SIST EN 13704:2002
(Main)Chemical disinfectants - Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
Chemical disinfectants - Quantitative suspension test for the evaluation of sporicidal activity of chemical disinfectants used in food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)
This European Standard specifies a test method (phase 2/step 1) (see annex H) and the minimum requirements for sporicidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation in hard water and that are used in food, industrial, domestic and institutional areas, excluding areas and situations where disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in the above considered areas.
This European Standard applies at least to the following :
a) processing, distribution and retailing of :
1) food of animal origin :
- milk and milk products ;
- meat and meat products ;
- fish, seafood, and related products ;
- eggs and egg products ;
- animal feeds ;
- etc. ;
2) food of vegetable origin :
- beverages ;
- fruits, vegetables and derivatives (including sugar, distillery, etc.) ;
- flour, milling and baking ;
- animal feeds ;
- etc. ;
b) institutional and domestic areas :
- catering establishments ;
- public areas ;
- public transports ;
- schools ;
- nurseries ;
- shops ;
- sports rooms ;
- waste containers (bins, etc.) ;
- hotels ;
- dwellings ;
- clinically non sensitive areas of hospitals ;
- offices ;
- etc. ;
c) other industrial areas :
- packaging material ;
- biotechnology (yeast, proteins, enzymes, etc.) ;
- pharmaceutical ;
- cosmetics and toiletries ;
- textiles ;
- space industry, computer industry ;
- etc.
Using this European Standard, it is not possible to determine the sporicidal activity of undiluted product as some dilution is always produced by adding the inoculum and interfering substance. Products can only be tested at a concentration of 80 % or less.
NOTE The method described is intended to determine the activity of commercial formulations or active substances on spores in the conditions in which they are used.
Chemische Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Diese Europäische Norm legt ein Prüfverfahren (Phase 2, Stufe 1) (siehe Anhang H) und Anforderungen an die sporizide Mindestwirkung von chemischen Desinfektionsmitteln fest, die in hartem Wasser eine homogene, physikalisch stabile Lösung ergeben, und die im Lebensmittelbereich, in der Industrie, im Haushalt und in öffentlichen Einrichtungen benutzt werden, ausgenommen davon sind Bereiche und Situationen, in denen die Desinfektion medizinisch indiziert ist und Produkte, die auf lebenden Geweben benutzt werden, mit Ausnahme derjenigen für die Handhygiene in den oben angegebenen Bereichen.
Diese Europäische Norm gilt grundsätzlich für den folgenden Anwendungsbereich:
a) Verarbeitung, Vertrieb und Verkauf von:
1) Lebensmitteln tierischer Herkunft:
3 Milch und Milchprodukte;
3 Fleisch und Fleischprodukte;
3 Fisch, Meerestiere und daraus hergestellte Zubereitungen;
3 Eier und Eiprodukte;
3 Tiernahrung
3 usw.
2) Lebensmitteln pflanzlicher Herkunft:
3 Getränke;
3 Früchte, Gemüse und daraus hergestellte Zubereitungen (einschließlich Zucker, Destillationsprodukte usw.);
3 Mehl, gemahlene und gebackene Produkte;
3 Tiernahrung
3 usw.
b) Öffentliche Einrichtungen und Haushaltsbereiche:
3 Verpflegungseinrichtungen;
3 Öffentliche Bereiche;
3 Öffentliche Verkehrsmittel;
3 Schulen;
3 Kindergärten;
3 Geschäfte;
3 Sportstätten;
3 Abfallbehälter (Mülltonnen usw.);
3 Hotels;
3 Wohngebäude;
3 Klinisch nicht relevante Bereiche von Krankenhäusern;
3 Büroräume
3 usw.
c) Andere industrielle Bereiche:
3 Verpackungsmaterial;
3 Biotechnologie (Hefe, Proteine, Enzyme usw.);
3 Hersteller von Pharmazeutika;
3 Hersteller von Kosmetika und Toilettenartikeln;
3 Hersteller von Textilien;
3 Weltraumforschungsindustrie und Computerindustrie
3 usw.
Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité sporicide des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)
La présente Norme européenne décrit une méthode d'essai (phase 2, étape 1) (voir annexe H) et les prescriptions minimales relatives a l'activité sporicide des désinfectants chimiques qui forment une préparation homogene physiquement stable dans l'eau dure et qui sont utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans le domaine domestique et en collectivité, a l'exception des domaines et des situations ou la désinfection est médicalement prescrite et a l'exclusion des produits utilisés sur des tissus vivants (excepté ceux destinés a l'hygiene des mains dans les domaines indiqués ci-dessus).
La présente Norme européenne s'applique au moins aux domaines suivants :
a) fabrication, distribution et vente au détail de :
1) produits alimentaires d'origine animale :
3 lait et produits laitiers ;
3 viande et produits dérivés de la viande ;
3 poisson, fruits de mer et leurs produits dérivés ;
3 ?ufs et produits avicoles ;
3 produits alimentaires pour animaux ;
3 etc. ;
2) produits alimentaires d'origine végétale :
3 boissons ;
3 fruits, légumes et leurs dérivés (y compris le sucre, la distillerie, etc.) ;
3 farine, minoterie et boulangerie ;
3 produits alimentaires pour animaux ;
3 etc. ;
b) domaines domestiques et collectivité :
3 établissements de restauration ;
3 lieux publics ;
3 transports publics ;
3 écoles ;
3 haltes-garderies ;
3 magasins ;
3 salles de sport ;
3 conteneurs pour déchets (poubelles, etc.) ;
3 hôtels ;
3 lieux d'habitation ;
3 zones cliniquement non sensibles des hôpitaux ;
3 bureaux ;
3 etc. ;
c) autres zones industrielles :
3 matériaux d'emballage ;
3 biotechnologie (levure, protéines, enzymes, etc.) ;
3 pharmaceutique ;
3 cosmétique et produits d'hygiene corporelle ;
3 textiles ;
3 industrie spatiale, industrie informatique ;
3 etc..
Kemična razkužila - Kvantitativni suspenzijski preskus za ocenjevanje sporocidnega delovanja kemičnih razkužil v živilski in drugih industrijah in javnih ustanovah - Preskusna metoda in zahteve (faza 2, stopnja 1)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 13704:2002
01-september-2002
.HPLþQDUD]NXåLOD.YDQWLWDWLYQLVXVSHQ]LMVNLSUHVNXV]DRFHQMHYDQMH
VSRURFLGQHJDGHORYDQMDNHPLþQLKUD]NXåLOYåLYLOVNLLQGUXJLKLQGXVWULMDKLQMDYQLK
XVWDQRYDK3UHVNXVQDPHWRGDLQ]DKWHYH ID]DVWRSQMD
Chemical disinfectants - Quantitative suspension test for the evaluation of sporicidal
activity of chemical disinfectants used in food, industrial, domestic and institutional areas
- Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der
sporiziden Wirkung chemischer Desinfektionsmittel in den Bereichen Lebensmittel,
Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen
(Phase 2, Stufe 1)
Désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité
sporicide des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire,
dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et
prescriptions (phase 2, étape 1)
Ta slovenski standard je istoveten z: EN 13704:2002
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
SIST EN 13704:2002 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 13704:2002
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SIST EN 13704:2002
EUROPEAN STANDARD
EN 13704
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2002
ICS 71.100.35
English version
Chemical disinfectants - Quantitative suspension test for the
evaluation of sporicidal activity of chemical disinfectants used in
food, industrial, domestic and institutional areas - Test method
and requirements (phase 2, step 1)
Désinfectants chimiques - Essai quantitatif de suspension Chemische Desinfektionsmittel - Quantitativer
pour l'évaluation de l'activité sporicide des désinfectants Suspensionsversuch zur Bestimmung der sporiziden
chimiques utilisés dans le domaine de l'agro-alimentaire, Wirkung chemischer Desinfektionsmittel in den Bereichen
dans l'industrie, dans les domaines domestiques et en Lebensmittel, Industrie, Haushalt und öffentliche
collectivité - Méthode d'essai et prescriptions (phase 2, Einrichtungen - Prüfverfahren und Anforderungen (Phase 2,
étape 1) Stufe 1)
This European Standard was approved by CEN on 11 November 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, Malta, 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
© 2002 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 13704:2002 E
worldwide for CEN national Members.
