Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in veterinary area on porous surfaces without mechanical action - Test method and requirements (phase 2, step 2)

This European Standard specifies a test method and the minimum requirements for bactericidal activity of chemical disinfectants and antiseptic products that form a homogeneous, physically stable preparation when diluted with hard water - or in the case of ready-to-use products - with water.
This European Standard applies to products that are used in the veterinary area on porous surfaces without mechanical action i.e. in the breeding, husbandry, production, !veterinary care facilities", transport and disposal of all animals except when in the food chain following death and entry to the processing industry.
EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 2 test.
NOTE 3 This method cannot be used to evaluate the activity of products against mycobacteria or bacterial spores.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf porösen Oberflächen ohne mechanische Wirkung - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Diese Europäische Norm legt ein Prüfverfahren und die Mindestanforderungen an die bakterizide Wirkung von chemischen Desinfektionsmitteln und Antiseptika fest, die bei Verdünnung mit Wasser standardisierter Härte oder – im Fall gebrauchsfertiger Produkte – mit Wasser eine homogene, physikalisch stabile Zubereitung bilden.
Diese Europäische Norm gilt für Produkte, die im Veterinärbereich auf porösen Oberflächen ohne mechanische Wirkung angewendet werden, d. h. bei der Aufzucht, Haltung, Produktion !in veterinärmedizinischen Gesundheitseinrichtungen," und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die Nahrungsmittelkette eintreten.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1 Das beschriebene Verfahren ist für die Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter den Bedingungen, unter denen sie verwendet werden, bestimmt.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
ANMERKUNG 3 Dieses Verfahren kann nicht angewendet werden, um die Wirkung von Produkten gegen Mykobakterien oder bakterielle Sporen zu bewerten.

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire sur des surfaces poreuses sans action mécanique - Méthode d'essai et prescriptions (phase 2, étape 2)

La présente Norme européenne spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité bactéricide des produits antiseptiques et désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans de l'eau dure ou – dans le cas de produits prêts à l’emploi – dans l’eau.
La présente Norme européenne s'applique aux produits utilisés dans le domaine vétérinaire sur des surfaces poreuses sans action mécanique, à savoir la reproduction, l'élevage, la production, !les établissements de soins vétérinaires", le transport et l’abattage de tous les animaux, sauf au cours de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L'EN 14885 spécifie en détail la relation entre les différents essais et les « recommandations d'emploi ».
NOTE 1 La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives dans les conditions dans lesquelles elles sont utilisées.
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 2.
NOTE 3 Cette méthode ne peut pas être utilisée pour évaluer l'activité des produits vis-à-vis des mycobactéries ou des spores bactériennes.

Kemična razkužila in antiseptiki - Kvantitativni preskus na poroznih površinah brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2) (vključno z dopolnilom A1)

Standard EN 16437:2014+A1 določa preskusno metodo ter minimalne zahteve za baktericidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo ali, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Ta evropski standard se uporablja za izdelke v veterini na poroznih površinah brez mehanskega delovanja, tj. pri vzreji, živinoreji, proizvodnji, v veterinarskih ambulantah, pri prevozu in odstranjevanju vseh živali, razen če so v prehrambeni verigi po smrti in so del predelovalne industrije. EN 14885 podrobno določa razmerje med različnimi preskusi in priporočili za uporabo.

General Information

Status
Published
Publication Date
15-Oct-2019
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
30-Jan-2020

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SLOVENSKI STANDARD
SIST EN 16437:2014+A1:2019
01-december-2019
Nadomešča:
SIST EN 16437:2014
Kemična razkužila in antiseptiki - Kvantitativni preskus na poroznih površinah
brez mehanskega delovanja za vrednotenje baktericidnega delovanja kemičnih

razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)

(vključno z dopolnilom A1)

Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of

bactericidal activity of chemical disinfectants and antiseptics used in veterinary area on

porous surfaces without mechanical action - Test method and requirements (phase 2,

step 2)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur

Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika

für den Veterinärbereich auf porösen Oberflächen ohne mechanische Wirkung -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l'évaluation

de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le

domaine vétérinaire sur des surfaces poreuses sans action mécanique - Mét
Ta slovenski standard je istoveten z: EN 16437:2014+A1:2019
ICS:
11.220 Veterinarstvo Veterinary medicine
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
SIST EN 16437:2014+A1:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
SIST EN 16437:2014+A1:2019
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 71.100.35 Supersedes EN 16437:2014
English Version
Chemical disinfectants and antiseptics - Quantitative
surface test for the evaluation of bactericidal activity of
chemical disinfectants and antiseptics used in veterinary
area on porous surfaces without mechanical action - Test
method and requirements (phase 2, step 2)

Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -

quantitatif de surface pour l'évaluation de l'activité Quantitativer Oberflächenversuch zur Bestimmung der

bactéricide des antiseptiques et des désinfectants bakteriziden Wirkung chemischer Desinfektionsmittel

chimiques utilisés dans le domaine vétérinaire sur des und Antiseptika für den Veterinärbereich auf porösen

surfaces poreuses sans action mécanique - Mét Oberflächen ohne mechanische Wirkung -

Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

This European Standard was approved by CEN on 30 November 2013 and includes Amendment 1 approved by CEN on 9 August

2019.

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-CENELEC 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-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16437:2014+A1:2019 E

worldwide for CEN national Members.
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
Contents Page

!European foreword" .......................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Requirements ................................................................................................................................................... 5

5 Test method ...................................................................................................................................................... 6

5.1 Principle ............................................................................................................................................................. 6

5.2 Materials and reagents .................................................................................................................................. 7

5.3 Apparatus and glassware .......................................................................................................................... 10

5.4 Preparation of test organism suspension and product test solutions ...................................... 11

5.5 Procedure for assessing the bactericidal activity of the product ............................................... 13

5.6 Experimental data and calculation ........................................................................................................ 17

5.7 Verification of methodology ..................................................................................................................... 21

5.8 Expression of results and precision ...................................................................................................... 22

5.9 Interpretation of results – conclusion .................................................................................................. 22

5.10 Test report ...................................................................................................................................................... 23

Annex A (informative) Referenced strains in national collections ......................................................... 25

Annex B (informative) Neutralisers - Examples of neutralisers of the residual antimicrobial

activity of chemical disinfectants and antiseptics ............................................................................ 26

Annex C (informative) Graphical representations of dilution-neutralization method ................... 28

Annex D (informative) Example of a typical test report ............................................................................. 32

Bibliography ................................................................................................................................................................. 37

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
!European foreword"

This document (EN 16437:2014+A1:2019) 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 April 2020 and conflicting national standards shall be

withdrawn at the latest by April 2020.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document includes Amendment 1 approved by CEN on 9 August 2019.

The start and finish of text introduced or altered by amendment is indicated in the text by tags ! ".

This document supersedes !EN 6437:2014".

!This document has been amended to update the scope and to correct a mistake in Tryptone Soya

Agar and Broth formulations. The maximum contact time has also been extended and an obligatory

contact time removed. The changes detailed above have no impact on the test results obtained using the

previous version. Those results are still valid."

According to the CEN/CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,

Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of

North Macedonia, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United

Kingdom.
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
Introduction

This document specifies a surface test for establishing whether a chemical disinfectant or antiseptic, for

use on porous surfaces without mechanical action, in the veterinary area. has or does not have

bactericidal activity under the laboratory conditions defined by this document, which influence the

action of disinfectants in practical use.

The laboratory test takes into account practical conditions of application of the product including pre-

drying test organisms and interfering substances on a surface, contact time and temperature, i.e.

conditions which may influence its action in practical situations.

The conditions are intended to cover general purposes and to allow reference between laboratories and

product types. Each utilization concentration of the chemical disinfectant or antiseptic, found by this

test corresponds to the chosen experimental conditions. However, for some applications, the

instructions of use of a product can differ and therefore additional test conditions need to be used.

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
1 Scope

This European Standard specifies a test method and the minimum requirements for bactericidal activity

of chemical disinfectants and antiseptic products that form a homogeneous, physically stable

preparation when diluted with hard water - or in the case of ready-to-use products - with water.

This European Standard applies to products that are used in the veterinary area on porous surfaces

without mechanical action i.e. in the breeding, husbandry, production, !veterinary care facilities",

transport and disposal of all animals except when in the food chain following death and entry to the

processing industry.

EN 14885 specifies in detail the relationship of the various tests to one another and to “use

recommendations”.

NOTE 1 The method described is intended to determine the activity of commercial formulations or active

substances under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 2 test.

