Measurement of antibacterial activity on plastics and other non-porous surfaces

ISO 22196:2011 specifies a method of evaluating the antibacterial activity of antibacterial-treated plastics, and other non-porous, surfaces of products (including intermediate products). It is not intended to be used to evaluate the effects and propagation of bacteria on non-porous surfaces without antibacterial treatments. ISO 846 describes tests to evaluate the effects and propagation of bacteria on non-porous surfaces, which are different from those covered by ISO 22196. Secondary effects of antibacterial treatments, such as the prevention of biodeterioration and odour, are not covered by the standard, which is not intended to be used or referenced as a method to document or claim biodegradability of, for instance, plastics materials. Building materials are excluded, except where they are used in the same manner as treated articles. Antibacterial-treated textile products are excluded, even if the surfaces are coated or laminated (such products are covered by ISO 20743). Photocatalytic materials and products are excluded (such materials and products are covered by ISO 27447).

Mesurage de l'action antibactérienne sur les surfaces en plastique et autres surfaces non poreuses

General Information

Status
Published
Publication Date
20-Jul-2011
Current Stage
9093 - International Standard confirmed
Start Date
12-Sep-2016
Completion Date
14-Jun-2021
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INTERNATIONAL ISO
STANDARD 22196
Second edition
2011-08-01
Measurement of antibacterial activity on
plastics and other non-porous surfaces
Mesurage de l'action antibactérienne sur les surfaces en plastique et
autres surfaces non poreuses
Reference number
ISO 22196:2011(E)
ISO 2011
---------------------- Page: 1 ----------------------
ISO 22196:2011(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2011

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or

ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
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Published in Switzerland
ii © ISO 2011 – All rights reserved
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ISO 22196:2011(E)
Contents Page

Foreword ............................................................................................................................................................iv

Introduction.........................................................................................................................................................v

1 Scope......................................................................................................................................................1

2 Normative references............................................................................................................................1

3 Terms and definitions ...........................................................................................................................2

4 Materials .................................................................................................................................................2

4.1 Bacteria to be used for the tests..........................................................................................................2

4.2 Reagents, culture media and solutions ..............................................................................................3

5 Apparatus...............................................................................................................................................4

6 Sterilization of apparatus and storage of stock cultures..................................................................5

6.1 Dry-heat sterilization.............................................................................................................................5

6.2 High-pressure steam sterilization........................................................................................................5

6.3 Preparation of glassware......................................................................................................................5

6.4 Maintenance of stock cultures.............................................................................................................5

7 Procedure...............................................................................................................................................6

7.1 Pre-culture of bacteria ..........................................................................................................................6

7.2 Preparation of test specimens .............................................................................................................6

7.3 Preparation of test inoculum................................................................................................................6

7.4 Inoculation of test specimens..............................................................................................................6

7.5 Incubation of the inoculated test specimens .....................................................................................8

7.6 Recovery of bacteria from test specimens.........................................................................................8

7.7 Determining the viable bacteria count by the pour plate culture method.......................................8

8 Expression of results............................................................................................................................9

8.1 Determination of the number of viable bacteria.................................................................................9

8.2 Conditions for a valid test ....................................................................................................................9

8.3 Calculation of the antibacterial activity...............................................................................................9

8.4 Effectiveness of the antibacterial agent............................................................................................10

9 Repeatability and reproducibility.......................................................................................................10

10 Test report............................................................................................................................................10

Annex A (normative) Quality of biological materials...................................................................................11

A.1 General .................................................................................................................................................11

A.2 Chemical composition of 1/500 nutrient broth (1/500 NB) ..............................................................11

Annex B (informative) Repeatability and reproducibility............................................................................12

B.1 Background..........................................................................................................................................12

B.2 Summary ..............................................................................................................................................12

B.3 Experiment ...........................................................................................................................................12

B.4 Results and discussion ......................................................................................................................13

Bibliography......................................................................................................................................................15

© ISO 2011 – All rights reserved iii
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ISO 22196:2011(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies

(ISO member bodies). The work of preparing International Standards is normally carried out through ISO

technical committees. Each member body interested in a subject for which a technical committee has been

established has the right to be represented on that committee. International organizations, governmental and

non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the

International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards

adopted by the technical committees are circulated to the member bodies for voting. Publication as an

International Standard requires approval by at least 75 % of the member bodies casting a vote.

