Surface active agents -- Microbiology -- Microbiological test methods for liquid hand dishwashing

This document provides microbiological test methods for enumeration and detection of aerobic mesophilic bacteria, detection of Escherichia coli and Pseudomonas aeruginosa in liquid hand dishwashing.

Agents de surface -- Microbiologie -- Méthodes d’essai microbiologique pour les détergents liquides de lavage de vaisselle à la main

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Published
Publication Date
14-Jan-2019
Current Stage
6060 - International Standard published
Start Date
06-Dec-2018
Completion Date
15-Jan-2019
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ISO 21703:2019 - Surface active agents -- Microbiology -- Microbiological test methods for liquid hand dishwashing
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INTERNATIONAL ISO
STANDARD 21703
First edition
2019-01
Surface active agents — Microbiology
— Microbiological test methods for
liquid hand dishwashing
Agents de surface — Microbiologie — Méthodes d’essai
microbiologique pour les détergents liquides de lavage de vaisselle
à la main
Reference number
ISO 21703:2019(E)
ISO 2019
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ISO 21703:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
ii © ISO 2019 – All rights reserved
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ISO 21703:2019(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 2

5 Diluents and culture media ....................................................................................................................................................................... 2

5.1 General ........................................................................................................................................................................................................... 2

5.2 Neutralizing diluent and diluents ........................................................................................................................................... 2

5.2.1 General...................................................................................................................................................................................... 2

5.2.2 Neutralizing diluent: Fluid casein digest — soy lecithin — polysorbate 20

medium (SCDLP 20 broth) ...................................................................................................................................... 3

5.2.3 Diluent for the bacterial suspension (Tryptone sodium chloride solution) ................. 3

5.3 Culture medium for counting ..................................................................................................................................................... 3

5.3.1 General...................................................................................................................................................................................... 3

5.3.2 Soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA) ........................... 3

5.3.3 Other medium for counting .................................................................................................................................... 4

5.4 Culture medium for detection .................................................................................................................................................... 4

5.4.1 General...................................................................................................................................................................................... 4

5.4.2 Enrichment broth: Eugon LT 100 broth ....................................................................................................... 5

5.5 Culture media for isolation and identification ............................................................................................................. 5

5.5.1 MacConkey agar medium ......................................................................................................................................... 5

5.5.2 Levine eosin-methylene blue agar medium (EMB agar medium) ......................................... 6

5.5.3 Cetrimide agar medium ............................................................................................................................................. 6

5.5.4 Pseudomonas agar medium for detection of pyocyanin (Pseudomonas agar P) ..... 7

6 Apparatus and glassware ............................................................................................................................................................................ 7

7 Strains of microorganisms ......................................................................................................................................................................... 7

8 Handling of the product and laboratory samples .............................................................................................................. 8

9 Procedure..................................................................................................................................................................................................................... 8

9.1 General recommendation .............................................................................................................................................................. 8

9.2 Preparation of the initial suspension .................................................................................................................................. 8

9.3 Enumeration methods ...................................................................................................................................................................... 8

9.3.1 Dilutions for enumeration ....................................................................................................................................... 8

9.3.2 Plate-count methods .................................................................................................................................................... 8

10 Method for detection and identification ...................................................................................................................................11

10.1 Enrichment ..............................................................................................................................................................................................11

10.2 Isolation .....................................................................................................................................................................................................11

10.3 Detection of aerobic mesophilic bacteria ......................................................................................................................11

10.4 Detection and Identification of Escherichia coli ...................................................................................................11

10.4.1 General...................................................................................................................................................................................11

10.4.2 Gram’s stain .......................................................................................................................................................................11

10.4.3 Culture on Levine eosin-methylene blue agar medium (EMB agar medium) ..........11

10.4.4 Expression of results .................................................................................................................................................12

10.5 Detection and identification of Pseudomonas aeruginosa ........................................................................12

10.5.1 General...................................................................................................................................................................................12

10.5.2 Gram’s stain .......................................................................................................................................................................12

10.5.3 Oxidase test .......................................................................................................................................................................12

10.5.4 Culture on Pseudomonas agar medium for detection of pyocyanin ................................12

10.5.5 Expression of results .................................................................................................................................................12

11 Neutralization of the antimicrobial properties of the product .........................................................................13

11.1 General ........................................................................................................................................................................................................13

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11.2 Preparation of inoculum ..............................................................................................................................................................13

11.3 Suitability of counting methods ............................................................................................................................................13

11.3.1 Principle ...............................................................................................................................................................................13

11.3.2 Suitability test of the pour-plate method ................................................................................................13

11.3.3 Suitability of the surface spread method .................................................................................................13

11.3.4 Suitability of the membrane filtration method ...................................................................................14