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SIST EN 13704:2002
EN 13704:2002 (E)
Contents
page
Foreword.3
Introduction .4
1 Scope .5
2 Normative references .6
3 Terms and definitions.6
4 Requirements .7
5 Test method.7
5.1 Principle.7
5.2 Materials and reagents .8
5.3 Apparatus and glassware .10
5.4 Preparation of spore test suspension and test solutions .11
5.5 Procedure .12
5.6 Calculation and expression of results.15
5.7 Conclusion.17
5.8 Test report .18
Annex A (normative) Preparation of Bacillus spore stock suspensions .20
A.1 Material and reagents .20
A.2 Preparation of Bacillus spore stock suspensions .20
Annex B (normative) Validation of dilution-neutralization and membrane filtration methods .21
B.1 Principle.21
B.2 Preparation of spore suspension.21
B.3 Preparation of product test solution.21
B.4 Test for validation .21
B.5 Validation.24
Annex C (informative) Preparation of Clostridium sporogenes spore stock suspension.25
C.1 Culture media and reagents.25
C.2 Apparatus and glassware .26
C.3 Preparation of regenerated media and incubation conditions .26
C.4 Preparation of Clostridium spore stock suspension .26
Annex D (informative) Neutralizers.27
Annex E (informative) Rinsing liquids.28
Annex F (informative) Example of a typical test report .29
Annex G (informative) Referenced strains in national collections.32
G.1 Bacillus subtilis.32
G.2 Bacillus cereus.32
G.3 Clostridium sporogenes.32
Annex H (informative) Information on the application and interpretation of European standards on
chemical disinfectants and antiseptics .33
H.1 General guidelines for the application and interpretation of test methods in accordance with
European Standards for chemical disinfectants and antiseptics.33
H.2 Guide to interpretation of tests for chemical disinfectants and antiseptics .34
2
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SIST EN 13704:2002
EN 13704:2002 (E)
Foreword
This European Standard has been prepared by Technical Committee CEN/TC 216 "Chemical disinfectants and
antiseptics", the secretariat of which is held by AFNOR.
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 August 2002, and conflicting national standards shall be withdrawn at the latest by
August 2002.
In this standard the annexes A and B are normative. The annexes C, D, E, F, G and H are informative.
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, Malta, Netherlands, Norway, Portugal, Spain,
Sweden, Switzerland and the United Kingdom.
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SIST EN 13704:2002
EN 13704:2002 (E)
Introduction
This European Standard describes a suspension test method for establishing whether a chemical disinfectant has
or does not have a sporicidal activity in the fields described in clause 1.
The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time,
temperature, in suspension, etc.) reflect parameters which are found in practical situations including conditions
which can influence the action of disinfectants. Each utilization concentration found from this test corresponds to
defined experimental conditions.
The conditions are intended to cover general purposes and to allow reference between laboratories and product
types.
However for some applications the recommendations of use of a product can differ and therefore additional test
conditions need to be used.
4
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SIST EN 13704:2002
EN 13704:2002 (E)
1 Scope
This European Standard specifies a test method (phase 2/step 1) (see annex H) and the minimum requirements for
sporicidal activity of chemical disinfectant products that form a homogeneous, physically stable preparation in hard
water and that are used in food, industrial, domestic and institutional areas, excluding areas and situations where
disinfection is medically indicated and excluding products used on living tissues except those for hand hygiene in
the above considered areas.
This European Standard applies at least to the following :
a) processing, distribution and retailing of :
1) food of animal origin :
milk and milk products ;
meat and meat products ;
fish, seafood, and related products ;
eggs and egg products ;
animal feeds ;
etc. ;
2) food of vegetable origin :
beverages ;
fruits, vegetables and derivatives (including sugar, distillery, etc.) ;
flour, milling and baking ;
animal feeds ;
etc. ;
b) institutional and domestic areas :
catering establishments ;
public areas ;
public transports ;
schools ;
nurseries ;
shops ;
sports rooms ;
waste containers (bins, etc.) ;
hotels ;
5
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SIST EN 13704:2002
EN 13704:2002 (E)
dwellings ;
clinically non sensitive areas of hospitals ;
offices ;
etc. ;
c) other industrial areas :
packaging material ;
biotechnology (yeast, proteins, enzymes, etc.) ;
pharmaceutical ;
cosmetics and toiletries ;
textiles ;
space industry, computer industry ;
etc.
Using this European Standard, it is not possible to determine the sporicidal activity of undiluted product as some
dilution is always produced by adding the inoculum and interfering substance. Products can only be tested at a
concentration of 80 % or less.
