prEN 16756

Antimicrobial wound dressings - Requirements and test methods

This standard specifies requirements and test methods for the antimicrobial activity of antimicrobial wound dressings. It is designed for microbicidal and microbistatic dressings.
Test methods specifically for microbial binding are not included in the standard.

Antimikrobielle Wundauflagen - Anforderungen und Prüfverfahren

Diese Norm legt Anforderungen und Prüfverfahren für die antimikrobielle Wirkung antimikrobieller Wundauflagen fest. Sie ist vorgesehen für mikrobizide und mikrobistatische Auflagen.
Prüfverfahren speziell für mikrobielle Bindungen sind nicht in dieser Norm enthalten.

Pansements antimicrobiens - Exigences et méthodes d'essai

La présente norme spécifie les exigences et méthodes d’essai applicables à l’activité antimicrobienne de pansements antimicrobiens. Elle s’applique aux pansements microbicides et microbiostatiques.
Les méthodes d’essai spécialement applicables à la fixation microbienne ne sont pas incluses dans la norme.

Antimikrobni sanitetni material - Zahteve in preskusne metode

General Information

• Status: Not Published
• Publication Date: 21-Mar-2016
• Withdrawal Date: 21-Sep-2016
• ICS: 11.120.20 - Wound dressings and compresses
• Technical Committee: CEN/TC 205 - Non-active medical devices
• Drafting Committee: CEN/TC 205/WG 15 - Antimicrobial wound care dressings
• Current Stage: 4098 - Decision to abandon - Enquiry
• Start Date: 14-Aug-2015
• Completion Date: 14-Apr-2025
• Directive: 93/42/EEC - Council Directive 93/42/EEC of 14 June 1993 concerning medical devices
• Mandate: M/023 - Standardization mandate to CEN/CENELEC concerning the development of European standards relating to medical devices
• Ref Project: oSIST prEN 16756:2014 - Antimicrobial wound dressings - Requirements and test methods

Overview

prEN 16756 - "Antimicrobial wound dressings - Requirements and test methods" (CEN, draft 2014) defines laboratory requirements and standardized test methods to evaluate the antimicrobial activity of wound dressings. The standard is designed to assess both microbicidal and microbistatic dressings under simulated clinical conditions (test fluids, temperatures, organisms, contact times). It does not include specific methods for microbial binding.

Key topics and technical requirements

• Classification
  • Microbicidal: ≥ 3‑log reduction in viable cells (standard test conditions, 24 h against specified organisms).
  • Microbistatic: prevents further growth of the initial inoculum over 24 h.
• Performance requirements
  • Results are reported as antimicrobial activity (A). Table-based pass/fail criteria distinguish microbicidal (A ≥ 3) from microbistatic (0 ≤ A < 3).
• Test methods (Annex H)
  • Direct contact test method
  • Shaking method
  • Two‑compartment method
  • Procedures for sample preparation, equilibration, saturation volume (use of Simulated Wound Fluid), recovery and plate-count enumeration, calculations and reporting
• Controls and validation
  • Use of a negative control dressing (same product without antimicrobial treatment, or equivalent sterile gauze if unavailable)
  • Validation of dilution-neutralization (Annex A) including neutralizer selection, toxicity checks, and neutralization effectiveness
  • Reference strains, media and simulated wound fluids are specified in informative annexes (E, F, G)
• Reporting and information to be supplied
  • Annex/method used, dressing details, mean counts at time 0 and time t, antimicrobial activity A, and a full test report (example tables provided)

Practical applications and users

• Medical device manufacturers developing or claiming antimicrobial wound dressings can use prEN 16756 to:
  • Validate product claims (microbicidal vs microbistatic)
  • Design and document internal and regulatory test data
• Independent/contract microbiology laboratories performing conformity or performance testing for tendering and regulatory submissions
• Regulatory and procurement bodies assessing product evidence and compliance with EU medical device requirements (see Annex ZA for relationship to Directive essential requirements)
• R&D teams and quality assurance groups setting specifications, optimizing formulations (e.g., silver dressings), or comparing product performance across types

Related standards and keywords

• Normative reference: EN 12353 (preservation of test organisms)
• Informative annexes provide strains, media, apparatus, and rationale to align laboratory practice
• SEO keywords: prEN 16756, antimicrobial wound dressings, test methods, microbicidal, microbistatic, neutralizer validation, simulated wound fluid, direct contact test

prEN 16756 offers a structured, reproducible approach to demonstrating antimicrobial activity of wound dressings suitable for product development, laboratory testing, and regulatory documentation.

