SIST EN 17422:2022
(Main)Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of teat disinfectants used in the veterinary area - Test method and requirements (phase 2 step 2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of teat disinfectants used in the veterinary area - Test method and requirements (phase 2 step 2)
This procedure specifies a test method and the minimum requirements for bactericidal activity of teat disinfectants that form a homogeneous, physically stable preparation when diluted with hard water - or in the case of ready-to-use products - with water.
This method applies to teat disinfectants that are used in the veterinary area on teat skin without mechanical action as pre-milking and/or post-milking teat disinfectants.
NOTE 1 The method described is intended to determine the activity of commercial formulations under the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 2 test.
NOTE 3 Two types of synthetic skin were assessed in a ring trial with no significant difference in performance. Other synthetic skins may become available and may be used if it can be shown that they give comparable results to the two referenced in this standard.
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Beurteilung von Zitzendesinfektionsmittel für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die bakterizide Wirkung von Zitzendesinfektionsmitteln fest, die bei Verdünnung in Wasser standardisierter Härte – oder im Falle gebrauchsfertiger Produkte – in Wasser als homogenes und physikalisch stabiles Präparat vorliegen.
Dieses Verfahren ist anzuwenden für Zitzendesinfektionsmittel, die auf der Zitzenhaut ohne mechanische Einwirkung als Desinfektionsmittel vor und/oder nach dem Melken im Veterinärbereich zum Einsatz kommen, d. h. bei der Aufzucht, Haltung, Produktion, in veterinärischen Pflegeeinrichtungen, beim Transport von Tieren sowie bei der Tierkörperbeseitigung aller Tiere mit Ausnahme der Tiere, die nach der Tötung direkt als Nahrungsmittel verwendet oder der weiterverarbeitenden Industrie zugeführt werden.
ANMERKUNG 1 Das beschriebene Verfahren soll der Bestimmung der Wirkung handelsüblicher Zubereitungen unter den Bedingungen dienen, unter denen sie in der Praxis angewendet werden.
ANMERKUNG 2 Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l’évaluation des désinfectants de trayons utilisés dans le domaine vétérinaire - Méthode d’essai et prescriptions (phase 2, étape 2)
Le présent document spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité bactéricide des désinfectants de trayons 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.
Cette méthode s’applique aux désinfectants de trayons utilisés sur la peau des trayons sans action mécanique comme désinfectants pré traite et/ou post traite dans le domaine vétérinaire, à savoir pour 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.
NOTE 1 La méthode décrite a pour objet de déterminer l’activité de formulations commerciales dans leurs conditions d’emploi.
NOTE 2 Cette méthode correspond à un essai de phase 2, étape 2.
Kemična razkužila in antiseptiki - Kvantitativni površinski preskus brez mehanskega delovanja za vrednotenje razkužil za seske v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)
Ta postopek določa preskusno metodo in minimalne zahteve za baktericidno delovanje kemičnih razkužil za seske, ki tvorijo homogen, fizikalno stabilen pripravek, razredčen s trdo vodo oziroma ga razredčimo z vodo pri proizvodih, ki so pripravljeni za uporabo.
Ta metoda se uporablja za kemična razkužila za seske, ki se uporabljajo v veterinarski medicini kot razkužila za seske pred in/ali po molži brez mehanskega delovanja.
OPOMBA 1: Opisana metoda je namenjena določevanju dejavnosti komercialnih mešanic pod pogoji, v katerih se uporabljajo.
OPOMBA 2: Ta metoda ustreza 2. stopnji preskusa faze 2.
