števila somatskih kolifagov (ISO 10705-2:2000)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 10705-2:2001
01-december-2001
1DGRPHãþD
SIST ISO 10705-2:2000
Kakovost vode - Ugotavljanje prisotnosti in števila bakteriofagov - 2. del -
Ugotavljanje števila somatskih kolifagov (ISO 10705-2:2000)
Water quality - Detection and enumeration of bacteriophages - Part 2: Enumeration of
somatic coliphages (ISO 10705-2:2000)
Wasserbeschaffenheit - Nachweis und Zählung von Bakteriophagen - Teil 2: Zählung
von somatischen Coliphagen (ISO 10705-2:2000)
Qualité de l'eau - Détection et dénombrement des bactériophages - Partie 2:
Dénombrement des coliphages somatiques (ISO 10705-2:2000)
Ta slovenski standard je istoveten z: EN ISO 10705-2:2001
ICS:
07.100.20 Mikrobiologija vode Microbiology of water
SIST EN ISO 10705-2:2001 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 10705-2:2001
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SIST EN ISO 10705-2:2001
EUROPEAN STANDARD
EN ISO 10705-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2001
ICS 07.100.20
English version
Water quality - Detection and enumeration of bacteriophages -
Part 2: Enumeration of somatic coliphages (ISO 10705-2:2000)
Qualité de l'eau - Détection et dénombrement des Wasserbeschaffenheit - Nachweis und Zählung von
bactériophages - Partie 2: Dénombrement des coliphages Bakteriophagen - Teil 2: Zählung von somatischen
somatiques (ISO 10705-2:2000) Coliphagen (ISO 10705-2:2000)
This European Standard was approved by CEN on 22 June 2001.
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 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 Management Centre has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece,
Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, 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
© 2001 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 10705-2:2001 E
worldwide for CEN national Members.
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SIST EN ISO 10705-2:2001
EN ISO 10705-2:2001 (E)
CORRECTED 2001-11-07
Foreword
The text of the International Standard from Technical Committee ISO/TC 147 "Water quality"
of the International Organization for Standardization (ISO) has been taken over as an
European Standard 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 February 2002, and conflicting national
standards shall be withdrawn at the latest by February 2002.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,
Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United
Kingdom.
Endorsement notice
The text of the International Standard ISO 10705-2:2000 has been approved by CEN as a
European Standard without any modifications.
NOTE: Normative references to International Standards are listed in annex ZA (normative).
2
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SIST EN ISO 10705-2:2001
EN ISO 10705-2:2001 (E)
Annex ZA (normative)
Normative references to international publications
with their relevant European publications
This European Standard incorporates by dated or undated reference, provisions from other
publications. These normative references are cited at the appropriate places in the text and the
publications are listed hereafter. For dated references, subsequent amendments to or revisions
of any of these publications apply to this European Standard only when incorporated in it by
amendment or revision. For undated references the latest edition of the publication referred to
applies (including amendments).
NOTE Where an International Publication has been modified by common modifications,
indicated by (mod.), the relevant EN/HD applies.