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

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

ISO 22196 was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 6, Ageing, chemical

and environmental resistance.

This second edition cancels and replaces the first edition (ISO 22196:2007). The main change is the

extension of the scope of the standard to include non-porous surfaces other than plastics (for details, see the

Introduction).
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ISO 22196:2011(E)
Introduction

Antibacterial materials and products have been widely and rapidly accepted by general consumers as fulfilling

a relatively new function, which is distinguishable from the more traditional function of material protection.

Antibacterial products created by incorporating an antibacterial agent (biocide) can suppress the growth of

bacteria on the surfaces of products when conditions exist where growth can occur. They can keep surfaces

clean and sanitary and can also have an advantage in minimizing the impact on the environment by

minimizing diffusion of the agent. This technology is significant for the quality of life, not only in developed

countries but also in developing countries.

Antibacterial products have been widely used in plastics, coating materials, ceramics, natural and artificial

leather, stainless steel, rubber, etc. The products involved cover a variety of categories, such as electrical

appliances, personal items, household goods, nursing-care articles, pet accessories and aircraft-interior

fittings.

The scope of the first edition of ISO 22196 was limited to plastics surfaces. In this second edition, the scope

has been extended to include surfaces made of other non-porous materials, thus making the second edition

[11]

applicable to products of the kinds listed above. The test method, which is based on JIS Z 2801 , has

remained unchanged.
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INTERNATIONAL STANDARD ISO 22196:2011(E)
Measurement of antibacterial activity on plastics and other non-
porous surfaces
1 Scope

WARNING — Handling and manipulation of microorganisms which are potentially hazardous requires

a high degree of technical competence and may be subject to current national legislation and

regulations. Only personnel trained in microbiological techniques should carry out such tests.

Appropriate practices for disinfection, sterilization and personal hygiene must be strictly observed.

This International Standard specifies a method of evaluating the antibacterial activity of antibacterial-treated

plastics, and other non-porous, surfaces of products (including intermediate products).

It is not intended to be used to evaluate the effects and propagation of bacteria on non-porous surfaces

[1]

without antibacterial treatments. ISO 846 describes tests to evaluate the effects and propagation of bacteria

on non-porous surfaces, which are different from those covered by this International Standard (see e.g.

ISO 846:1997, method C).

Secondary effects of antibacterial treatments, such as the prevention of biodeterioration and odour, are not

covered by this International Standard, which is not intended to be used or referenced as a method to

document or claim biodegradability of, for instance, plastics materials. In the case of plastics, biodegradation

[2] [3] [4]
is covered in ISO 14851 , ISO 14852 and ISO 14855 and related standards.

Building materials are excluded, except where they are used in the same manner as treated articles.

Antibacterial-treated textile products are excluded, even if the surfaces are coated or laminated (such

[5]
products are covered by ISO 20743 ).

Photocatalytic materials and products are excluded (such materials and products are covered by

[6]
ISO 27447 ).

The results obtained should include a reference to this International Standard and the conditions used.

Results obtained with this International Standard indicate antibacterial activity under the specified

experimental conditions used, and do not reflect activity under other circumstances where a variety of factors,

such as temperature, humidity, different bacterial species, nutrient conditions, etc., have to be considered. A

minimum diffusion of the antibacterial agents/chemicals into the test inoculum is necessary with this procedure.

It is recommended that workers consult ISO 7218.
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.

ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations
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ISO 22196:2011(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
antibacterial

term describing a state where growth of bacteria on the surfaces of products is suppressed or describing the

effect of an agent which suppresses the growth of bacteria on the surfaces of products

3.2
antibacterial agent

agent that inhibits the growth of bacteria on the surfaces of products, used either as a surface treatment or as

a compounded ingredient
3.3
antibacterial activity

difference in the logarithm of the viable cell counts found on an antibacterial-treated product and an untreated

product after inoculation with and incubation of bacteria
3.4
antibacterial effectiveness

ability of an antibacterial agent to inhibit the growth of bacteria on the surface of materials treated with an

antibacterial agent, as determined by the value of the antibacterial activity
4 Materials
4.1 Bacteria to be used for the tests
Both of the following species of bacteria shall be used:
a) Staphylococcus aureus;
b) Escherichia coli.

The bacterial strains to be used are shown in Table 1. If bacterial strains obtained from culture collections

other than those shown in Table 1 are used, they shall be obtained from a member agency of the World

Federation for Culture Collections (WFCC) or of the Japan Society for Culture Collections (JSCC) and shall be

the same strains as those shown in Table 1. Prepare stock cultures of these species in accordance with the

supplier's directions.
Table 1 — Bacterial strains to be used
Name Strain
ATCC 6538P
CIP 53.156
Staphylococcus aureus DSM 346
NBRC 12732
NCIB 8625
ATCC 8739
CIP 53.126
Escherichia coli DSM 1576
NBRC 3972
NCIB 8545
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ISO 22196:2011(E)

If required, other species can also be used, in which case the species and the reason for their use shall be

included in the test report.
4.2 Reagents, culture media and solutions
Water shall be distilled or deionized and have a conductivity of < 1 µS/cm.

All reagents shall be of analytical grade and/or of a grade appropriate for microbiological purposes.

4.2.1 Nonionic surfactant
Polyoxyethylene sorbitan monooleate shall be used.
4.2.2 Biological materials
The following biological materials are required:
⎯ lecithin;
⎯ D-glucose;
⎯ yeast extract;
⎯ meat extract (see Annex A);
⎯ peptone (see Annex A);
⎯ casein peptone;
⎯ soybean peptone;
⎯ tryptone.
4.2.3 Culture medium
4.2.3.1 General

The culture medium specified below shall be used. The medium may be obtained from commercial suppliers,

in which case it shall be prepared for use in accordance with the manufacturer's instructions.

The quantity of the culture medium can be changed provided the same composition is retained.

4.2.3.2 Suspension medium — 1/500 nutrient broth (1/500 NB)

Prepare nutrient broth by dissolving 3,0 g of meat extract, 10,0 g of peptone and 5,0 g of sodium chloride in

1 000 ml of distilled or deionized water. Dilute the nutrient broth with distilled or deionized water to a 500-fold

volume and adjust the pH to a value between 6,8 and 7,2 with sodium hydroxide or hydrochloric acid. Sterilize

by autoclaving (see 6.2). If it is not used immediately after preparation, store it at 5 °C to 10 °C. A 1/500 NB

that has been kept for one week or longer after preparation shall not be used.
4.2.3.3 Nutrient agar

Prepare nutrient agar by dissolving 5,0 g of meat extract, 10,0 g of peptone, 5,0 g of sodium chloride and

15,0 g of agar powder in 1 000 ml of distilled or deionized water. Heat, with stirring, on a hotplate or in a

boiling-water bath until the agar has dissolved. Adjust the pH to a value between 7,0 and 7,2 (at 25 °C) with

sodium hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not used immediately after

preparation, store it at 5 °C to 10 °C. Nutrient agar that has been kept for one month or longer after

preparation shall not be used.
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ISO 22196:2011(E)
4.2.3.4 Plate count agar