OPOMBA 3: Pri krožnem preskušanju sta bili ocenjeni dve vrsti sintetične kože brez pomembne razlike v učinkovitosti. Ob odstopanju od tega standarda se lahko uporabljajo druge sintetične kože, če je mogoče dokazati, da dajejo rezultate, primerljive z obema, navedenima v tem standardu.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN 17422:2022
01-november-2022
Kemična razkužila in antiseptiki - Kvantitativni površinski preskus brez
mehanskega delovanja za vrednotenje razkužil za seske v veterini - Preskusna
metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of
teat disinfectants used in the veterinary area - Test method and requirements (phase 2
step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur
Beurteilung von Zitzendesinfektionsmittel für den Veterinärbereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l’évaluation
des désinfectants de trayons utilisés dans le domaine vétérinaire - Méthode d’essai et
prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 17422:2022
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
SIST EN 17422:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN 17422:2022
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SIST EN 17422:2022
EN 17422
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2022
EUROPÄISCHE NORM
ICS 71.100.35
English Version
Chemical disinfectants and antiseptics - Quantitative
surface test for the evaluation of teat disinfectants used in
the veterinary area - Test method and requirements
(phase 2 step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface pour l'évaluation des Quantitativer Oberflächenversuch zur Beurteilung von
désinfectants de trayons utilisés dans le domaine Zitzendesinfektionsmittel für den Veterinärbereich -
vétérinaire - Méthode d'essai et prescriptions (phase 2, Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
étape 2)
This European Standard was approved by CEN on 8 May 2022.
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, Türkiye 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
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17422:2022 E
worldwide for CEN national Members.
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SIST EN 17422:2022
EN 17422:2022 (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 . 6
5.3 Apparatus and glassware . 9
5.4 Preparation of test organism suspension and product test solutions . 10
5.5 Procedure for assessing the bactericidal activity of the product . 12
5.6 Experimental data and calculation . 15
5.7 Verification of methodology . 18
5.8 Expression of results and precision. 18
5.9 Interpretation of results – conclusion . 19
5.10 Test report . 20
Annex A (informative) Referenced strains in national collections . 22
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of
chemical disinfectants and antiseptics and rinsing liquids . 23
Annex C (informative) Example of a typical test report . 24
Bibliography . 27
2
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SIST EN 17422:2022
EN 17422:2022 (E)
European foreword
This document (EN 17422:2022) 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 December 2022, and conflicting national standards shall
be withdrawn at the latest by December 2022.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
3
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SIST EN 17422:2022
EN 17422:2022 (E)
Introduction
This document specifies a surface test for establishing whether a teat disinfectant, for use on teat skin
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
applying test organisms and interfering substances on a synthetic skin test surface, contact time and
temperature, i.e. conditions that may influence its action in practical situations.
In this document, synthetic human skin is used as the test surface.
4
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SIST EN 17422:2022
EN 17422:2022 (E)
1 Scope
This document specifies a test method and the minimum requirements for bactericidal activity of teat
disinfectants that form a homogeneous, physically stable preparation when diluted with hard water - or
in the case of ready-to-use products - with water.
This method applies to teat disinfectants that are used on teat skin without mechanical action as pre-
milking and/or post-milking teat disinfectants in the veterinary area - 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 into processing industry.
NOTE 1 The method described is intended to determine the activity of commercial formulations under the
conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 2 test.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 1656, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area — Test method
and requirements (phase 2, step 1)
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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obpuirements
4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) (post-milking disinfectant) or 3 decimal log
(lg) (pre-milking disinfectant) reduction from a water control, when tested in accordance with Table 1
and Clause 5 under simulated soiling (10,0 g / l milk powder for post-milking teat disinfectants, 3,0 g / l
bovine albumin for pre-milking teat disinfectants).
5
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SIST EN 17422:2022
EN 17422:2022 (E)
Table 1 — Requirements
Bactericidal activity on synthetic skin
Test Conditions
without mechanical action
Escherichia coli
Test organism
Staphylococcus aureus
Test temperature 30 °C ± 1 °C
Pre-milking teat Post-milking teat
disinfectants disinfectants
Minimum contact time
30 s ± 5 s 1 min ± 5 s
Pre-milking teat Post-milking teat
disinfectants disinfectants
Maximum contact time
3 min ± 10 s 5 min ± 10 s
Other contact times may be selected at intervals of 30 s for contact times up to
1 min and at intervals of 1 min for contact times > 1 min
Interfering substance
10,0 g/l milk powder
Post-milking teat disinfectants
3,0 g/l bovine albumin
Pre-milking teat disinfectants
5 Test method
5.1 Principle
A test suspension of bacteria mixed with interfering substance is inoculated onto a synthetic skin test
surface and maintained at 30 °C for a period of conditioning.
