Home Microbiological AnalysisUpdates on ISO Standards from our Expert Barbara Gerten

Updates on ISO Standards from our Expert Barbara Gerten

Barbara Gerten
Senior Scientist Traditional Microbiology
Merck KGaA, Darmstadt, Germany

Barbara is a microbiologist with many years of industry experience who has been at Merck KGaA, Darmstadt, Germany since 2008. She is a member of several ISO/CEN committees and Chair of IDF Standing Committee for Harmonization of Microbiological Methods and Chair of German DIN NAL Working Group “Microbiology in the Food Chain”.

(EN) ISO 23419:2022-06

Microbiology of the food chain — Whole genome sequencing for typing and genomic characterization of bacteria — General requirements and guidance

Next generation sequencing (NGS) provides rapid, economical and high-throughput access to microbial whole genome sequences and is being applied to an expanding number of problems in food microbiology.

This document provides guidance for both the laboratory and bioinformatic components of whole genome sequences and associated metadata for bacterial foodborne microorganisms sampled along the food chain (e.g. ingredients, food, feed, production environment). Although microbiology of the food chain includes viruses and fungi, this document is only intended for bacteria. This document is intended to be applicable to all currently available next generation DNA sequencing technologies.

This document specifies the minimum requirements for generating and analysing whole genome sequencing (WGS) data of bacteria obtained from the food chain. This process can include the following stages:

handling of bacterial cultures;
axenic genomic DNA isolation;
library preparation, sequencing, and assessment of raw DNA sequence read quality and storage;
bioinformatics analysis for determining genetic relatedness, genetic content and predicting phenotype, and bioinformatics pipeline validation;
metadata capture and sequence repository deposition;
validation of the end-to-end WGS workflow (fit for purpose for intended application).

This document is applicable to bacteria isolated from:

products intended for human consumption;
products intended for animal feed;
environmental samples from food and feed handling and production areas;
samples from the primary production stage.

ISO 4833-1:2013/Amd 1:2022-01

Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — Part 1: Colony count at 30 °C by the pour plate technique — Amendment 1: Clarification of scope

This amendment clarifies the scope of the document which specifies a horizontal method for enumeration of microorganisms that are able to grow and form colonies in a solid medium after aerobic incubation at 30 °C.

The method described in this document is applicable to:

products intended for human consumption;
products intended for feeding animals (including pets);
environmental samples in the area of food and feed production and handling;
all samples from the primary production stage.

This technique is suitable for, but not limited to, the enumeration of microorganisms in test samples

with a minimum of 10 colonies counted on a plate. This corresponds to a level of contamination that is expected to be higher than 10 cfu/ml for liquid samples or higher than 100 cfu/g for solid samples.

This technique is especially suitable for:

products that require a reliable count when a low limit of quantification is specified,
products expected to contain spreading colonies that can obscure colonies of other organisms, e.g. milk and milk products likely to contain spreading Bacillus species;
products expected to contain bacteria that are sensitive to oxygen, e.g. some lactic acid bacteria that develop during shelf life or modified atmosphere storage.

(EN) ISO 4833-2:2013/AMD 1:2022-01

Microbiology of the food chain — Horizontal method for the enumeration of microorganisms — Part 2: Colony count at 30 °C by the surface plating technique — Amendment 1: Clarification of scope

This amendment clarifies the scope of the document which specifies a horizontal method for enumeration of microorganisms that are able to grow and form colonies on the surface of a solid medium after aerobic incubation at 30 °C.

The method described in this document is applicable to:

products intended for human consumption;
products intended for feeding animals (including pets);
environmental samples in the area of food and feed production and handling;
all samples from the primary production stage.

This technique is suitable for, but not limited to, the enumeration of microorganisms in test samples with a minimum of 10 colonies counted on a plate. This corresponds to a level of contamination that is expected to be higher than 100 cfu/ml for liquid samples or higher than 1 000 cfu/g for solid samples.

This technique is especially suitable for:

products containing heat-sensitive organisms that are likely to form a significant proportion of the total flora (e.g. psychrotrophic organisms in chilled and frozen foods, dried foods, other foods that can contain heat-sensitive organisms);
products containing obligately aerobic bacteria that are likely to form a significant proportion of the total flora (e.g. Pseudomonas species.);
products that contain small particles that can prove difficult to distinguish from colonies in a pour plate;
products whose intense colour prevents the recognition of colonies in a pour plate;
products for which a distinction between different types of colony is desired as part of the assessment of food quality.

In addition to the manual spread plating technique, this document also describes the use of a spiral plater, an automated method of performing surface colony counts (see Annex A of EN ISO 4833-2:2013).

(EN) ISO 20836:2021-11

Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of microorganisms — Thermal performance testing of thermal cyclers

This document is part of a family of International Standards under the general title Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of food borne pathogens:

(EN) ISO 22174, General requirements and definitions;
(EN) ISO 20837, Requirements for sample preparation for qualitative detection;
(EN) ISO 20836, Thermal performance testing of thermal cyclers;
(EN) ISO 20838, Requirements for amplifications and detection for qualitative methods.

This document describes a method for performance testing for standard thermal cyclers and real-time thermal cyclers that allows laboratories to evaluate if the thermal cycler used is suitable for the intended use and meets the specifications set by the laboratory.