Publication Year Title EN Year
ISO 3696 1987 Water for analytical laboratory use - EN ISO 3696 1995
Specification and test methods
ISO 5667-1 1980 Water quality — Sampling — Part 1: EN 25667-1 1993
Guidance on the design of sampling
programmes
ISO 5667-2 1991 Water quality — Sampling — Part 2: EN 25667-2 1993
Guidance on sampling techniques
ISO 5667-3 1994 Water quality - Sampling - Part 3: EN ISO 5667-3 1995
Guidance on the preservation and
handling of samples
3
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SIST EN ISO 10705-2:2001
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SIST EN ISO 10705-2:2001
INTERNATIONAL ISO
STANDARD 10705-2
First edition
2000-04-01
Water quality — Detection and enumeration
of bacteriophages —
Part 2:
Enumeration of somatic coliphages
Qualité de l'eau — Détection et dénombrement des bactériophages —
Partie 2: Dénombrement des coliphages somatiques
Reference number
ISO 10705-2:2000(E)
©
ISO 2000
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
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ii © ISO 2000 – All rights reserved
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
Contents Page
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Safety precautions.2
5 Principle.2
6 Diluent, culture media and reagents.2
6.1 Basic materials.2
6.2 Diluent.2
7 Apparatus and glassware .2
8 Microbiological reference cultures .4
9 Sampling.4
10 Preparation of test material .5
10.1 Culturing and maintenance of host strains .5
10.2 Calibration of absorbance measurements for counts of viable microorganisms .6
11 Procedure .6
11.1 Preparation of inoculum cultures .6
11.2 Standard procedure.7
11.3 Method for samples with high bacterial background flora .7
11.4 Samples with low phage counts .7
11.5 Presence/absence test .8
11.6 Quality assurance.8
12 Expression of results .9
12.1 Plaque count procedures (11.2 to 11.4).9
12.2 Presence/absence test (11.5).9
13 Test report .10
Annex A (normative) Culture media, reagents and diluent.11
Annex B (informative) General description of somatic bacteriophages .14
Annex C (informative) Culturing of bacteriophage ��X174.15
��
Bibliography.16
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(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 3.
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 part of ISO 10705 may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
International Standard ISO 10705-2 was prepared by Technical Committee ISO/TC 147, Water quality,
Subcommittee SC 4, Microbiological methods.
ISO 10705 consists of the following parts, under the general title Water quality — Detection and enumeration of
bacteriophages:
� Part 1: Enumeration of F-specific RNA bacteriophages
� Part 2: Enumeration of somatic coliphages
� Part 3: Concentration methods
� Part 4: Enumeration of bacteriophages infecting Bacteroides fragilis
Annex A forms a normative part of this part of ISO 10705. Annexes B and C are for information only.
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SIST EN ISO 10705-2:2001
INTERNATIONAL STANDARD ISO 10705-2:2000(E)
Water quality — Detection and enumeration of bacteriophages —
Part 2:
Enumeration of somatic coliphages
1 Scope
This part of ISO 10705 specifies a method for the detection and enumeration of somatic coliphages by incubating the
sample with an appropriate host strain. The method is applicable to all kinds of water, sediments and sludge extracts,
where necessary after dilution. The method is also applicable to shellfish extracts.
In the case of low phage numbers, a preconcentration step may be necessary for which a separate International
Standard will be developed.
NOTE It is desirable for International Standards to be adopted as widely as possible. This part of ISO 10705 includes
reference to alternative procedures which obviate the need for expensive materials or equipment which may not be readily
available in developing countries. Use of these alternatives will not affect the performance of this method.
2 Normative references
The following normative documents contain provisions which, through reference in this text, constitute provisions of
this part of ISO 10705. For dated references, subsequent amendments to, or revisions of, any of these publications
do not apply. However, parties to agreements based on this part of ISO 10705 are encouraged to investigate the
possibility of applying the most recent editions of the normative documents indicated below. For undated
references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain
registers of currently valid International Standards.
ISO 31-0:1992, Quantities and units — Part 0: General principles.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
ISO 5667-1:1980, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes.
ISO 5667-2:1991, Water quality — Sampling — Part 2: Guidance on sampling techniques.
ISO 5667-3:1994, Water quality — Sampling — Part 3: Guidance on the preservation and handling of samples.
ISO 6887:1983, Microbiology — General guidance for the preparation of dilutions for microbiological examination.
ISO 8199:1988, Water quality — General guide to the enumeration of micro-organisms by culture.
ISO/IEC Guide 2, Standardization and related activities — General vocabulary.
3 Terms and definitions
For the purposes of this part of ISO 10705, the terms and definitions given in ISO/IEC Guide 2 and the following
apply.
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
3.1
somatic coliphage
bacterial virus which is capable of infecting selected Escherichia coli host strains (and related strains) by
attachment to the bacterial cell wall as the first step of the infection process
NOTE Somatic coliphages produce visible plaques (clearance zones) in a confluent lawn of host bacteria grown under
appropriate culture conditions.