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2014
Antimikrobni sanitetni material - Zahteve in preskusne metode
Antimicrobial wound dressings - Requirements and test methods
Antimikrobielle Wundauflagen - Anforderungen und Prüfverfahren
Pansements antimicrobiens - Exigences et méthodes d'essai
Ta slovenski standard je istoveten z: prEN 16756
ICS:
11.120.20 Sanitetni materiali, obveze in Wound dressings and
komprese compresses
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
DRAFT
NORME EUROPÉENNE
EUROPÄISCHE NORM
June 2014
ICS 11.120.20
English Version
Antimicrobial wound dressings - Requirements and test methods
Pansements antimicrobiens - Exigences et méthodes Antimikrobielle Wundauflagen - Anforderungen und
d'essai Prüfverfahren
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee CEN/TC 205.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2014 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16756:2014 E
worldwide for CEN national Members.

Contents
Page
Foreword . iv
Introduction .v
1 Scope .6
2 Normative references .6
3 Terms and definitions .6
4 Classification .7
5 Requirements .7
5.1 Microbicidal dressings .7
5.2 Microbistatic dressings .7
5.3 Performance Table.7
6 Information to be supplied .7
Annex A (normative) Validation of dilution-neutralisation .9
A.1 Principle .9
A.2 Neutralizer selection .9
A.3 Preparation of microbial suspension .9
A.4 Test validation .9
A.5 Neutralizer toxicity . 10
A.6 Test organism viability . 11
A.7 Neutralisation Effectiveness . 11
A.8 Interpretation of data . 12
Annex B (informative) Neutralizers . 13
Annex C (informative) Rationale . 14
C.1 Title . 14
C.2 Microbial strains . 14
C.3 Cutting dressings . 15
C.4 Positive control . 15
C.5 Microbistatic requirements . 15
C.6 Performance Requirements . 15
C.7 Test method Information . 15
C.8 Media . 16
C.9 Incubation Temperatures . 16
C.10 Dressing Classification . 16
Annex D (informative) Apparatus . 17
Annex E (informative) Reagents and culture media . 18
E.1 General . 18
E.2 Water . 18
E.3 Tryptone Soya Agar . 18
E.4 Maximum recovery diluent . 18
E.5 Simulated Wound Fluid (SWF) . 18
E.6 Sabouraud agar (SAB) . 19
E.7 Neutralizer . 19
Annex F (informative) Reference strains. 20
F.1 Storage of strains . 20
F.2 Bacterial strains . 20
F.3 Yeasts and molds . 20
F.4 Other Strains . 20
Annex G (informative) Preparation of microbial suspensions . 21
G.1 Stock Cultures . 21

G.2 Working Culture . 21
Annex H (normative) Test methods . 22
H.1 Introduction . 22
H.2 Equilibration of reagents . 22
H.3 Neutralizer . 22
H.4 Choice of experimental conditions . 22
H.5 Sample Size . 23
H.6 Direct contact test method . 23
H.7 Shaking method . 24
H.8 Two compartment method . 25
H.9 Recovery and enumeration of test organisms . 26
H. 10 Calculation and expression of results . 26
H.11 Test report . 29
Annex I (informative) Figures and illustrations . 30
Annex ZA (informative) Relationship between this European Standard and the Essential
Requirements of EU Directive 93/92/EEC on medical devices . 32
Bibliography . 33

iii
Foreword
This document (prEN 16756:2014) has been prepared by Technical Committee CEN/TC 205 “Non-active
medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association, and supports essential requirements of EU Directive(s).
For relationship with EU Directive(s), see informative Annex ZA, which is an integral part of this document.