After this conditioning time, the test surface is immersed in the product or dilutions of the product at
30 °C for a defined period of time specified in Table 1. At the end of that contact time, neutralizer is added
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 are determined quantitatively.
The number of bacteria on a surface treated with water in place of the disinfectant is also determined and
the reduction is calculated.
5.2 Materials and reagents
5.2.1 Test organisms
The bactericidal activity shall be evaluated using the following strains as test organisms:
- Escherichia coli
- Staphylococcus aureus
NOTE Refer to Annex A for strain references in other culture collections.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3).
6
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SIST EN 17422:2022
EN 17422:2022 (E)
5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this method 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.
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.
A ready-to-use medium may be used if it complies with the required specification.
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 [2]) 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.
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;
Sodium chloride (NaCl) 5,0 g;
Agar 15,0 g;
Distilled Water to 1 000,0 ml.
Sterilize in the autoclave as above. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2
when measured at (20 ± 1) °C.
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;
Distilled Water to 1 000,0 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 ± 1) °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2. It shall be
sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given
in Table B.1.
7
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SIST EN 17422:2022
EN 17422:2022 (E)
5.2.2.6 Standardized hard water
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 distilled water and
2 2
dilute to 1 000 ml. Sterilize by membrane filtration or in the autoclave. Autoclaving - if used - may cause
a loss of liquid. In this case make up to 1 000 ml with water under aseptic conditions. Store the solution
in the refrigerator (5.3.2.8) at 2 °C to 8 °C for no longer than one month.
Prepare solution B:
Dissolve 35,02 g sodium bicarbonate (NaHCO ) in distilled water and dilute to 1 000 ml. Sterilize by
3
membrane filtration. Store the solution in the refrigerator (5.3.2.8) at 2 °C to 8 °C for no longer than one
week.
Place 600 ml to 700 ml of sterile distilled water in a sterile 1 000 ml volumetric flask and add 6,0 ml of
solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with sterile distilled water. The pH of the
hard water shall be 7,0 ± 0,2 when measured at (20 ± 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.
5.2.2.7 Interfering substances
5.2.2.7.1 General
Interfering substances shall be sterile and prepared at 2 times the final concentration in the test.
5.2.2.7.2 Skimmed milk
Prepare a solution of 20 g milk-powder guaranteed free of antibiotics and additives in 1 000 ml water
(5.2.2.2).
+3 +3
Heat for 30 min at 105 °C (or 5 min at 121 °C).
0 0
The final concentration in the test procedure (5.5) is 10 g/l milk powder.
5.2.2.7.3 Bovine albumin solution
Dissolve 0,6 g of bovine albumin fraction 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 the refrigerator (5.3.2.8) at (5 ± 3) °C and use within
one month.
The final concentration of the bovine albumin in the test procedure (5.5.2) is 3 g/l.
8
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SIST EN 17422:2022
EN 17422:2022 (E)
5.2.3 Test surface - synthetic skin
1
5.2.3.1 VITRO-SKIN®
Handle skin with sterile tweezers/implements. Eight pieces are used for each test: 3 for the test dilutions,
1 for the water control, 1 for control B, 2 for control C (1 inoculated and 1 un-inoculated) and 1 for a
sterility check.
VITRO-SKIN®: Mark with a pencil and ruler on the outside of the sealed skin packet eight 2 × 2 cm
squares (per test). Cut using sterile scissors and re-hydrate for 16 to 24 h before use (follow instructions
provided from the supplier). When ready to start the test, place each piece in a sterile wide necked vessel,
with lid on.