11.4 Suitability of the detection method ....................................................................................................................................14

11.4.1 Procedure ............................................................................................................................................................................14

11.4.2 Interpretation ..................................................................................................................................................................14

12 Test report ................................................................................................................................................................................................................15

Annex A (informative) Neutralizers of antimicrobial activity of preservatives and rinsing

liquids ...........................................................................................................................................................................................................................16

Annex B (informative) Other culture media ..............................................................................................................................................17

Bibliography .............................................................................................................................................................................................................................19

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ISO 21703:2019(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.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

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. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

This document was prepared by Technical Committee ISO/TC 91, Surface active agents.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/members .html.
© ISO 2019 – All rights reserved v
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INTERNATIONAL STANDARD ISO 21703:2019(E)
Surface active agents — Microbiology — Microbiological
test methods for liquid hand dishwashing
1 Scope

This document provides microbiological test methods for enumeration and detection of aerobic

mesophilic bacteria, detection of Escherichia coli and Pseudomonas aeruginosa in liquid hand

dishwashing.
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

ISO 21148, Cosmetics — Microbiology — General instructions for microbiological examination

3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at https: //www .electropedia .org/
3.1
liquid hand dishwashing
liquid detergent which is used for dishwashing by hand
3.2
product

portion of an identified liquid hand dishwashing product received in the laboratory for testing

3.3
sample

portion of the product (at least 1 g or 1 ml) which is used in the test to prepare the initial suspension

3.4
initial suspension

suspension (or solution) of the sample in a defined volume of an appropriate enrichment broth

3.5
sample dilution
dilution of the initial suspension
3.6
aerobic mesophilic bacteria

mesophilic bacteria growing aerobically under the conditions specified in this document

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ISO 21703:2019(E)
3.7
Pseudomonas aeruginosa

gram-negative rod (bacilli), motile; smooth colonies pigmented (light brown or greenish)

Note 1 to entry: The main characteristics for identification are growth on selective cetrimide agar medium,

oxidase positive, production of diffusible fluorescent pigments and production of a soluble phenazine pigment

(pyocyanin) in suitable media.
3.8
Escherichia coli
gram-negative rod (bacilli), motile, smooth colonies

Note 1 to entry: The main characteristics for identification are catalase positive, oxidase negative, fermentation

of lactose, production of indole, growth on selective medium containing bile salts with characteristic colonies.

3.9
enrichment broth

non-selective liquid medium containing suitable neutralizers and/or dispersing agents and validated

for the product under test
4 Principle

This document provides enumeration and detection of aerobic mesophilic bacteria on a non-selective

agar medium and enrichment medium, detection of Escherichia coli and Pseudomonas aeruginosa by

presence or absence of bacterial growth after enrichment.

Microorganisms to be tested might be different from country to country according to the practices or

regulations. Users can choose enumeration and/or detection methods for those microorganisms which

are mentioned in this document based on their needs.

In order to ensure product quality and safety for consumers, it is advisable to perform appropriate

microbiological risk analysis so as to determine the type of the product to which this document is

applicable. For example, products considered to present a low microbiological risk include those with

low water activity, those with extreme pH values, etc. (see ISO 29621).

Alternative microbiological methods may be substituted for the tests presented here provided that

their equivalence has been demonstrated or the method has been otherwise validated.

The possible inhibition of microbial growth by the sample shall be neutralized to allow the detection

of viable microorganism. In all cases the neutralization of the antimicrobial properties of the product

shall be checked.
5 Diluents and culture media
5.1 General

General recommendations for microbiological examinations are specified in ISO 21148.

The following neutralizer, diluents and culture media are suitable for enumeration of aerobic mesophilic

bacteria. Other diluents, neutralizers and culture media may be used if they have been demonstrated to

be suitable for use.
5.2 Neutralizing diluent and diluents
5.2.1 General

The diluent is used to disperse the sample. It may contain neutralizers if the specimen to be tested

has antimicrobial properties. The efficacy of the neutralization shall be demonstrated before the

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determination or detection of the count. Information related to the suitable neutralizers is given in

Annex A.

5.2.2 Neutralizing diluent: Fluid casein digest — soy lecithin — polysorbate 20 medium

(SCDLP 20 broth)
5.2.2.1 Composition
Pancreatic digest of casein, 20,0 g
Soy lecithin, 5,0 g
Polysorbate 20, 40,0 ml
Water, 1 000 ml
5.2.3 Diluent for the bacterial suspension (Tryptone sodium chloride solution)
5.2.3.1 Composition
Tryptone, pancreatic digest of casein, 1,0 g
Sodium chloride, 8,5 g
Water, 1 000 ml
5.2.3.2 Preparation

Dissolve the components in the water by mixing while heating. Dispense into suitable containers.

Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall be equivalent to 7,0 ± 0,2

when measured at room temperature.
5.3 Culture medium for counting
5.3.1 General

Culture media may be prepared as follows, or from dehydrated culture media according to the

instructions of the manufacturer. Ready-to-use media may be used when their composition and/or

growth yields are comparable to those of the formulae given herein.
5.3.2 Soybean-casein digest agar medium (SCDA) or tryptic soy agar (TSA)
5.3.2.1 Composition
Pancreatic digest of casein, 15,0 g
Papaic digest of soybean meal, 5,0 g
Sodium chloride, 5,0 g
Agar, 15,0 g
Water, 1 000 ml
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ISO 21703:2019(E)
5.3.2.2 Preparation

Dissolve the components or the dehydrated complete medium in the water by mixing while heating.

Dispense the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After

sterilization and cooling down, the pH shall be equivalent to 7,3 ± 0,2 when measured at room

temperature.
5.3.3 Other medium for counting
5.3.3.1 Eugon LT 100 agar medium
5.3.3.2 Composition
Pancreatic digest of casein, 15,0 g
Papaic digest of soybean meal, 5,0 g
L-cystine, 0,7 g
Sodium chloride, 4,0 g
Sodium sulphite, 0,2 g
Glucose, 5,5 g
Egg lecithin, 1,0 g
Polysorbate 80, 5,0 g
Octoxynol 9, 1,0 g
Agar, 15,0 g
Water, 1 000 ml
5.3.3.3 Preparation

Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their complete

dissolution. Dissolve the other components by mixing while heating. Mix gently to avoid foam. Dispense

the medium into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization

and cooling down, the pH shall be equivalent to 7,0 ± 0,2 when measured at room temperature.

5.4 Culture medium for detection
5.4.1 General
An enrichment broth shall be used for bacterial detection.

The enrichment broth is used to disperse the sample and to increase the initial microbial population. It

may contain neutralizers if the specimen to be tested has antimicrobial properties.

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5.4.2 Enrichment broth: Eugon LT 100 broth
5.4.2.1 Eugon LT 100 broth
5.4.2.2 General

This medium contains ingredients which neutralize inhibitory substances present in the sample:

lecithin and polysorbate 80, dispersing agent: octoxynol 9.
5.4.2.3 Composition
Pancreatic digest of casein, 15,0 g
Papaic digest of soybean meal, 5,0 g
L-cystine, 0,7 g
Sodium chloride, 4,0 g
Sodium sulfite, 0,2 g
Glucose, 5,5 g
Egg lecithin, 1,0 g
Polysorbate 80, 5,0 g
Octoxynol 9, 1,0 g
Water, 1 000 ml
5.4.2.4 Preparation

Dissolve successively polysorbate 80, octoxynol 9 and egg lecithin into boiling water until their

complete dissolution. Dissolve the other components by mixing while heating. Dispense the medium

into suitable containers. Sterilize in the autoclave at 121 °C for 15 min. After sterilization, the pH shall

be equivalent to 7,0 ± 0,2 when measured at room temperature.
5.5 Culture media for isolation and identification
5.5.1 MacConkey agar medium
5.5.1.1 Composition
Pancreatic digest of gelatin, 17,0 g
Pancreatic digest of casein, 1,5 g
Peptic digest of animal tissue, 1,5 g
Lactose, 10,0 g
Bile salts mixture, 1,5 g
Sodium chloride, 5,0 g
Agar, 13,5 g
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ISO 21703:2019(E)
Neutral red, 30,0 mg
Crystal violet, 1,0 mg
Water, 1 000 ml
5.5.1.2 Preparation

Dissolve all solid components in the water and boil for 1 min to effect solution. Dispense in suitable

containers and sterilize at 121 °C for 15 min. The pH, after sterilization and cooling down, shall be

equivalent to 7,1 ± 0,2 when measured at room temperature.
5.5.2 Levine eosin-methylene blue agar medium (EMB agar medium)
5.5.2.1 Composition
Pancreatic digest of gelatin, 10,0 g
Potassium dihydrogen phosphate (KH PO ), 2,0 g
2 4
Agar, 15,0 g
Lactose, 10,0 g
Eosine Y, 400 mg
Methylene blue, 65 mg
Water, 1 000 ml
5.5.2.2 Preparation

Dissolve the pancreatic digest of gelatin, the dibasic potassium phosphate, and the agar in the water,

with warming, and allow to cool. Just prior to use, liquefy the gelled agar solution, add the remaining

ingredients, as solutions, in the following amounts, and mix; for each 100 ml of the liquefied agar

solution
— 5 ml of 20 % lactose solution,
— 2 ml of 2 % eosin Y solution, and
— 2 ml of 0,033 % methylene blue solution.