Introduction
This standard describes a range of test methods for establishing whether a wound dressing exerts
antimicrobial activity.
The laboratory tests attempt to simulate conditions of application, through the use of appropriate test fluids,
temperature, organisms, and contact times reflecting the parameters found in clinical situations. Conditions
which may influence the action of wound dressings having antimicrobial properties should be included.
The conditions are intended to cover general purposes and to allow comparison between laboratories and
product types.
v
1 Scope
This standard specifies requirements and test methods for the antimicrobial activity of antimicrobial wound
dressings. It is designed for microbicidal and microbistatic dressings.
Test methods specifically for microbial binding are not included in the standard.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
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.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
antimicrobial dressing
wound dressing which can be shown to exert antimicrobial activity when tested to the appropriate tests in
Annex H of this standard
3.2
negative control dressing
wound dressing which is the same dressing as the dressing to be tested but without the antimicrobial
treatment
Note 1 to entry: If this is not available, then use a non-medicated dressing from the same product group preferably
from the same manufacturer. If no similar product is available, a sterile gauze swab of the same weight shall be used
3.3
plate count method
method in which the number of microorganisms present after incubation is calculated by counting the number
of colonies according to a ten-fold dilution method
3.4
neutralizer
chemical agents used to inactivate, neutralize, or quench the antimicrobial properties of antimicrobial agents
3.5
test dressing
wound dressing which is to be tested to assess its antimicrobial activity
3.6
saturation volume
volume of fluid (SWF) absorbed by the dressing as determined by the method in H.4.3
Note 1 to entry: This volume of fluid is added to the dressing to prevent the dressing absorbing all of the challenge
media (ISWF).
4 Classification
Classification of antimicrobial dressings is not presently standardized. The following two classes are thought
to be useful in the context of this standard when selecting appropriate test methods as described in Table 1.
Microbicidal – capability of the dressing to produce at least a 3-log reduction in the number of viable cells
from the challenge organisms when tested under the conditions in Annex H.
Microbistatic – capability of the dressing to prevent further growth of the initial inoculum but which does not
achieve at least a 3 log reduction when tested under the conditions in Annex H.
If a manufacturer is claiming compliance with this standard, they must comply with the requirements in
Monographs 6 and 7.
NOTE The editing panel recommend that the usefulness of these definitions be reviewed during the development of
the standard.
5 Requirements
5.1 Microbicidal dressings
When tested using the relevant method in Annex H, antimicrobial dressings shall demonstrate at least a 3-log
reduction in activity as determined by that particular test method over 24 hours and against all three test
organisms.
5.2 Microbistatic dressings
When tested using the relevant method in Annex H, microbistatic dressings shall prevent further growth of the
initial inoculum over 24 hours and against all three test organisms.
5.3 Performance Table
Table 1 shows the performance requirements for antimicrobial dressing pending on the two classifications.
Table 1 — Performance requirements for antimicrobial dressings
Performance requirements
Microbicidal Microbistatic
A ≥ 3 0 ≤ A < 3
A = Antimicrobial activity (see Annex H.10)
6 Information to be supplied
The following information shall be supplied on request:
 the Annex number of the test method used;
 details of the test dressing, negative control dressing, and the mean values of C C , T , and T
0 t 0 t;
 the antimicrobial activity A;
 the full test report (see Tables 2 and 3 as examples).
Table 2 — Example for a test report (blank)
Test dressing ______________________________________________________________
Control dressing ______________________________________________________________
Test method Annex ________________________________________________________
Exposure period ______________________________________________________________
Organism Dressing Time 0 Log Time XX Log Antimicrobial
10 10
counts per counts per Activity (A)
sample (Ms) sample (Ms)
Control
S. aureus
Test
Control
P. aeruginosa
Test
Control
C. albicans
Test
Result _______________________________________
NOTE  The standard test time is (24 ± 1) h. Any additional exposure times should be provided in the same
format.
Table 3 — Example for a test report (filled out)
Test dressing Silver foam_____________________________________________________
Control dressing Silver-free foam_________________________________________________
Test method Annex H.7_____________________________________________________
Exposure period 24 hours_______________________________________________________
Organism Dressing Time 0 Log Time 24 h Log Antimicrobial
10 10
counts per counts per Activity (A)
sample (Ms) sample (Ms)
Control  6.3  6.7 - 0.4
S. aureus
Test  6.3  2.1  4.2
Control  6.4  7.0 - 0.6
P. aeruginosa
Test  6.1  1.2  4.9
Control  6.1  6.2 - 0.1
C. albicans
Test  6.1 < 1.6 > 4.5
Result Microbicidal_____________________________