NOTE Two types of synthetic skin were assessed in a ring trial with no significant difference in performance.
One has been chosen as the test surface because it is commercially available. Other synthetic skins can become
available and can be used if it is shown that they give comparable results to the one referenced in this standard.
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;
b) by dry heat, in a hot air oven.
2
5.3.2 Usual microbiological equipment ,and in particular, the following:
5.3.2.1 Apparatus for sterilization (dry and moist heat):
+3
a) for moist heat sterilization, an autoclave capable of being maintained at ( ) °C for a minimum
121
0
holding time of 15 min;
+3
b) for dry heat sterilization, a hot air oven capable of being maintained at (180 ) °C for a minimum
0
+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 (20 ± 1) °C, (30 ± 1) °C, and (45 ± 1) °C.
5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C, (36 ± 1) °C or (37 ± 1) °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar-
media.
5.3.2.5 Stopwatch
1
VITRO-SKIN® can be obtained from IMS Inc., 110 Marginal Way, PMB 155, Portland ME 04101-2497 USA. This
information is given for the convenience of users of this document and does not constitute an endorsement by CEN
of the product named.
2
Disposable sterile equipment is an acceptable alternative to reusable glassware.
9
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SIST EN 17422:2022
EN 17422:2022 (E)
5.3.2.6 Shakers
3
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
(5.2.2.7.3).
5.3.2.8 Refrigerator, capable of being controlled at (5 ± 3) °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 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)
5.4.1.1 General
NOTE The test and validation suspensions are the same in this document.
For each test, 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, prepare a subculture from the stock culture by
streaking onto TSA slopes or plates and incubate. 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.
If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used
for subsequent sub-culturing, 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.
3
Vortex® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
10
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SIST EN 17422:2022
EN 17422:2022 (E)
5.4.1.4 Test suspension (“N”)
a) Take 10 ml of diluent and place in a 100 ml flask with 5 g of glass beads. Take the working culture
and transfer loopfuls of the cells into the diluent. 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. Aspirate the suspension from the glass beads
and transfer to a tube.
9 4 9
b) Adjust the number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using diluent,
estimating the number of cfu/ml by any suitable means. Maintain this test suspension in the water
bath at (20 ± 1) °C and use within 2 h. The use of a spectrophotometer for adjusting the number of
cells is highly recommended (about 620 nm wavelength – cuvette 10 mm path length). To achieve
reproducible results of this measurement it may be necessary to dilute the test suspension.
NOTE A colourimeter is a suitable alternative.
-7 -8
c) For counting prepare 10 and 10 dilutions of the test suspension using diluent.
Mix [5.3.2.6 a)].
Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate
technique.
1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml melted TSA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size - on an appropriate number (at least two) of surface dried plates containing
TSA.
For incubation and counting see 5.4.1.5.
5.4.1.5 Incubation and counting of the test suspension
Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates that are not countable (for any reason).
Count the plates and determine the total number of cfu. Incubate the plates for a further 20 h to 24 h. Do
not recount plates that no longer show well-separated colonies. Recount the remaining plates. If the
number has increased, use only the higher number for further evaluation.
Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and
determine the Vc values according to 5.6.2.2.
Calculate the numbers of cfu per 1 ml in the test suspension N (5.6.2.3).
5.4.2 Product test solution
Product test solutions shall be prepared in hard water (5.2.2.6) at a minimum of three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.8.2). The product, as received, may be used as one of the product test solutions. Dilutions of
ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2)
instead of hard water.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a
volumetric flask and filling up with hard water (5.2.2.6). Subsequent dilutions (i.e. lower concentrations)
shall be prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.6).
For liquid products, dilutions of the product shall be prepared with hard water (5.2.2.6) in volumetric
flasks (5.3.2.12) on a volume/volume basis.
4
cfu/ml = colony forming unit per millilitre
11
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EN 17422:2022 (E)
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure.