Prepare plate count agar by dissolving 2,5 g of yeast extract, 5,0 g of tryptone, 1,0 g of glucose and 15,0 g of

agar powder in 1 000 ml of distilled or deionized water. Heat, with stirring, on a hotplate or in a boiling-water

bath until the agar has dissolved. Adjust the pH to a value between 7,0 and 7,2 (at 25 °C) with sodium

hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not used immediately after preparation,

store it at 5 °C to 10 °C. Plate count agar that has been kept for one month or longer after preparation shall

not be used.
4.2.3.5 Slant culture medium

Warm 6 ml to 10 ml of nutrient agar and pour into a screw-capped test tube. Sterilize by autoclaving (see 6.2).

After sterilization, place the test tube at an angle of about 15° to the horizontal and allow the contents to

solidify. If it is not used immediately after preparation, store it at 5 °C to 10 °C. Slant culture medium kept for

one month or longer after preparation shall not be used.

4.2.3.6 Soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate

(SCDLP broth)

Prepare SCDLP broth by dissolving 17,0 g of casein peptone, 3,0 g of soybean peptone, 5,0 g of sodium

chloride, 2,5 g of disodium hydrogen phosphate, 2,5 g of glucose and 1,0 g of lecithin in 1 000 ml of distilled or

deionized water. Mix thoroughly and add 7,0 g of nonionic surfactant. Adjust the pH to a value between 6,8

and 7,2 (at 25 °C) with sodium hydroxide or hydrochloric acid. Sterilize by autoclaving (see 6.2). If it is not

used immediately after preparation, store it at 5 °C to 10 °C. SCDLP broth kept for one month or longer after

preparation shall not be used.

NOTE SCDLP is the default neutralizer in the majority of circumstances. Information about selection and evaluation

[7] [8]

of alternative antibacterial neutralizing agents can be found in ASTM E1054 and EN 1040 .

4.2.3.7 Phosphate buffer solution

Prepare phosphate buffer solution by placing 34,0 g of potassium dihydrogen phosphate in a 1 000 ml

volumetric flask. Add 500 ml of distilled or deionized water and mix to dissolve. Adjust the pH to a value

between 6,8 and 7,2 (at 25 °C) with sodium hydroxide. Add distilled or deionized water to make up to 1 000 ml.

Sterilize by autoclaving (see 6.2). Phosphate buffer solution kept for one month or longer after preparation

shall not be used.
4.2.3.8 Phosphate-buffered physiological saline

Prepare physiological saline by placing 8,5 g of sodium chloride in 1 000 ml of distilled or deionized water and

mixing to dissolve. Dilute the phosphate buffer solution prepared in 4.2.3.7 with the physiological saline to an

800-fold volume. Sterilize the phosphate-buffered physiological saline solution by autoclaving (see 6.2). If this

solution is not used immediately after preparation, store it at 5 °C to 10 °C. Phosphate-buffered physiological

saline kept for one month or longer after preparation shall not be used.
5 Apparatus
Unless otherwise specified, use the following apparatus and materials:

5.1 Dry-heat sterilizer, capable of maintaining the temperature at a value between 160 °C and 180 °C

within ±2 °C of the set point at equilibrium conditions.

5.2 Autoclave, capable of maintaining a temperature of (121 ± 2) °C and a pressure of (103 ± 5) kPa.

5.3 Hotplate with stirrer, or hot-water bath.
5.4 pH-meter, capable of measuring to ±0,2 units.
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ISO 22196:2011(E)
5.5 Balance, capable of weighing to ±0,01 g.
5.6 Pipetters, sterile, with 1 000 µl tips.

5.7 Incubator, capable of maintaining the temperature within ±1 °C of the set point at equilibrium conditions.

5.8 Vortex mixer, if required (see 7.6.1).
5.9 Sonicator, if required (see 7.6.1).
5.10 Inoculating loops, 4 mm in ring diameter, sterile.