Annex A
(normative)
Validation of dilution-neutralisation
A.1 Principle
The effectiveness of antimicrobial agents incorporated into wound dressings is measured by their ability to kill
microorganisms within a specified contact time. Consequently, accurate determination of antimicrobial activity
requires complete and immediate neutralisation of the antimicrobial agent. Inefficient or incomplete
neutralisation will permit killing or inactivation of microorganisms to continue beyond the experimental
exposure time, resulting in an overestimation of antimicrobial activity. A neutralizer is therefore required for
each antimicrobial dressing to inactivate or quench the microbicidal properties of the antimicrobial agent.
Neutralising agents may also be incorporated into agar to increase the neutralising capacity for the test
methods detailed in Annex H. Agar containing neutralising agents may be used for the enumeration of
microbial test suspensions providing its neutralisation effectiveness has been validated and it does not have
an inhibitory effect on organism viability.
A.2 Neutralizer selection
Refer to Annex B for examples of suitable neutralizers for certain antimicrobial agents. ASTM E1054-08 also
documents examples of appropriate neutralizers for antimicrobial agents.
The neutralizer selected should, where possible, minimize the neutralizer volume to allow the use of 1ml pour
plates to be used from the neat dilution, therefore maximising the sensitivity of the test.
A.3 Preparation of microbial suspension
NOTE The reference strains detailed in Annex F should be used in the neutralisation assay.
a) Prepare the microbial suspensions in accordance with Annex G.
b) Separately dilute each microbial suspension in MRD such that when a 100 µl volume is inoculated, the
final concentration within each neutralizer validation suspension contains 30 to 100 cfu/ml (see
Table A.1).
c) Using standard microbiological techniques perform an inoculum count on each suspension to confirm the
inoculum concentration.
A.4 Test validation
The neutralisation evaluation consists of three experimental tests (i.e. neutralizer toxicity, test organism
viability and neutralisation effectiveness test) for each test strain. At least three replicates must be performed
for each experimental condition. Table A.1 details the specific requirements of each experimental test which
are dependent on the chosen test method.
NOTE Each experimental test must simulate the test conditions as those detailed in Annex H (e.g. sample volumes,
dilution ratios and incubation conditions must replicate those in the chosen test method).

Table A.1 — Requirements for the neutralisation validation
Test Method
Requirements Direct Contact Method Shaking Method Two Compartment
(see H.6) (see H.7 Method (see H.8)
Neutralizer toxicity 8.9 ml Neutralizer 4.4 ml Neutralizer 4.4 ml Neutralizer
+ 100 µl inoculum + 100 µl inoculum + 100 µl inoculum
+ 1 ml MRD + 0.5 ml SWF + 0.5 ml SWF
Test organism viability 8.9 ml MRD 4.4ml MRD 4.4 ml MRD
+ 100 µl inoculum + 100µl inoculum + 100 µl inoculum
+ 1 ml MRD + 0.5ml SWF + 0.5 ml SWF
Neutralisation Dressing sample as per Dressing sample as per Dressing sample as per
effectiveness H.5 H.5 H.5
+ 5 x 0.1 ml SWF + 5 ml SWF + the + 2.5 ml SWF + the
as per H.6.3 determined SV as per determined SV as per
H.4.3 H.4.3
Incubate (32 ± 2) °C for Incubate (32 ± 2) °C for Incubate (32 ± 2) °C for
desired contact time desired contact time desired contact time

8.9 ml Neutralizer 4.4 ml Neutralizer 4.4 ml Neutralizer
+ 100 µl inoculum + 100 µl inoculum + 100 µl inoculum
+ Dressing sample + 0.5 ml SWF sample + 0.5 ml SWF sample