NOTE The method described is intended to determine the activity of commercial formulations or active substances on
spores in the conditions in which they are used.
2 Normative references
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).
EN 1040, Chemical disinfectants and antiseptics – Basic bactericidal activity – Test method and requirements
(phase 1).
3 Terms and definitions
For the purposes of this European Standard, the following terms and definitions apply.
3.1
product (for chemical disinfection and/or antisepsis)
chemical agent or formulation used as a chemical disinfectant or antiseptic [EN 1040]
3.2
sporicide (in this standard)
product which kills spores of the genus Bacillus
NOTE The adjective derived from « sporicide » is « sporicidal ».
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3.3
sporicidal activity (in this standard)
3
capability of the product to produce at least a reduction of 10 in the number of bacterial spores belonging to
reference strain of Bacillus subtilis under conditions defined by this European Standard
3.4
clean conditions
conditions representative of surfaces which have received a satisfactory cleaning programme and/or are known to
contain minimal levels of organic and/or inorganic materials
4 Requirements
The product, when diluted in hard water and tested in accordance with clause 5 under simulated clean conditions
(0,3 g/l bovine albumin see 3.4) according to its practical applications and under the required test conditions (20 °C,
3
60 min, 1 selected reference strain), shall demonstrate at least a 10 reduction in viable counts.
The sporicidal activity shall be evaluated using the test organism Bacillus subtilis.
The determined sporicidal concentration of the test product is suggested as being suitable for practical situations of
use.
Where appropriate, additional specific sporicidal activity shall be determined under other conditions of time,
temperature and additional strains (see 5.2.1 and 5.5.1) in accordance with 5.5.1 in order to take into account
intended specific use conditions.
NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the
initial test conditions of 20 °C, 60 min, 1 selected reference strain.
5 Test method
5.1 Principle
5.1.1 A test suspension of bacterial spores in a solution of interfering substance, simulating clean conditions, is
added to a prepared sample of the product under test diluted in hard water. The mixture is maintained at
20 °C ± 1 °C for 60 min ± 10 s (required test conditions).
At this contact time, an aliquot is taken ; the sporicidal action in this portion is immediately neutralized or
suppressed by a validated method. The method of choice is dilution-neutralization. If a suitable neutralizer cannot
be found, membrane filtration is used. The number of surviving bacterial spores in each sample are determined and
the reduction in viable counts is calculated.
5.1.2 The test is performed using spores of Bacillus subtilis. Additional and optional exposure times,
temperatures and strains are specified.
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5.2 Materials and reagents
5.2.1 Test organisms
The sporicidal activity shall be evaluated by using spores of the following strain :
1)
Bacillus subtilis ATCC 6633 .
If required for specific applications, additional strains may be chosen from, for example :
1)
Bacillus cereus ATCC 12826 ;
1)
Clostridium sporogenes CIP 7939 .
NOTE 1 See annex G for corresponding strain numbers in some other culture collections.
NOTE 2 See annex C for particular culture and handling conditions for Clostridium sporogenes.
If additional strains are used, they shall be incubated under optimum growth conditions (temperature, time,
atmosphere) and noted in the test report.
If the additional strains selected do not correspond to the specified strains, their suitability for supplying inocula of
sufficient concentration shall be verified. If the additional strains tested are not classified at a reference centre their
identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national
culture under a reference for 5 years.
5.2.2 Culture media and reagents
5.2.2.1 General
The reagents shall be of analytical grade and/or appropriate for microbiological purposes.
NOTE To improve reproducibility, it is recommended that commercially available dehydrated material is used for the
preparation of culture media. The manufacturer’s instructions relating to the preparation of these products should be rigorously
followed.
5.2.2.2 Water
The water shall be free from substances that are toxic or inhibiting to the bacterial spores or to the bacteria. It shall
be freshly glass distilled water and not demineralized water.
Sterilize in the autoclave (see 5.3.1).
NOTE 1 If the water is sterilized during the sterilization of the reagents, this is not necessary.
NOTE 2 If distilled water of adequate quality is not available, water for injectable preparation (European Pharmacopoeia) can
be used.