NOTE 3 This method cannot be used to evaluate the activity of products against mycobacteria or bacterial

spores.
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.

EN 12353, Chemical disinfectants and antiseptics - Preservation of test organisms used for the

determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal

(including bacteriophages) activity

EN 14885, Chemical disinfectants and antiseptics - Application of European Standards for chemical

disinfectants and antiseptics
3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14885 apply.

4 Requirements

The product shall demonstrate at least a 4 decimal log (lg) reduction from a water control, when tested

in accordance with Table 1 and Clause 5 under simulated soiling (3,0 g/l bovine albumin).

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
Table 1 — Obligatory and additional test conditions
Test Conditions Bactericidal activity on porous surfaces
without mechanical action in the
veterinary area
Test organism Enterococcus hirae
a) obligatory Proteus vulgaris
Pseudomonas aeruginosa
Staphylococcus aureus
b) additional any relevant test organism
Test temperature 10 °C ± 1 °C
a) obligatory
b) additional 4 °C ± 1 °C; 20 °C ± 1 °C; 40 °C ± 1 °C
!Contact time The contact time(s) shall be selected from
the values given below"
!minimum 1 min ± 5 s"
!range 5 min ± 10 s, 15 min + 10 s, 30 min ± 10 s;
60 min ± 10 s and then up to 330
min ± 10 s at 30 min intervals"
!maximum 360 min ± 10 s "
Interfering substance 3,0 g/l bovine albumin
a) obligatory
b) additional any relevant substance
!Deleted text"
NOTE For the additional conditions, the concentration defined as a result can be
lower than the one obtained under the obligatory test conditions.

Any additional specific bactericidal activity shall be determined in accordance with 5.2.1 and 5.5.1.1 in

order to take into account intended specific use conditions.
5 Test method
5.1 Principle

A test suspension of bacteria mixed with interfering substance is inoculated onto the test surface and

dried. After the drying time the test surface is immersed into a sample of the product as delivered

and/or diluted with hard water (for ready to use products: water) ensuring that the test surface is

totally covered for one minute. The test surface is removed from the product solution and maintained at

a specified temperature for a defined period of time specified in Clause 4 and 5.5.1.1. At the end of that

contact time, the test surface is transferred to a neutralizer so that the action of the disinfectant is

immediately neutralized. The bacteria are removed from the surface by ultrasound treatment. The

numbers of surviving bacteria which can be recovered from the surface is determined quantitatively.

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)

The number of bacteria on a surface treated with water in place of the disinfectant is also determined

and the reduction is calculated.

The test is performed using Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and

Staphylococcus aureus as test organisms (Clause 4, Table 1).

Additional and optional contact times and temperatures are specified (Clause 4, Table 1). Additional

interfering substances and test organisms may be used.
5.2 Materials and reagents
5.2.1 Test organisms
The bactericidal activity shall be evaluated using the following strains :
Enterococcus hirae ATCC 10541;
Proteus vulgaris ATCC 13315;
Pseudomonas aeruginosa ATCC 15442;
Staphylococcus aureus ATCC 6538.
NOTE See Annex A for strain references in some other culture collections.

The required incubation temperature for these organisms is 36 °C + 1 °C or 37 °C + 1 °C (5.3.2.3).

The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test

and its control and validation.

If additional test organisms are used, they shall be incubated under optimum growth conditions

(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms

selected do not correspond to the specified strains, their suitability for supplying the required inocula

shall be verified. If these additional test organisms 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 collection under a reference for five years.
5.2.2 Culture media and reagents
5.2.2.1 General

All weights of chemical substances given in this European Standard refer to the anhydrous salts.

Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for

consequent molecular weight differences.

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be

free from substances that are toxic or inhibitory to the test organisms.

If additional strains do not grow on the media (5.2.2.3) or cannot be used with diluent (5.2.2.4)

additional media shall be used and shall be reported as well as additional incubation conditions.

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.
Ready-to-use media may be used if it complies with the required specification.

The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections (ATCC). This

information is given for the convenience of users of this European Standard and does not constitute an endorsement by CEN of

this product.
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
For each culture medium and reagent a time limitation for use should be fixed.
5.2.2.2 Water

The water shall be freshly glass-distilled water and not demineralized water. If distilled water of

adequate quality is not available, water for injections (see bibliographic reference [1]) may be used.

Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used e.g. for

preparation of culture media and subsequently sterilized.
NOTE See 5.2.2.6 for the procedure to prepare hard water.
5.2.2.3 Tryptone Soya Agar (TSA)
Tryptone soya agar, consisting of:
Tryptone, pancreatic digest of casein 15,0 g
Soya peptone, papaic digest of soybean meal 5,0 g
!Deleted text"
Sodium chloride (NaCl) 5,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at 20 °C ± 1 °C.

In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3), it may be necessary to add

neutralizer to the TSA. Annex B gives guidance on the neutralisers that may be used. It is recommended

not to use a neutralizer that causes opalescence in the agar.
5.2.2.4 Diluent
Tryptone Sodium chloride solution, consisting of:
Tryptone, pancreatic digest of casein 1,0 g
Sodium chloride (NaCl) 8,5 g
Water (5.2.2.2) to 1 000 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the diluent shall be equivalent to

7,0 ± 0,2 when measured at 20 °C ± 1 °C.
5.2.2.5 Neutralizer

The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.2. The neutralizer shall be sterile. The neutralizer is added to diluent (5.2.2.4) and TSB (5.2.2.8).

NOTE Information on neutralisers that have been found to be suitable for some categories of products is

given in Annex B.
5.2.2.6 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:

— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride

(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.7) or in the

autoclave [5.3.2.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to 1

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)

000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator (5.3.2.8)

for no longer than one month;

— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to

1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8)

for no longer than one week;

— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (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 (5.2.2.2). The pH of the

hard water shall be 7,0 ± 0,2, when measured at 20 °C ± 1 °C. 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 (HCl).

The hard water shall be freshly prepared under aseptic conditions and used within 12 h.

NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water

produces a different final water hardness in each test tube. In any case the final hardness expressed as calcium

carbonate (CaC03) is in the test tube lower than 375 mg/l.
5.2.2.7 Interfering substances
5.2.2.7.1 General

The interfering substance shall be chosen according to the conditions of use laid down for the product.

The interfering substance shall be sterile and prepared at 2 times its final concentration in the test.

For the additional interfering substances, the ionic composition (e.g. pH, calcium and/or magnesium

hardness) and chemical composition (e.g. mineral substances, protein, carbohydrates, lipids,

detergents) shall be defined.

NOTE The term “interfering substance” is used even if it contains more than one substance.

5.2.2.7.2 Soiling (bovine albumin solution)

Dissolve 0,6 g of bovine albumin V (suitable for microbiological purposes) in 90 ml of water (5.2.2.2) in

a 100 ml volumetric flask. Make up to the mark with water (5.2.2.2).

Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.

The final concentration of the bovine albumin in the test procedure (5.5) is 3 g/l.

5.2.2.8 Tryptone Soya Broth (TSB) with neutralizer
Tryptone Soya broth, consisting of:
Tryptone, pancreatic digest of casein 17,0 g
Soya peptone, papaic digest of soybean meal 3,0 g
Sodium chloride (NaCl) 5,0 g
Dipotassiumhydrogenphosphate (K HPO ) 2,5 g
2 4
!Glucose 2,5 g"
Water (5.2.2.2) to 1 000 ml

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH of the medium shall be equivalent to

7,3 ± 0,2 when measured at 20 °C ± 1 °C.
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)

An adequate neutralizer shall be added according to its chemical properties before or after autoclaving

(5.2.2.5). TSB with neutralizer should be filled into glass tubes in portions of 10 ml.

5.2.3 Test surface

Poplar wood: Size: 10mm wide, 20 mm long and 0,6 mm −1,0 mm thick with visually smooth cut edges.

Cut from sliced veneer, stored at least one year before use, from untreated wood of the European poplar

tree.

Prior to use put the surfaces into a glass Petri dish in a single layer and sterilize in the autoclave [5.3.2.1

a)] for 15 min.

Test surfaces should be kept in a sterile vessel until use. The surfaces should be used only once.

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:

a) by moist heat, in an autoclave [5.3.2.1 a)];
b) by dry heat, in a hot air oven [5.3.2.1 b)].
5.3.2 Usual microbiological laboratory equipment
and in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)

a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum

holding time of 15 min;

b) for dry heat sterilization, a hot air oven capable of being maintained at (180 ) °C for a minimum

+5 +5

holding time of 30 min, at (170 ) °C for a minimum holding time of 1 h or at (160 ) °C for a

0 0
minimum holding time of 2 h.