A.5 Neutralizer toxicity
This is performed to demonstrate that the chosen neutralizer does not have any inhibitory effect on the
challenge organisms.
a) Transfer the required volume of suitable neutralizer (see E.7) to a test tube.
b) Inoculate a 100 µl volume of microbial suspension prepared in A.3.
c) Add the appropriate volume of MRD or SWF to the neutralizer/challenge organism suspension such that
the resulting suspension contains 30 to 100 cfu/ml of the challenge organism. Mix contents.
d) Allow the suspension to stand for an appropriate time (see note below).
e) After the exposure time, transfer 1ml volumes of the suspension to duplicate agar pour plates; agar
spread plates may be used by transferring a 0.1 ml volume onto duplicate plates, taking into account the
extra 10-fold dilution. If neutralizers are incorporated into agar for the test method, then use the same
medium for plating the suspension.
f) Repeat this procedure an additional two times, for a total of three replicates.
g) Incubate the agar plates under the same conditions as those used in the chosen test method.
h) Following incubation, count the number of colonies on each agar plate and determine the cfu/ml as
detailed in A.8.
NOTE Allow the resulting suspension to stand for the longest exposure period representative of that used in chosen
test method. Unless prior knowledge and experience in the use of the chosen neutralizer is known, a validation exercise
may be performed to determine optimal standing time for effective neutralisation and absence of toxicity.
A.6 Test organism viability
This is performed to determine the population of the challenge organism used in the neutralisation assay.
Perform the test as detailed in A.5 but replace the neutralizer with required volume of MRD as detailed in
Table A.1.
A.7 Neutralisation Effectiveness
This is performed to show the neutralizer is effective at inactivating or quenching the microbicidal properties of
an antimicrobial agent.
a) Aseptically cut a sample of dressing to the size specified in Table A.1 and transfer to a sterile petri-dish.
b) For the direct contact method only, hydrate the dressing sample with an identical volume of saturation
fluid (SWF) to that determined in the test method (see H.4.3).
c) Depending on the chosen test method, manipulate the dressing as detailed in Table A.1:
 Direct contact method: add 5 x 0.1 ml volumes of SWF across the dressing sample as described in
H.6
 Shaking method: Transfer dressing sample to 5 ml of SWF (+ saturation volume) contained within
an Erlenmeyer flask
 Two compartment method: Transfer dressing sample to 2.5 ml of SWF (+ saturation volume)
contained within a 6 well plate.
d) Incubate the dressing sample for the desired contact time, under the same conditions as those used in
the chosen test method (see Annex H).
e) Following the desired contact time, remove the dressing sample from the incubator.
f) Transfer an appropriate volume of neutralizer (see Table A.1) to a test tube. For the Direct Contact
Method, transfer the appropriate volume of neutralizer to a sterile stomacher bag.
g) Inoculate a 100 µl volume of microbial suspension prepared in A.3.
h) Depending on the chosen test method, perform the following step:
 Direct contact method: Transfer the dressing sample to the neutralizer/challenge organism
suspension. Process the dressing in a stomacher blender as detailed in H.6;
 Shaking method/Two compartment method: Remove a 0.5 ml volume of SWF from the
appropriate test vessel and transfer to the neutralizer/challenge organism suspension such that the
resulting suspension contains 30 to 100 cfu/ml of the challenge organism. Mix contents.
i) Allow the resulting suspension to stand for an appropriate time (see note below).
After the exposure time, transfer 1ml volumes of the suspension to duplicate agar pour plates; agar
spread plates may be used by transferring a 0.1 ml volume onto duplicate plates, taking into account the
extra 10-fold dilution. If neutralizers are incorporated into agar for test method, then use the same
medium for plating the suspension.
j) Repeat this procedure an additional two times, for a total of three replicates.
Incubate the agar plates under conditions appropriate for each test organism.
Following incubation, count the number of colonies on each agar plate and determine the cfu/ml as detailed in
A.8.
NOTE Allow the resulting suspension to stand for the longest exposure period representative of that used in chosen
test method. Unless prior knowledge and experience in the use of the chosen neutralizer is known, a validation exercise
may be performed.
A.8 Interpretation of data
a) Apply the following formula to calculate the number N of surviving challenge organisms resulting from
each replicate of each test:
P1+P2
N= (CFU /ml)
where
P1 = counts on plate 1
P2 = counts on plate 2
b) Compare the mean cfu/ml from A.7 (Neutralisation effectiveness) and A.5 (Neutralizer toxicity) to A.6
(Organism viability).
c) Neutralisation is considered adequate if A.7 recovery population is ≥ 70 % of A.6 organism viability
population (see Note below).
d) The chosen neutralising medium is considered non-toxic if A.5 recovery population is within 70 % of A.6
organism viability population (see Note below).