1)
ATCC 6633 and ATCC 12826 are the collection numbers of strains supplied by the American Type Culture Collections. CIP
7939 is the collection number of spores supplied by the Collection de l'Institut Pasteur. This information is given for the
convenience of users of this standard and does not constitute an endorsement by CEN of the product named. Corresponding
strains supplied by other culture collections may be used if they can be shown to lead to the same results.
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5.2.2.3 Glucose Yeast Extract Agar (GYA)
For counting of viable Bacillus spores :
Amino-acids, without vitamins, obtained by acid hydrolysis of casein. 1,0 g
Soluble starch. 1,0 g
Glucose . 2,5 g
Yeast extract . 5,0 g
FeSO . 0,1 g
4
MnSO H O 0,000 1 g
4, 2
Agar .15,0 g
Water (see 5.2.2.2) 1 000,0 ml
Sterilize in the autoclave (see 5.3.1). After sterilization the pH of the medium shall be equivalent to 6,8 ± 0,2 when
measured at 20 °C.
5.2.2.4 Neutralizer
The neutralizer shall be validated for the product under test in accordance with annex D. The neutralizer shall be
sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in annex D.
5.2.2.5 Rinsing liquid (for membrane filtration)
The liquid shall be sterile, compatible with the filter membrane and capable of filtration through the filter membrane
under the test conditions described in annex B.
NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in
annex E.
5.2.2.6 Hard water for dilution of products
Hard water for dilution of products shall be prepared as follows :
Solution A : Dissolve 19,84 g anhydrous magnesium chloride (MgCl ) or an equivalent of hydrated
2
magnesium chloride and 46,24 g anhydrous calcium chloride (CaCl ) or an equivalent of hydrated calcium
2
chloride in water (see 5.2.2.2) and dilute to 1000 ml.
Sterilize in the autoclave (see 5.3.1). Store the solution at 2 °C - 8 °C for no longer than one month.
Solution B : Dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (see 5.2.2.2) and dilute to 1 000 ml.
3
Sterilize by membrane filtration (see 5.3.2.7). Store the solution at 2 °C - 8 °C for no longer than one week.
Hard Water :
For the preparation of 1 litre, place at least 600 ml water (see 5.2.2.2) in a 1000 ml volumetric flask (see 5.3.2.12)
and add 6,0 ml of solution A, then 8,0 ml of solution B.
Mix and dilute to 1 000 ml with water (see 5.2.2.2).
The pH of the hard water shall be 7,0 ± 0,2.
If necessary adjust the pH by using a solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH)
or approximately 36,5 g/l (about 1 mol/l) of hydrochloric acid (HCI).
The hard water shall be freshly prepared under aseptic conditions and used within 12 hours.
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NOTE When preparing the product test solutions (see 5.4.2) the addition of the product to this hard water produces a
different final water hardness in each test tube.
In any case the final hardness is lower than 300 mg/kg of calcium carbonate (CaCO ) in the test tube.
3
5.2.2.7 Interfering substance
5.2.2.7.1 General
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test.
5.2.2.7.2 Bovine albumin solution
Bovine albumin solution for the test conditions shall be prepared as follows :
dissolve 0,30 g of bovine albumin (Cohn fraction V for Dubos medium) in 100 ml of water (see 5.2.2.2) ;
sterilize by membrane filtration.
The final concentration of the bovine albumin in the test procedure (see 5.5.2) is 0,3 g/l.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and reagents or
the sample, except those which are supplied sterile, by one of the following methods :
3
a) in the autoclave (see 5.3.2.1) by maintaining it at (121 ) °C for a minimum holding time of 15 min ;
0
b) in the dry heat sterilizer (see 5.3.2.1) by maintaining it at 180 °C for a minimum holding time of 30 min, at
170 °C for a minimum holding time of 1 h, or at 160 °C for a minimum holding time of 2 h.
2)
5.3.2 Usual microbiological laboratory equipment and, in particular, the following :
5.3.2.1 Apparatus for sterilization
3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum holding time
0
of 15 min ;
b) for dry heat sterilization, a hot air oven capable of being maintained at 180 °C for a minimum holding time of
30 min, at 170 °C for a minimum holding time of 1 h, or at 160 °C a minimum holding time of 2 h.
5.3.2.2 Water baths, capable of being controlled at 20 °C ± 1 °C, 45 °C ± 1 °C and at additional test
temperatures ± 1 °C (see 5.5.1).