5.11 Cover film, that does not affect bacterial growth or absorb water, made of polyethylene, polypropylene

or polyester [poly(ethylene terephthalate)]. Film that is 0,05 mm to 0,10 mm thick is recommended.

NOTE Films cut from Stomacher bags are also suitable.
5.12 Screw-capped test tubes.
5.13 Petri dishes, sterile, 90 mm to 100 mm in diameter.
5.14 Gauze or absorbent cotton.
5.15 1 000 ml volumetric flask.

5.16 Stoppered Erlenmeyer flasks or media bottles, as required for preparation of media.

6 Sterilization of apparatus and storage of stock cultures
6.1 Dry-heat sterilization

Place objects to be sterilized in a dry-heat sterilizer, using the following minimum times for the given

temperature:
Temperature Minimum sterilization time
180 °C 30 minutes
170 °C 60 minutes
160 °C 120 minutes
6.2 High-pressure steam sterilization

Put the objects to be sterilized in an autoclave and maintain at (121 ± 2) °C for at least 15 min.

6.3 Preparation of glassware

Wash well with alkali or neutral detergent, then rinse well with distilled or deionized water. Sterilize using dry

heat or an autoclave prior to use.
6.4 Maintenance of stock cultures

Stock cultures shall be stored at 5 °C to 10 °C on an appropriate medium and transferred monthly. After five

transfers or if more than one month has passed between transfers, the stock culture shall be discarded and

replaced with a fresh culture obtained from the institute or culture collection concerned.

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ISO 22196:2011(E)
7 Procedure
7.1 Pre-culture of bacteria

Using a sterile inoculating loop, transfer bacteria from the stock culture to the slant culture medium (4.2.3.5)

and incubate at (35 ± 1) °C for 16 h to 24 h. From this culture, use a sterile inoculating loop to transfer bacteria

onto fresh slant culture medium and incubate at (35 ± 1) °C for 16 h to 20 h.
7.2 Preparation of test specimens

Testing shall be performed on at least three specimens from each treated test material. At least six specimens

of the untreated material are required. Half of the untreated test specimens are used to measure viable cells

immediately after inoculation and half are used to measure viable cells after incubation for 24 h.

NOTE Use of more than three replicate specimens of the treated test material can help reduce variability, especially

for materials that show smaller antimicrobial effects.

When testing a series of antibacterial treatments for a single polymer, each antibacterial treatment may be

compared to the same single set of untreated specimens if all tests are conducted at the same time using the

same test inoculum.

Prepare flat (50 ± 2) mm × (50 ± 2) mm specimens of the treated and untreated test materials. Specimens

should be no more than 10 mm in thickness. If it is difficult or impossible to cut the product into a square of

this size, then test specimens of other sizes and shapes may be used, as long as they can be covered with a

2 2

film of surface area between 400 mm and 1 600 mm . It is preferable to prepare test specimens from the final

product itself. However, if the shape of the product prevents this, then the test specimens may be prepared in

a format suitable for testing using the same raw materials and processing methods as are normally used for

the product. If the test specimen differs from the 50 mm × 50 mm square dimensions, the actual dimensions

used shall be stated in the test report.

When preparing specimens, take care to avoid contamination with microorganisms or extraneous organic

debris. Similarly, do not allow specimens to come into contact with each other. If metal apparatus is used to

avoid cross-contamination, it is necessary to ensure that the metal does not have any antibacterial effect. If

necessary, test specimens can be cleaned/disinfected/sterilized prior to testing (e.g. by wiping with 70 %

ethanol in water).