The concentration of the product stated in the test report shall be the desired test concentration. Record
the test concentration in terms of mass per volume or volume per volume and details of the product
sample as received.
5.5 Procedure for assessing the bactericidal activity of the product
5.5.1 General
5.5.1.1 Experimental conditions
Besides the specified temperature, contact time, interfering substance and test organisms, additional
experimental conditions may be selected according to the practical use considered for the product
(Clause 4):
a) temperature (in °C):
The obligatory temperature to be tested is specified in Clause 4, Table 1; the allowed deviation for
the temperature is ± 1 °C. It is not necessary to test teat disinfectant products at other temperatures.
b) contact time t (in min):
The minimum, maximum and additional contact times to be tested are specified in Clause 4, Table 1;
the allowed deviation for each chosen contact time is ±10 s for contact times above 1 min and ±5 s
for contact times of 1 min and below.
c) interfering substance:
The interfering substances to be tested are specified in Clause 4, Table 1.
d) test organisms are: Escherichia coli and Staphylococcus aureus (Clause 4, Table 1 and 5.2.1).
Additional test organisms may be tested.
5.5.1.2 Neutralization
To determine a suitable neutralizer carry out the validation of the dilution-neutralization method (5.5.2.4
and 5.5.2.5) using a suitable neutralizer, chosen according to laboratory experience and/or published
data.
If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into
account the information given in Annex B.
The neutralization shall have been shown to be effective within 10 s when tested using EN 1656.
NOTE In special circumstances, because of difficulties in obtaining adequate neutralization, neutralizer may be
added to the TSA (5.2.2.3).
5.5.1.3 General instructions for the validation and control procedures
The neutralization shall be controlled and validated - only for the highest product test concentration - for
each of the test organisms and for each experimental condition (interfering substance, contact time).
These procedures (water control, neutralizer control and method validation) shall be performed at the
same time as the test and with the same neutralizer used in the test.
If a neutralizer has been added to TSA (5.5.1.2) used for the validation and control procedures the TSA
used for the test shall con
...
SLOVENSKI STANDARD
oSIST prEN 17422:2019
01-september-2019
Kemična razkužila in antiseptiki - Kvantitativni preskus brez mehanskega
delovanja za vrednotenje razkužil za seske v veterini - Preskusna metoda in
zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of
teat disinfectants used in the veterinary area - Test method and requirements (phase 2
step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur
Beurteilung von Zitzendesinfektionsmittel für den Veterinärbereich - Prüfverfahren und
Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface pour l’évaluation
des désinfectants de trayons utilisés dans le domaine vétérinaire - Méthode d’essai et
prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 17422
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
oSIST prEN 17422:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 17422:2019
DRAFT
EUROPEAN STANDARD
prEN 17422
NORME EUROPÉENNE
EUROPÄISCHE NORM
July 2019
ICS 71.100.35
English Version
Chemical disinfectants and antiseptics - Quantitative
surface test for the evaluation of teat disinfectants used in
the veterinary area - Test method and requirements
(phase 2 step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface pour l'évaluation des Quantitativer Oberflächenversuch zur Beurteilung von
désinfectants de trayons utilisés dans le domaine Zitzendesinfektionsmittel für den Veterinärbereich -
vétérinaire - Méthode d'essai et prescriptions (phase 2, Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
étape 2)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.
If this draft becomes a European Standard, 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.
This draft European Standard was established by CEN 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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
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. prEN 17422:2019 E
worldwide for CEN national Members.
