ISO 21703:2019

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

General Information

• Status: Published
• Publication Date: 14-Jan-2019
• ICS: 07.100.99 - Other standards related to microbiology
• ICS: 71.100.40 - Surface active agents
• Technical Committee: ISO/TC 91 - Surface active agents
• Drafting Committee: ISO/TC 91 - Surface active agents
• Current Stage: 9092 - International Standard to be revised
• Start Date: 04-Nov-2024
• Completion Date: 13-Dec-2025

Standards Content (Sample)

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 2019
© 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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ii © ISO 2019 – All rights reserved

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
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
iv © ISO 2019 – All rights reserved

Foreword
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bodies (ISO member bodies). The work of preparing International Standards is normally carried out
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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).
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.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.
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
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
2 © ISO 2019 – All rights reserved

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
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.
4 © ISO 2019 – All rights reserved

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
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
6 © ISO 2019 – All rights reserved

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
1)
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 thi
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