The finished medium may not be clear. Dispense in suitable containers and sterilize at 121 °C for 15 min.

The pH, after sterilization and cooling down, shall be equivalent to 7,1 ± 0,2 when measured at room

temperature.
5.5.3 Cetrimide agar medium
5.5.3.1 Composition
Pancreatic digest of gelatin, 20,0 g
Magnesium chloride, 1,4 g
Potassium sulfate, 10,0 g
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ISO 21703:2019(E)
Cetrimide (cetyltrimethylammonium bromide), 0,3 g
Agar, 13,6 g
Glycerin, 10,0 ml
Water, 1 000 ml
5.5.3.2 Preparation

Dissolve all solid components in the water and add the glycerin. Heat, with frequent agitation, and boil

for 1 min to effect dissolution.
Dispense in suitable flasks and sterilize at 121 °C for 15 min.

After sterilization and cooling down, the pH shall be equivalent to 7,2 ± 0,2 when measured at room

temperature.
5.5.4 Pseudomonas agar medium for detection of pyocyanin (Pseudomonas agar P)
5.5.4.1 Composition
Pancreatic digest of gelatin, 20,0 g
Anhydrous magnesium chloride, 1,4 g
Anhydrous potassium sulfate, 10,0 g
Agar, 15,0 g
Glycerin, 10,0 ml
Water, 1 000 ml
5.5.4.2 Preparation

Dissolve all solid components in the water and add the glycerin. Heat, with frequent agitation, and boil

for 1 min to effect dissolution.
Dispense in suitable flasks and sterilize at 121 °C for 15 min.

After sterilization and cooling down, the pH shall be equivalent to 7,2 ± 0,2 when measured at room

temperature.
6 Apparatus and glassware
The laboratory equipment, apparatus and glassware specified in ISO 21148.
7 Strains of microorganisms

For testing the efficacy of neutralizers, the representative of Gram negative is used. Pseudomonas

aeruginosa ATCC (American Type Culture Collection) 9027 or Escherichia coli ATCC 8739 or equivalent

strain may be used.

1) ATCC®9027 and ATCC®8739 are a trademark of American Type Culture Collection. This information is given

for the convenience of users of this document and does not constitute an endorsement by ISO of the product named.

Equivalent products may be used if they can be shown to lead to the same results.

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ISO 21703:2019(E)

The culture should be reconstituted according to the procedures provided by the supplier of

reference strain.
8 Handling of the product and laboratory samples

If necessary, store products to be tested at room temperature. Do not incubate, refrigerate or freeze

products and samples before or after analysis. Sampling of products should be carried out, as described

in ISO 21148. Analyse samples as specified in ISO 21148 and in accordance with the following procedure.

9 Procedure
9.1 General recommendation

Use sterile material, equipment and aseptic techniques to prepare the sample, initial suspension and

dilutions.

In the case of the preparation of an initial suspension, the time which elapses between the end of the

preparation and the moment the inoculum comes into contact with the culture medium shall not exceed

45 min, unless specifically mentioned in the established protocols or documents.
9.2 Preparation of the initial suspension

The initial suspension is prepared by adding at least 1 g or 1 ml of the well-mixed product to at least

9 ml of neutralizing diluents (5.2.2) or diluent (5.2.3).

The initial suspension is usually 1:10 dilution. More dilutions may be required if high levels of

contamination are expected and/or if anti-microbial properties are still present in 1:10 dilution.

Avoid the development of foam as it might impact the final results.
9.3 Enumeration methods
9.3.1 Dilutions for enumeration

Usually, the initial suspension is the first counted dilution. If needed, additional serial dilutions

(e.g. 1:10 dilution) may be performed from the initial suspension using the same diluent (according

to the expected level of contamination of the product). Generally, counting is performed using at least

two Petri dishes. But it is possible to use only one Petri dish in case of routine testing, or if counts are

performed on successive dilutions of the same sample or according to previous results.

9.3.2 Plate-count methods
9.3.2.1 Pour-plate method

In Petri dishes 85 mm to 100 mm in diameter, add 1 ml of the initial suspension and/or sample dilution

prepared and pour 15 ml to 20 ml of the melted agar medium (5.3.2) kept in a water bath at no more

than 48 °C. If larger Petri dishes are used, the amount of agar medium is increased accordingly.

Mix the initial suspension and/or sample dilution with the medium carefully rotating the plates

sufficiently to disperse them. Allow the mixture in the Petri dishes to solidify on a horizontal surface at

room temperature.
9.3.2.2 Surface spread method

In Petri dishes 85 mm to 100 mm in diameter, put 15 ml to 20 ml of the melted agar medium (5.3.2)

kept in a water bath at no more than 48 °C. If larger Petri dishes are used, the volume of the agar is

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