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Contents Page
European foreword . 3
Introduction. 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions. 5
4 Requirements . 6
5 Test method . 6
5.1 Principle . 6
5.2 Materials and reagents . 6
5.2.1 Test organisms . 6
5.2.2 Culture media and reagents . 7
5.2.3 Test surface - synthetic skin . 8
5.3 Apparatus and glassware . 9
5.3.1 General . 9
5.4 Preparation of test organism suspension and product test solutions . 10
5.4.1 Test organism suspension (test and validation suspension) . 10
5.4.2 Product test solution . 11
5.5 Procedure for assessing the bactericidal activity of the product . 11
5.5.1 General . 11
5.5.2 Test procedure . 12
5.6 Experimental data and calculation . 14
5.6.1 Explanation of terms and abbreviations . 14
5.6.2 Calculation . 15
5.7 Verification of methodology . 17
5.7.1 General . 17
5.7.2 Control of weighted mean counts . 17
5.7.3 Basic limits . 17
5.8 Expression of results and precision. 18
5.8.1 Reduction. 18
5.8.2 Control of active and non-active product test solution (5.4.2) . 18
5.8.3 Limiting test organism and bactericidal concentration . 18
5.8.4 Precision, repetitions . 18
5.9 Interpretation of results – conclusion . 18
5.9.1 General . 18
5.9.2 Bactericidal activity for teat disinfection . 19
5.10 Test report . 19
Annex A (informative) Referenced strains in national collections . 21
Annex B (informative) Examples of neutralizers of the residual antimicrobial activity of
chemical disinfectants and antiseptics and rinsing liquids . 22
Annex C (informative) Example of a typical test report . 23
Bibliography . 26
2
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European foreword
This document (prEN 17422:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
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Introduction
This document specifies a surface test for establishing whether a teat disinfectant, for use on teat skin
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
applying test organisms and interfering substances on a synthetic skin surface, contact time and
temperature, i.e. conditions which may influence its action in practical situations. The method is based
on EN 16437 Chemical disinfectants and antiseptics - Quantitative surface test for the evaluation of
bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area on porous
surfaces without mechanical action - Test method and requirements (phase 2, step 2).
In this document synthetic human skin is used as the test surface.
4
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1 Scope
This procedure specifies a test method and the minimum requirements for bactericidal activity of teat
disinfectants that form a homogeneous, physically stable preparation when diluted with hard water - or
in the case of ready-to-use products - with water.
This method applies to teat disinfectants that are used on teat skin without mechanical action as pre-
milking and/or post-milking teat disinfectants in the veterinary area - 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 into processing industry.
NOTE 1 The method described is intended to determine the activity of commercial formulations under the
conditions in which they are used.
NOTE 2 This method corresponds to a phase 2 step 2 test.
NOTE 3 Two types of synthetic skin were assessed in a ring trial with no significant difference in performance.
One has been chosen as the test surface because it is commercially available. Other synthetic skins can become
available and can be used if it shown that they give comparable results to the one referenced in this standard.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 1656, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area — Test method
and requirements (phase 2, step 1)
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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obpuirements
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4 Requirements
The product shall demonstrate at least a 4 decimal log (lg) (post-milking disinfectant) or 3 decimal log
(lg) (pre-milking disinfectant) reduction from a water control, when tested in accordance with Table 1
and Clause 5 under simulated soiling (10,0 g/l milk powder or 3,0 g/l bovine albumin).
Table 1 — Requirements
Bactericidal activity on synthetic skin
Test Conditions
without mechanical action
- Escherichia coli
Test organism
- Staphylococcus aureus
Test temperature 30°C ± 1°C
Minimum Maximum
Contact time
Up to 1 min at For times > 1 min at
intervals of 30 s intervals of 1 min
Post milking teat disinfectants 1 min ± 5 s 5 min ± 10 s
Pre-milking teat disinfectants 30 s ± 5 s 3 min ± 10 s
Interfering substance
10,0 g/l milk powder
Post milking teat disinfectants
3,0 g/l bovine albumin
Pre-milking teat disinfectants
5 Test method
5.1 Principle
A test suspension of bacteria mixed with interfering substance is inoculated onto a synthetic skin surface
and maintained at 30 °C for a period of conditioning.
After this conditioning time, the skin surface is immersed in the product or dilutions of the product at
30 °C for a defined period of time specified in Table 1. At the end of that contact time, neutralizer is added
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 are determined quantitatively.