NOTE As per USP 36, p.227 “Validation of microbial recovery from pharmacopoeial articles”.

Annex B
(informative)
Neutralizers
Table B.1 — Examples of active agents and possible neutralizers
Antimicrobial Agent Neutralising Agent
Silver Sodium thiosulphate / sodium thioglycolate”
Quaternary ammonium agents Lecithin / polysorbate
(Benzalkonium chloride)
Chlorhexidine Polysorbate 80 / egg lecithin
Polyhexamethylene biguanide (PHMB) Polysorbate 80 / asolectin.
Honey Lecithin
Iodine Sodium thiosulphate and lecithin

NOTE The above list is not exhaustive and other neutralizers may be tried see EN 1040 [1] for more examples.
Annex C
(informative)
Rationale
NOTE This annex is intended to explain how and why certain decisions have been made concerning this standard.
C.1 Title
The use of the term 'antimicrobial' has been specifically chosen to enable the standard to include methods
other than microbicidal when available.
C.2 Microbial strains
The standard presently includes tests for a range of bacterial strains and the yeast Candida albicans. Future
editions will consider the inclusion of other yeasts and moulds.
The strains used in this standard (see Annex F) have been selected based on their use in other antimicrobial
standards and are good examples of their species.
Specifically:
Staphylococcus aureus is the most commonly isolated organisms from acute and chronic wounds. The strain
ATCC 6538 is chosen since it is a clinical isolate and is it also specified in ISO 22196 and ISO 20743
(antimicrobial testing of plastics and textiles, respectively).
Pseudomonas aeruginosa is commonly isolated from both acute and chronic wounds. The strain ATCC 9027
is chosen since it is a clinical isolate and it is specified in disinfectant standards EN 1040 [1], 1276 and 13727.
Candida albicans is part of the normal microflora (and therefore an opportunistic pathogen) of various
ecological niches on the human body including skin. It acts as an opportunistic pathogen in
immunocomprimised patients or those on broad spectrum antibiotics and is therefore a key infection-causing
organism across many different wound types. The strain ATCC 10231 is chosen as the most commonly
isolated yeast from chronic wounds.
Whilst the Working Group understand there is interest in effectiveness against antibiotic resistant
organisms, there is no requirement in this standard to use such organisms, as this mechanism is only related
to antibiotics
Users of this standard (manufacturers, purchasers, test houses) may use different microbial species in
addition to those specified herein as long as appropriate validation has been undertaken.
The tests in this standard could also, with amendment, be used for other microorganisms.
Test reports should contain explanatory text as to how and why alternative microorganisms have been used,
and provide details of any amendments to the methods.
C.3 Cutting dressings
To obtain appropriate samples for the tests in this standard it is frequently necessary to cut the dressings
under aseptic conditions. Depending on the dressing design and construction, this action may disrupt the
dressing matrix and release antimicrobial agents which would not necessarily be released from an intact
dressing, potentially enhancing the dressings performance during the test, which might not be reflected in the
performance of an intact dressing.
The Working Group acknowledge that this is potentially problematical, but that at the time of writing, it reflects
the state of the art in dressings testing.
In the future it may be possible to deal with this issue by including a test for an intact dressing using a wound
model such as that described by Walker et al[8].
The Working Group are interested in any proposals for test methods for intact dressings for future revisions.
C.4 Positive control
The Working Group acknowledge that the selection of an appropriate positive control is difficult.
C.5 Microbistatic requirements
The requirements for a microbistatic dressing to meet the following requirements:
A ≥ 0 and A < 3
is based on the following rationale:
If A >= 0 then the growth on the test dressing is the same as or lower than that seen on the control. If A is < 0,
then there has been growth with the test dressing.
A value of <3 shows that the dressing has not achieved the 3-log reduction necessary to meet the
requirements for a microbicidal effect
C.6 Performance Requirements
The method is designed to show antimicrobial activity in vitro. Whilst the method is not correlated with clinical
activity, there is an expectation that a product demonstrating microbicidal activity in vitro is able to exert a
clinical effect in vivo under suitable conditions.
Peterson & Shanholtzer[9] note that the selection of a 3-log reduction for the minimum bactericidal
concentration (MBC) (i.e., the lowest concentration which results in a 99.9% reduction in the initial inoculum)
is an arbitary endpoint, but is well accepted and has been used as the reference value for many years.
C.7 Test method Information
C.7.1 General
This annex contains explanatory text about each of the test methods used in this standard, and explains the
type of dressing they should be used for.
NOTE the test methods are not directly comparable.
Caution is also urged when comparing different classes of dressings (for example, comparing alginates with
foams), as at present no data is available to support this.
C.7.2 Direct Contact method
The method is based upon a method referenced in Gallant-Behn et al[3]. It is suitable for most dressing types,
but the recovery for super absorbent dressings may be limited.
C.7.3 Shaking Method
The method is based upon a method in Parsons et al[4]. It is suitable for most dressing types, but the volume
of fluid required may not represent the clinical situation for super absorbent dressings.