5.3.2.2 Water bath, capable of being controlled at 4 °C ± 1 °C, 10 °C ± 1 °C, 20 °C ± 1 °C, 40 °C ± 1 °C

(5.5.1) and 45 °C ± 1 °C (to maintain melted TSA, 5.2.2.3, 5.5.2.2 and 5.5.2.3).

5.3.2.3 Incubator, capable of being controlled at 36 °C ± 1 °C or 37 °C ± 1 °C (5.2.1). The same

temperature shall be used for incubation performed during a test and its control and validation.

5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.

A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar-

media (5.2.2.3).

DES-IN-TEST Supply Walbrunnenstrasse D-70599 Stuttgart Tel. ++49 (0) 711 45 54 06. This information is given for the

convenience of the users of this European Standard and does not constitute an endorsement by CEN of this product.

3) Disposable sterile equipment is an acceptable alternative to reusable glassware.

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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)
5.3.2.5 Stopwatch
5.3.2.6 Shakers
a) Electromechanical agitator, e.g. Vortex® mixer ;
b) Mechanical shaker.

5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to

be filtered with a filter holder of at least 50 ml volume and suitable for use with filters of diameter

47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.6) and bovine albumin

(5.2.2.7).
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.

5.3.2.9 Graduated pipettes of nominal capacities 10 ml, 1 ml, 0,1 ml and 0,05 ml or calibrated

automatic pipettes.
5.3.2.10 Petri dishes, (plates) 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 Temperature controlled cabinet, capable of being controlled at 10 °C ± 1 °C.

5.3.2.14 Ultrasonic bath, capable of operating at a frequency 30 kHz to 55 kHz, maximal output

1 000 W.
5.4 Preparation of test organism suspension and product test solutions
5.4.1 Test organism suspension (test and validation suspension)
NOTE Test and validation suspension are the same in this European Standard.
5.4.1.1 General

For each test organism, one suspension shall be prepared: this is used as the bacterial “test suspension”

to perform the test and the “validation suspension” to perform the controls and method validation.

5.4.1.2 Preservation and stock cultures of test organisms

The test organisms and their stock cultures shall be prepared and kept in accordance with EN 12353.

5.4.1.3 Working culture of test organisms

In order to prepare the working culture of test organisms (5.2.1), prepare a subculture from the stock

culture (5.4.1.2) by streaking onto TSA (5.2.2.3) slopes or plates and incubate (5.3.2.3). After 18 h to 24

h prepare a second subculture from the first subculture in the same way and incubate for 18 h to 24 h.

From this second subculture, a third subculture may be produced in the same way. The second and (if

produced) third subculture are the working cultures.

4) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this

European Standard and does not constitute an endorsement by CEN of this product.
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SIST EN 16437:2014+A1:2019
EN 16437:2014+A1:2019 (E)

If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used

for subsequent subculturing, provided that the subculture has been kept in the incubator (5.3.2.3)

during the 48 h period. In these circumstances, prepare a further 24 h subculture before proceeding.

Never produce and use a fourth subculture.

For additional strains, any departure from this method of culturing the bacteria or preparing the

suspensions shall be noted, giving the reasons in the test report.
5.4.1.4 Test suspension (“N”)

a) Take 10 ml of diluent (5.2.2.4) and place in a 100 ml flask with 5 g of glass beads (5.3.2.11). Take

the working culture (5.4.1.3) and transfer loopfuls of the cells into the diluent (5.2.2.4). The cells

should be suspended in the diluent by rubbing the loop against the wet wall of the flask to dislodge

the cells before immersing in the diluent. Shake the flask for 3 min using a mechanical shaker

[5.3.2.6 b)]. Aspirate the suspension from the glass beads and transfer to a tube.

9 5) 9

b) Adjust the number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using diluent

(5.2.2.4), estimating the number of cfu/ml by any suitable means. Maintain this test suspension in

the water bath at 20 °C ± 1 °C and use within 2 h. Adjust the temperature according to 5.5.1.1 a) and

5.5.1.4 only immediately before the start of the test.
The use of a spectrophotometer for adjusting the number of cells is h
...

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