Cleaning of the test specimen can cause softening, dissolution of the surface coating or elution of components,

so should be avoided. If cleaning is required due to gross contamination, the cleaning method shall be stated

in the test report.
7.3 Preparation of test inoculum

Using a sterile inoculating loop, transfer one loop of the test bacteria, pre-incubated as specified in 7.1, into a

small amount of 1/500 NB prepared in accordance with 4.2.3.2. Ensure that the test bacteria are evenly

dispersed, and estimate the number of bacteria using direct microscopic observation and a counting chamber

or another appropriate method (e.g. spectrophotometrically). Dilute this suspension with 1/500 NB, as

appropriate for the estimated bacterial concentration, to obtain a bacterial concentration that is between

5 5 5

2,5 × 10 cells/ml and 10 × 10 cells/ml, with a target concentration of 6 × 10 cells/ml. Use this solution as the

test inoculum. If the test inoculum is not used immediately, then chill it on ice (0 °C) and use it within 2 h of

preparation.
7.4 Inoculation of test specimens

The surface to be tested is the exposed outer surface of the product. Cross-sections of the product shall not

be used. Place each test specimen prepared in accordance with 7.2 into a separate sterile Petri dish with the

test surface uppermost. Pipette 0,4 ml of the test inoculum prepared in accordance with 7.3 onto the test

surface. Cover the test inoculum with a piece of film (5.11) that measures 40 mm × 40 mm and gently press

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ISO 22196:2011(E)

down on the film so that the test inoculum spreads to, but does not leak beyond, the edges of the film. After

the specimen has been inoculated and the cover film applied, replace the lid of the Petri dish (see Figure 1).

Dimensions in millimetres
Key
1 cover film
2 test inoculum (0,4 ml)
3 test specimen
4 Petri dish
5 lid of Petri dish
Figure 1 — Inoculation of test specimen and placement of cover film

Unless otherwise specified, the standard size of the cover film shall be a square of (40 ± 2) mm × (40 ± 2) mm

for the 50 mm × 50 mm test specimen. If the test specimen is not of a standard size, then the size of the film

shall be reduced in direct proportion. Do not, however, reduce the size of the film to less than 400 mm and

the edges of the cover film shall always be 2,5 mm to 5,0 mm inside the edge of the test specimen on all sides.

If the size of the cover film differs from 40 mm × 40 mm, the actual size used shall be stated in the test report.

The volume of inoculum used shall also be adjusted to be in proportion to the area of the cover film used, and

the volume shall be recorded in the test report.

It is essential that the test inoculum does not leak beyond the edges of the cover film. However, for some

surfaces (e.g. those that are very hydrophilic), this might be difficult to achieve. When leakage occurs, use

option 1 below. If leakage still occurs with option 1, use option 2. If one of these options is used to ensure that

leaking does not occur, it shall be described in the test report.

⎯ Option 1: Reduce the volume of test inoculum applied to the test surface, but do not use less than 0,1 ml.

When the volume of test inoculum is decreased, the concentration of the bacterial cells in the inoculum

shall be increased to provide the same number of bacterial cells as when the normal volume of test

inoculum is applied.

⎯ Option 2: Increase the viscosity of the test inoculum by adding an inert thickener such as agar.

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ISO 22196:2011(E)
7.5 Incubation of the inoculated test specimens

Unless otherwise specified, incubate the Petri dishes containing the inoculated test specimens (including half

of the untreated test specimens) at a temperature of (35 ± 1) °C and a relative humidity of not less than 90 %

for (24 ± 1) h. The antibacterial effectiveness of a product is evaluated based on the value of the antibacterial

activity obtained from the test at the incubation temperature specified. Other temperatures may be used if

agreed by the interested parties. If a temperature other than (35 ± 1) °C is used, it shall be included in the test

report.

NOTE If incubation temperatures of less than 35 °C are used, the total count of the viable bacteria might be reduced.

This might affect the antibacterial activity compared to measurements conducted using a 35 °C incubation temperature.

7.6 Recovery of bacteria from test specimens
7.6.1 Test specimens immediately after inoculation

Immediately after inoculation, process half of the untreated test specimens by adding 10 ml of either SCDLP

broth (4.2.3.6) or a suitable, validated neutralizer to the Petri dish containing the test specimen. The value

obtained from these test sp
...

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