4 Safety precautions
The host strain used in this standard is non-pathogenic to man and animals, and should be handled in accordance
with the normal (national or international) safety procedures for bacteriological laboratories. Somatic coliphages are
also non-pathogenic to man and animals, but some types are very resistant to drying. Appropriate precautions should
therefore be taken to prevent cross-contamination of test materials, particularly when examining or handling cultures
of high titre or when inoculating cultures of the host strain. Such procedures shall be carried out in a biohazard cabinet
or a separate area of the laboratory.
Chloroform is a carcinogenic substance. Observe relevant safety precautions or use an alternative method of equal
efficacy.
5Principle
The sample is mixed with a small volume of semi-solid nutrient medium. A culture of host strain is added and plated
on a solid nutrient medium. After this, incubation and reading of plates for visible plaques takes place. The results are
expressed as the number of plaque-forming particles, pfp (also termed plaque-forming units, pfu), per unit of sample
volume.
6 Diluent, culture media and reagents
6.1 Basic materials
Use ingredients of uniform quality and chemicals of analytical grade for the preparation of culture media and reagents
and follow the instructions given in annex A. For information on storage see ISO 8199, except where indicated in this
part of ISO 10705. Alternatively, use dehydrated complete media and follow strictly the manufacturer's instructions.
For the preparation of media, use glass-distilled water or deionized water free from substances which might inhibit
bacterial growth under the conditions of the test, and complying with ISO 3696.
NOTE Use of other grades of chemicals is permissible providing they are shown to be of equal performance in the test.
6.2 Diluent
For making sample dilutions, use peptone-saline solution (A.7) or another diluent complying with ISO 6887.
7 Apparatus and glassware
Usual microbiological laboratory equipment, including
7.1 Hot-air oven for dry-heat sterilization and an autoclave. Apart from apparatus supplied sterile, glassware
and other equipment shall be sterilized according to the instructions given in ISO 8199.
7.2 Incubator or water bath, thermostatically controlled at (36� 2) °C.
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
7.3 Incubator or water bath, thermostatically controlled at (36� 2) °C and equipped with a shaking device, for
example a rotating platform at (100� 10) r/min.
7.4 Water bath or heating block, thermostatically controlled at (45 � 1) °C.
7.5 Water bath or equivalent device for melting of agar media.
7.6 pH meter.
7.7 Counting apparatus with indirect, oblique light.
7.8 Deep freezer, thermostatically controlled at (�20� 5) °C.
7.9 Deep freezer, thermostatically controlled at (�70� 10) °C or liquid nitrogen storage vessel.
7.10 Spectrophotometer, capable of holding cuvettes of 1 cm optical path length or side-arm of nephelometric
flasks (7.17) and equipped with a filter for the range 500 nm to 650 nm with a maximum bandwidth of � 10 nm.
Usual sterile, microbiological laboratory glassware or disposable plasticsware according to ISO 8199 and including
7.11 Petri dishes of 9 cm or 14 cm to 15 cm diameter, vented.
7.12 Graduated pipettes of 0,1 ml, 1 ml, 5 ml and 10 ml capacity and Pasteur pipettes.
7.13 Glass bottles of suitable volume.
7.14 Culture tubes with caps or suitable alternative.
7.15 Measuring cylinders of suitable capacity.
7.16 Conical flasks of 250 ml to 300 ml capacity, with cotton wool plugs or suitable alternative.
7.17 Cuvettes of optical path length 10 mm or nephelometric conical flasks with cylindrical side-arms which fit
in the spectrophotometer (7.10) (see Figure 1); capacity 250 ml to 300 ml with cotton wool plugs or suitable
alternative.
7.18 Membrane filter units for decontamination, pore size 0,2 μm.
7.19 Plastics vials, lidded, of 1,5 ml to 3 ml capacity.
7.20 Refrigerator, temperature set at (5� 3) °C.
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
Figure 1 — Nephelometric conical flask for culturing the host strain
8 Microbiological reference cultures
For samples with low bacterial content (drinking water, unpolluted natural waters), use Escherichia coli strain C,
ATCC 13706. Samples containing large numbers of bacteria (polluted natural waters, wastewater) should be
[1] [2]
examined using the nalidixic acid resistant mutant E. coli strain CN (ATCC 700078 ), also known as WG5 .