The number of bacteria on a surface treated with water in place of the disinfectant is also determined and
the reduction is calculated.
5.2 Materials and reagents
5.2.1 Test organisms
1
The bactericidal activity shall be evaluated using the following strains as test organisms :
- Escherichia coli ATCC 10536
- Staphylococcus aureus ATCC 6538
NOTE The ATCC numbers are the collection numbers of strains supplied by the American Type Culture
Collections (ATCC).
Refer to 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).
1
The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection
(ATCC). This information is given for the convenience of users of this document and does not constitute an
endorsement by CEN of the product named.
6
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5.2.2 Culture media and reagents
5.2.2.1 General
All weights of chemical substances given in this method 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.
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.
A ready-to-use medium may be used if it complies with the required specification.
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 [2]) 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.
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;
Sodium chloride (NaCl) 5,0 g;
Agar 15,0 g;
Distilled Water to 1 000,0 ml.
Sterilize in the autoclave as above. After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2
when measured at (20 ± 1) °C.
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;
Distilled Water to 1 000,0 ml
Dispense in 9 ml portions and sterilize in the autoclave as above. After sterilization, the pH of the diluent
shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2. It shall be
sterile.
NOTE Information on neutralizers that have been found to be suitable for some categories of products is given
in Annex B.
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5.2.2.6 Standardized hard water
For the preparation of 1 l of hard water, the procedure is as follows:
Prepare solution A:
Dissolve 19,84 g magnesium chloride (Mg Cl ) and 46,24 g calcium chloride (Ca Cl ) in distilled water
2 2
and dilute to 1 000 ml. Sterilize by membrane filtration or in the autoclave. Autoclaving - if used - may
cause a loss of liquid. In this case make up to 1 000 ml with water under aseptic conditions. Store the
solution in the refrigerator (5.3.2.8) at 2-8 °C for no longer than one month.
Prepare solution B:
Dissolve 35,02 g sodium bicarbonate (NaHCO3) in distilled water and dilute to 1 000 ml. Sterilize by
membrane filtration. Store the solution in the refrigerator (5.3.2.8) at 2-8 °C for no longer than one week.
Place 600 ml to 700 ml of sterile distilled water in a sterile 1 000 ml volumetric flask and add 6,0 ml of
solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with sterile distilled water. The pH of the
hard water shall be 7,0 ± 0,2 when measured at (20 ± 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.
5.2.2.7 Interfering substances, shall be sterile and prepared at 2 times its final concentration in the
test.
5.2.2.7.1 Skimmed milk, guaranteed free of antibiotics and additives and prepared as follows:
— prepare a solution of 20 g milk-powder in 1 000 ml water (5.2.2.2).
+3 +3
— Heat for 30 min at 105 °C (or 5 min at 121 °C).
0 0
The final concentration in the test procedure (5.5) is 10g/l milk powder.
5.2.2.7.2 Bovine albumin solution
Dissolve 0,6 g of bovine albumin (Cohn fraction V for Dubos Medium) 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 the refrigerator (5.3.2.8) at 2-8 °C and use within one
month.
The final concentration of the bovine albumin in the test procedure (5.5.2) is 3 g/l.
5.2.3 Test surface - synthetic skin
2
5.2.3.1 Vitro skin
Handle skin with sterile tweezers/implements. Eight pieces are used for each test: 3 for the test dilutions,
1 for the water control, 1 for control B, 2 for control C (1 inoculated and 1 un-inoculated) and 1 for a
sterility check.
Vitroskin: Mark with a pencil and ruler on the outside of the sealed skin packet eight 2 × 2 cm squares
(per test). Cut using sterile scissors and re-hydrate for 16 to 24 h before use (follow instructions provided
from the supplier). When ready to start the test, place each piece in a sterile wide necked vessel, with lid
on.
2
Vitro skin may be obtained from IMS Inc., 110 Marginal Way, PMB 155, Portland ME 04101-2497 USA. This
information is given for the convenience of users of this document and does not constitute an endorsement by CEN
of the product named.