5.3.2.3 Incubator, capable of being controlled at 30 °C ± 1 °C.
, having an accuracy of calibration of ± 0,1 pH units at 25 °C.
5.3.2.4 pH-meter
5.3.2.5 Stopwatch
3)
5.3.2.6 Vortex mixer (electromechanical agitator, i.e. Vortex® mixer )
2)
Disposable equipment is an acceptable alternative to reusable glassware.
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5.3.2.7 Membrane filtration apparatus (if this method is used), constructed of a material compatible with the
product under test, with a filter holder which shall have a usable volume 50 ml minimum, and suitable for use with
filters of diameter 47 mm to 50 mm, of 0,45 μm pore size.
The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the
microorganisms over the membrane and in order to prevent overlong filtration, the device shall be set so as to
obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s.
5.3.2.8 Containers : Test tubes or flasks of suitable capacity.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic pipettes
may be used.
5.3.2.10 Petri dishes of size 90 mm to 100 mm
5.3.2.11 Glass beads (Diameter : 3 mm to 4 mm).
5.3.2.12 Volumetric flasks
5.3.2.13 Glass Roux bottles with straight neck
5.3.2.14 Microscope, preferably, a phase-contrast type, with magnification of at least x 400.
5.4 Preparation of spore test suspension and test solutions
5.4.1 Spore suspensions
5.4.1.1 Stock spore suspension of test organism
Bacillus subtilis spore stock suspension for the specific purposes of this standard can be purchased from a national
culture collection or prepared by the testing laboratory.
If the Bacillus subtilis spore stock suspension is prepared in the testing laboratory, the suspension shall be
prepared according to annex A.
As part of good laboratory practice (GLP), laboratories may want to include an internal reference product (e.g :
sodium hypochlorite) to check the resistance of the spores.
For the preparation of the stock spore suspensions of additionnal strains (see 5.2.1) refer to :
annex A for Bacillus cereus ATCC 12826 ;
annex C for Clostridium sporogenes CIP 7939.
5.4.1.2 Spore test suspension
To prepare the spore test suspension, dilute the spore stock suspension (see 5.4.1.1) with water (see 5.2.2.2). The
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number of spores in the test suspension must be adjusted to 1,5 x 10 to 5 x 10 cfu/ml, estimating the number of
units by any suitable mean.
Maintain the suspension test in the water bath at 20 °C ± 1 °C and use within 2 h.
Microscopic examination under 400 x magnification shall be carried out immediately after the preparation and just
before the test, to show the absence of vegetative cells and germinative spores.
3)
Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users
of this standard and does not constitute an endorsement by CEN of this product.
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SIST EN 13704:2002
EN 13704:2002 (E)
If there is any evidence of spore germination, the suspension shall be discarded.
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For counting of the spore test suspension prepare 10 and 10 dilutions of the test suspension (see 5.4.1.3) using
water (see 5.2.2.2). Mix (see 5.3.2.6). Take a sample of 1,0 ml of each dilution in duplicate and transfer each 1,0 ml
sample into separate Petri dishes (see 5.3.2.10) and add 12 ml to 15 ml melted GYA (see 5.2.2.3), cooled to
45 °C ± 1 °C.
5.4.1.3 Counting of spore test suspension
Incubate the Petri dishes at 30 °C ± 1 °C (see 5.3.2.3) for 3 days. Determine the higher number of colonies V for
c
4)
each plate. Calculate the number of cfu/ml (see 5.4.1.3).
in the test suspension (N) using the method given in 5.6.1.1.
5.4.2 Product test solution
Details of samples of the product as received shall be recorded.
Product test solutions shall be prepared in hard water (see 5.2.2.6) at three different concentrations to include one
concentration in the active range and one concentration in the non-active range. The concentration of the product
test solution shall be 1,25 times the required test concentration.
For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a volumetric
flask and filling up with hard water (see 5.2.2.6). Subsequent dilutions shall be prepared in volumetric flasks
(see 5.3.2.12) on a volume/volume basis in hard water (see 5.2.2.6).
For liquid products, dilutions of the product shall be prepared in hard water (see 5.2.2.6) on a volume/volume basis
using volumetric flasks (5.3.2.12).
For products supplied in a ready to use state, water (see 5.2.2.2) shall be used to prepare dilutions.
When the product is diluted in hard water it shall give a physically homogeneous stable preparation.
The
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