Use bacteriophage �X174 (ATCC 13706-B1) for the preparation of reference material (11.6.1).
NOTE The ATCC strains are available from the American Type Culture Collection, 10801 University Boulevard, Manassas,
VA 20110.
9 Sampling
Take samples and deliver them to the laboratory in accordance with ISO 8199, ISO 5667-1, ISO 5667-2 and
ISO 5667-3.
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
10 Preparation of test material
10.1 Culturing and maintenance of host strains
10.1.1 General
The culturing and maintenance of host strains involves several stages which are summarized in Figure 2.
For culturing of the host strains in the several stages, it is best to gently shake the cultures. In addition to increasing
the growth rate of bacteria, shaking ensures that all the cells are actively growing and no stationary-phase cells
develop, which could decrease the efficiency of plating. Therefore, inoculum cultures should be repeatedly shaked
by hand if a shaker is not available.
Figure 2 — Scheme for culture and maintenance of host strains
10.1.2 Preparation of stock cultures
Rehydrate the contents of a lyophilized ampoule of the reference culture of the host strains in a small amount (ca.
3 ml) of Modified Scholtens' Broth (M.S.B.) (A.1) using a Pasteur pipette (7.12). Transfer the suspension to a 300 ml
conical flask (7.16) containing (50 � 5) ml of MSB. Incubate for (20� 4) h at (36� 2) �C while gently shaking using an
incubator or water bath (7.3). Add 10 ml [i.e. a final concentration of 15 % to 20 % (volume fraction)] of sterile glycerol
(A.5) and mix well. Distribute into plastics vials (7.19) in ca. 0,5 ml aliquots and store at (�70� 10) �C or in liquid
nitrogen.
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
NOTE This first passage of the host strains should be stored as a reference in the laboratory.
10.1.3 Preparation of working cultures
Remove a vial of stock culture (10.1.2) from frozen storage, allow to equilibrate to room temperature (15 °C to 30 °C)
and inoculate on a plate of McConkey agar (A.6) or another lactose-containing medium in such a way that single
colonies are obtained. Incubate at (36� 2) �Cfor(20 � 4) h. The remaining content of the vial of stock culture can be
used to inoculate more plates on the same working day (if necessary), otherwise it should be treated as contaminated
waste.
Add (50 � 5) ml of MSB to a conical flask of 300 ml (7.16) and warm to at least room temperature (faster growth will
occur if the broth is prewarmed to 37�C). Select three to five lactose-positive colonies from the McConkey agar and
inoculate material from each of these colonies in the flask with MSB. Incubate for (5� 1) h at (36 � 2)�C while gently
shaking using an incubator or water bath (7.3). Add 10 ml of sterile glycerol (A.5) and mix well. Distribute in plastics
vials (7.19) in ca. 1,2 ml aliquots and store in a deep freezer at (�70 � 10) �C (7.9) for a maximum of two years.
NOTE If a great number of tests is anticipated, several conical flasks can be inoculated in parallel.
10.2 Calibration of absorbance measurements for counts of viable microorganisms
Remove a vial of working culture from the deep freeze (7.9) and allow to equilibrate to room temperature (15 °C to
30 °C). Add (50 � 5) ml of MSB to a nephelometric conical flask (7.17), warm to at least room temperature (faster
growth will occur if the broth is prewarmed to 37�C). Adjust the spectrophotometer reading to zero on the filled flask
side-arm. Alternatively, add (50� 5) ml of MSB (A.1) to a plain conical flask (7.16) and aseptically transfer a portion to
a cuvette (7.17). Using this cuvette, adjust the spectrophotometer reading to zero. Discard the broth transferred to the
cuvettes used to measure absorbance.
Inoculate MSB with 0,5 ml of working culture. Incubate at (36� 2) �C with gentle shaking in an incubator or water bath
(7.3) for up to 3,5 h. Every 30 min measure absorbance as indicated above and withdraw a 1 ml aliquot for viable
counts, ensuring that the flask is removed from the incubator for as short a time as possible.