C.7.4 Two compartment method
This method is based on cell culture method in Ågren and Mirastschijski[6]. The method is suitable for
releasing antimicrobial agents. The method allows the dressing to be in a moist environment although not
totally soaked in the test medium. Absorbent dressings are allowed to absorb test medium from underneath,
which mirrors the clinical situation.
C.8 Media
The Simulated Wound Fluid (SWF) is used for suspension of the test organisms when exposed to products
containing antimicrobial substances. It contains salts and proteins, which are known to interfere with some
antimicrobial substances used for dressings, and also to simulate wound like conditions. This formulation has
been chosen to include foetal calf serum as it provides a more nutritious environment for the microorganisms.
C.9 Incubation Temperatures
The incubation temperatures for the tests are based on temperatures found in wounds[15].
The incubation temperatures for the organism recovery of (30 – 35) °C for bacteria and (20 - 25) °C for yeasts
and molds (H.9) are based on the requirements in the European and United States Pharmacopoeias.
C.10 Dressing Classification
The committee note that it is presently difficult to assess whether a dressing is a 'releasing' or 'non-releasing'
dressing. Future editions will reconsider whether this would be a useful classification.
Annex D
(informative)
Apparatus
Usual microbiological laboratory apparatus and in particular the following:
1) Spectrophotometer, capable of measuring at a 600nm to 660nm wavelength, or McFarland's
nephelometer
2) Incubator, capable of being controlled at 32°C ± 2°C
3) Incubator, capable of being controlled at 23°C ± 2°C
4) Shaking incubator or shaker in incubator, capable of being controlled at 32°C ± 2°C & 100 ± 5rpm
5) Small size (up to 100ml) Paddle Blender (also known as a stomacher) and bags . ®
6) Mixer, (electromechanical agitator, e.g., Vortex mixer)
7) Cutting equipment, sterile, to prepare samples as per H.5.
8) Erlenmeyer flasks 50ml, sterile with caps or bungs
9) Various sterile containers, test tubes and flasks of suitable capacity including 60ml capacity tubes for
direct contact test samples;
10) Sterile Petri dishes, of size 90mm to 100mm
11) Forceps, sterile
12) Calibrated pH meter, having an accuracy of calibration of ± 0.1 pH units at 25°C
13) Autoclave, capable of being maintained at 121°C for a minimum holding time of 15m
14) Pipettes, of nominal capacities 10ml, 1ml and 0.1ml. Calibrated automatic pipettes may be used.
15) Sterile 6-well plates, untreated.
16) Sterile nylon mesh cell culture strainers, suitable for 6-well plates with membrane pore size of
100μm.
1 For example, Stomacher 80 Biomaster or Neutec Masticator 2 (Compact model – 80 ml).
2 Suitable cell strainers include the nylon mesh 100um Falcon cell strainer available from Corning, Cat No 08-771-19
Annex E
(informative)
Reagents and culture media
E.1 General
Reagents used in tests shall be of analytical quality and/or suited for microbiological purposes.
Dehydrated or ready-prepared products available on the commercial market are recommended for use in
preparing the culture media. The manufacturer's instructions for the preparation of these products should be
strictly followed.
E.2 Water
Shall be free from substances that are toxic or inhibiting to the bacteria. Demineralised or deionized water can
be used for the preparation of dehydrated media that will be sterilized.
E.3 Tryptone Soya Agar
For the maintenance of bacterial strains, and performance of viable counts
Tryptone, pancreatic digest of casein 15.0g
Soya peptone, papic digest of soya 5.0.g
NaCl          5.0g
Agar              15.0g
Water           to 1000 ml
Sterilize in the autoclave. After sterilisation the pH of the medium shall be equivalent to 7.3 ± 0.2 when
measured at 20°C ± 3°C.
E.4 Maximum recovery diluent
Peptone  1.0g
NaCl  8.5g
Water  to 1000ml
Sterilize in the autoclave. After sterilisation the pH of the medium shall be equivalent to 7.0 ± 0.2 when measured at 20°C