8
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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;
b) by dry heat, in a hot air oven
3
5.3.2 Usual microbiological equipment ,and in particular, the following:
5.3.2.1 Apparatus for sterilization (dry and moist heat):
+3
a) for moist heat sterilization, an autoclave capable of being maintained at (121 ) °C for a minimum
0
holding time of 15 min;
+3
) °C for a minimum
b) for dry heat sterilisation, a hot air oven capable of being maintained at (180
0
+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 (20 ± 1) °C, (30 ± 1) °C, and (45 ± 1) °C.
5.3.2.3 Incubator, capable of being controlled at (30 ± 1) °C, (36 ± 1) °C or (37 ± 1) °C.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.
A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar-
media.
5.3.2.5 Stopwatch
5.3.2.6 Shakers
4
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
(5.2.2.7.2).
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
3
Disposable sterile equipment is an acceptable alternative to reusable glassware.
4
Vortex® is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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5.3.2.13 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)
5.4.1.1 General
NOTE Test and validation suspension are the same in this document.
For each test, 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, prepare a subculture from the stock culture by
streaking onto TSA slopes or plates and incubate. 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.
If it is not possible to prepare the second subculture on a particular day, a 48 h subculture may be used
for subsequent sub-culturing, 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
5.4.1.4 Test suspension (“N”)
a) Take 10 ml of diluent and place in a 100 ml flask with 5 g of glass beads. Take the working culture
and transfer loopfuls of the cells into the diluent. 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. Aspirate the suspension from the glass beads
and transfer to a tube.
9 9
5
b) Adjust the number of cells in the suspension to 1,5 × 10 cfu /ml to 5,0 × 10 cfu/ml using diluent,
estimating the number of cfu/ml by any suitable means. Maintain this test suspension in the water
bath at (20 ± 1) °C and use within 2 h. The use of a spectrophotometer for adjusting the number of
cells is highly recommended (about 620 nm wavelength – cuvette 10 mm path length). To achieve
reproducible results of this measurement it may be necessary to dilute the test suspension.
NOTE A colourimeter is a suitable alternative.
−6 −7
c) For counting prepare 10 and 10 dilutions of the test suspension using diluent.
Mix [5.3.2.6 a)].
5
cfu/ml = colony forming unit per millilitre
10
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Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread plate
technique.
1) When using the pour plate technique, transfer each 1,0 ml sample into separate Petri dishes and add
15 ml to 20 ml melted TSA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of
approximately equal size - on an appropriate number (at least two) of surface dried plates containing
TSA.
For incubation and counting see 5.4.1.5.
5.4.1.5 Incubation and counting of the test suspension
Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates that are not countable (for any reason).
Count the plates and determine the total number of cfu. Incubate the plates for a further 20 h to 24 h. Do
not recount plates that no longer show well-separated colonies. Recount the remaining plates. If the
number has increased, use only the higher number for further evaluation.
Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and
determine the Vc values according to 5.6.2.2.
Calculate the numbers of cfu per 1 ml in the test suspension N using the formula given in Clause 5.6.1.
NOTE 0,05 ml of an equal parts mixture of the test suspension and interfering substance is added to the surface
therefore 0,025 ml of the suspension is added.
5.4.2 Product test solution
Product test solutions shall be prepared in hard water (5.2.2.6) at a minimum of three different
concentrations to include one concentration in the active range and one concentration in the non-active
range (5.8.2). The product, as received, may be used as one of the product test solutions. Dilutions of
ready-to-use products, i.e. products that are not diluted when applied, shall be prepared in water (5.2.2.2)
instead of hard water.
For solid products, dissolve the product as received by weighing at least 1,0 g ± 10 mg of the product in a
volumetric flask and filling up with hard water (5.2.2.6). Subsequent dilutions (i.e. lower concentrations)
shall be prepared in volumetric flask
...
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