�7 �5 �6 �7
Dilute aliquots to 10 and count colony-forming units (cfu) in 1 ml volumes of the 10 ,10 and 10 dilutions by the
standard pour-plate procedure in nutrient agar or Modified Scholtens' Agar (MSA) (A.2.1), in duplicate. Alternatively,
perform membrane filtration with 1 ml volumes of the same dilutions and count cfu by the standard membrane filter
procedure on nutrient agar or MSA (A.2.1), in duplicate. Incubate at (36� 2) �Cfor (20� 4) h (using 7.2). Count the
total number of colonies in/on each plate yielding between 30 and 300 colonies and calculate the number of cfu/ml
(consult ISO 8199 if necessary).
NOTE 1 This procedure should be carried out several times (approx. two to three times) to establish the relationship between
absorbance measurements and colony counts. Once sufficient data have been obtained, further work can then be based only
on absorbance measurements.
8
NOTE 2 If the cell density of approx. 10 cfu/ml is not reached within 3,5 h of incubation, 1 ml of working culture may be
inoculated instead of 0,5 ml.
11 Procedure
11.1 Preparation of inoculum cultures
Remove a vial of working culture from the deep freeze (7.9) and allow to equilibrate to room temperature (15 °C to
30 °C). Add (50 � 5) ml of MSB to a nephelometric conical flask (7.17) or plain conical flask (7.16), and prewarm to at
least room temperature (faster growth will occur if the broth is prewarmed to 37�C). Adjust the spectrophotometer
reading to zero as indicated in 10.2.
Inoculate 0,5 ml of working culture into MSB. Incubate at (36� 2) �C with gentle shaking in an incubator or water bath
(7.3). Measure absorbance every 30 min as indicated in 10.2. At an absorbance corresponding to a cell density of
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SIST EN ISO 10705-2:2001
ISO 10705-2:2000(E)
8
approximately 10 cfu/ml (based on data obtained in 10.2), take the inoculum culture from the incubator and quickly
cool the culture by placing it in melting ice. Use the inoculum culture within the same working day.
NOTE An alternative (but less controlled) way to prepare an inoculum culture is the following:
Inoculate 0,5 ml of working culture, thawed as indicated above, into (50 � 5) ml of MSB prewarmed at room temperature. Incubate
for (3 � 1) h at (36 � 2) °C with gentle shaking. Alternatively, inoculate typical colonies from an agar plate, or a loopful of growth
from an agar slant [incubated for not longer than (20 � 4) h at (36 � 2) °C and stored at (5 � 3 ) °C for not longer than a working
day], into (50 � 5) ml of MSB prewarmed at room temperature and incubate for (3 � 1) h at (36 � 2) °C with gentle shaking. Use
immediately or take the inoculum culture from the incubator and quickly cool to 5 °C to 10 °C, preferably by placing onto melting
ice. Use this inoculum culture within the same working day. Whatever the preparation procedure may be, the inoculum culture
8
should ideally have a count of approximately 10 cfu per ml.
11.2 Standard procedure
Prepare an inoculum culture as described in 11.1.
Melt bottles of 50 ml semi-solid Modified Scholtens' Agar (ssMSA) (A.3) in a boiling water bath (7.5) and place in a
water bath at (45� 1)�C. Aseptically add 300 μl of a calcium chloride solution (A.2.2) prewarmed at room
temperature and distribute 2,5 ml aliquots into culture tubes (7.14) with caps, placed in a water bath at (45� 1)�C.
To each culture tube, add 1 ml of the original sample (or diluted or concentrated sample) prewarmed at room
temperature. Examine each aliquot at least in duplicate.
Add 1 ml of inoculum culture to each culture tube containing the aliquots of sample and ssMSA, mix carefully avoiding
the formation of air bubbles and pour the contents on a layer of complete MSA (A.2.3) in a 9 cm Petri dish prewarmed
at room temperature. Distribute evenly and allow to solidify on a horizontal, cool surface. Dry the plates by incubating
with partially opened lids, then cover and incubate the plates upside-down at (36� 2)�Cfor
...
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