± 3°C.
E.5 Simulated Wound Fluid (SWF)
Depending on the test method, a sufficient quantity of SWF should be aseptically prepared as detailed below:
Maximum recovery diluent      50%
*
Foetal Calf Serum (supplier complement inactivated ) 50%
* - i.e., complement inactivated by the supplier.
For suspension of the test organisms when exposed to products containing antimicrobial substances.
E.6 Sabouraud agar (SAB)
A general medium with low pH for isolation of dermatophytes, other fungi and yeast. It is a simple medium
with peptone as nitrogen source and glucose as carbon and energy source. The low pH suppresses growth of
many bacteria and favours dermatophytes, other fungi and yeast.
Characteristic colony appearance, pigment and other characteristics develop well on this medium.
Peptone Neutral Bact. Peptone Oxoid L34 10 g
Agar bacteriological      18 g
Distilled water      1000 ml
Dissolve by heat by hand. When all is dissolved, add
Glucose puriss       40 g
Dissolve the glucose by hands as soon as possible in the hot, but not boiling agar, and mix.
Adjust pH to 5.8-6.0. Subsequently autoclave at 120 C for 15 m. Cool down before pouring plates. To be
stored in the dark.
Commercially available suppliers of SAB are also acceptable.
E.7 Neutralizer
The neutralizer shall be validated for the active substance in the product under test in accordance with
Annex A. 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 A.
Annex F
(informative)
Reference strains
F.1 Storage of strains
The strains shall be stored in accordance with the supplier's recommendations or EN 12353[2].
The identification and origin (culture collection) of the strains as well as the laboratory storage method shall be
recorded.
F.2 Bacterial strains
The following strains shall be used in all antibacterial activity tests:
- Pseudomonas aeruginosa ATCC 9027/NCIMB 8626/CCUG 22801
- Staphylococcus aureus ATCC 6538, CIP 4.38, DSM 799, NBRC 13276, CCUG 10778 or NCIMB
9518.
NOTE
- ATCC is the American Type Culture Collection (USA);
- CIP is the Pasteur Institute Collection (France);
- DSM is the German Collection of microorganisms and Cell Cultures (Germany);
- NBRC is the NITE Biological Resource Centre (Japan);
- CCUG is the Culture Collection, University of Gothenburg (Sweden);
- and NCIMB is the National Collection of Industrial Bacteria (UK).
F.3 Yeasts and molds
The yeast Candida albicans [NCPF 3179/ATCC 10231/CCUG 19915] shall be used as a challenge
organism.
F.4 Other Strains
If additional strains selected do not correspond to the specified strains, their suitability for producing inocula of
sufficient concentration shall be verified.
If additional strains tested are not classified at a reference centre (e.g. clinical isolates), their identification
characteristics shall be stated. In addition, they shall be held by the testing laboratory for 5 years to allow
repeat testing if necessary.
Future revisions of this standard will consider formalising the inclusion of other microorganisms as challenge
organisms.
Annex G
(informative)
Preparation of microbial suspensions
G.1 Stock Cultures
Stock cultures shall be kept in accordance with the requirements of EN 12353[2]
G.2 Working Culture
G.2.1 Subcultures from stocks
1. Prepare subcultures prior to starting testing. Following each incubation step, the resulting cultures
should be checked to ensure they are contamination free and colonial morphology is as expected for
the organism under culture. If contamination is apparent or suspected, re-start the subculturing
process.
2. Subcultures should be prepared using standard microbiological techniques from the stock culture
using agar plates or slants.
3. The working culture, used to prepare suspensions on the day of testing, should not be taken from the
first subculture but may be taken from the second or third subculture. The working culture should be
o o
incubated for 18-24 hours at 32 C ± 2 C.
4. If required, for the growth conditions for additio
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