The described method is based on a physical method that measures directly in the thermal cycler block in block-based thermal cyclers and in tubes in heated-chamber-based thermal cyclers. The described method provides a measurement uncertainty that is sufficiently low to allow meaningful comparison to specifications.

Furthermore, the method does meet the criteria of a metrological traceable calibration method in case it is used by ISO/IEC 17025-compliant laboratories.

This document specifies requirements for the installation, maintenance, temperature calibration and temperature performance testing of standard thermal cyclers and real-time thermal cyclers. It is applicable to the detection of microorganisms as well as any other applications in the food chain using polymerase chain reaction (PCR)-based methods.

This document has been established for food testing, but is also applicable to other domains using thermal cyclers (e.g. environmental, human health, animal health, forensic testing).

This first edition International Standard cancels and replaces the first edition Technical Specification (ISO/TS 20836:2005), which has been technically revised.

The main changes compared with the previous edition are as follows:

the Scope has been extended to include both thermal cyclers and real-time thermal cyclers;
the physical performance testing method has been described in more detail, and the biochemical performance testing method has been taken out;
information for laboratories regarding ISO/IEC 17025 has been included;
the performance testing method has been aligned with ISO/IEC 17025;
compliancy testing has been added;
in Annex C, two procedures to set PCR-method-based specifications have been added.

(EN) ISO 6888-1:2021

Microbiology of the food chain — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 1: Method using Baird-Parker agar medium

(EN) ISO 6888-2:2021

Microbiology of the food chain — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 2: Method using rabbit plasma fibrinogen agar medium

These recently published documents specify horizontal methods for the enumeration of coagulase-positive staphylococci by counting the colonies obtained on a solid medium after aerboc incubation at 34 °C to 38 °C.

Part 1 uses the solid medium Baird-Parker medium and part 2 uses the solid medium Rabbit plasma fibrinogen medium.

Both parts are applicable to:

products intended for human consumption;
products intended for animal feeding;
environmental samples in the area of food and feed production, handling, and
samples from the primary production stage.

Because of the large variety of products in the food chain, it is possible that these horizontal methods are not appropriate in every detail for all products. Nevertheless, it is expected that the required modifications are minimized so that they do not result in a significant deviation from this horizontal method.

Based on the information available at the time of publication of this document, the methods are not considered to be (fully) suited to the examination of fermented products or other products containing technological flora based on Staphylococcus spp. (e.g. S. xylosus) (such as cheeses made from raw milk and certain raw meat products) likely to be contaminated by:

staphylococci forming atypical colonies on a Baird-Parker agar medium;
background flora that can obscure the colonies being sought.

Nevertheless, (EN) ISO 6888-1 and (EN) ISO 6888-2 are given equivalent status.

Main Technical Changes Compared to the Previous Editions of Part 1 and Part 2

It is stated in the Introduction of both parts, that the main technical changes listed in the Foreword, introduced in these documents compared with the previous editions (of part 1 and part 2) are considered as minor. They have a minor impact on the performance characteristics of this method.

Results of the interlaboratory study and tested samples are described in Annex C of part 1 and part 2.

The second edition of ISO 6888-1:2021 cancels and replaces the first edition (ISO 6888-1:1999), which has been technically revised. It also incorporates the amendments ISO 6888-1:1999/Amd 1:2003 and ISO 6888-1:1999/Amd 2:2018.

This second edition of ISO 6888-1:2021 cancels and replaces the first edition (ISO 6888-2:1999), which has been technically revised. It also incorporates the Amendment ISO 6888-2:1999/Amd 1:2003.

The main changes of part 1 and part 2 compared with the previous editions are as follows:

the title has been changed to relate to the “food chain”;
the status of ISO 6888-1 and ISO 6888-2 have been clarified;
the documents have been aligned with ISO 7218:2007, i.e. and pour molten agar medium at 44 °C to 47 °C;
all occurrences, when appropriate, have been changed from “35 °C or 37 °C” to “34 °C to 38 °C”;
all occurrences of incubation time, when appropriate, have been changed from “18 h to 24 h” to “24 h ± 2 h”;
requirements have been added to use ISO 11133;
all available standards related to sampling techniques have been updated;
only for part 1: a description of typical and atypical colonies on Baird-Parker agar (BPA) medium has been updated;
only for part 1: the rabbit plasma fibrinogen agar (RPFA) medium has been added as alternative to the coagulase test for confirmation;
the flow diagram procedure in Annex A of part 1 and part 2 has been updated;
culture media and reagents with performance testing in Annex B of part 1 and part 2 have been added;
results of the interlaboratory study (for part 1 from ISO 6888-1:1999/Amendement1: 2003, Precision data; and for part 2 from ISO 6888-2:1999/Amendment 1:2003 Precision data) have been updated and added to Annec C of part 1 and part 2;
the Bibliography of part 1 and part 2

ISO/TS 21872-2:2020

Microbiology of the food chain — Horizontal method for the determination of Vibrio spp. — Part 2: Enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in seafood using nucleic acid hybridization

This document specifies a method for the direct enumeration of potentially enteropathogenic V. parahaemolyticus (tdh and/or trh positive) and/or the enumeration of total V. parahaemolyticus in seafood.

Potentially enteropathogenic strains of Vibrio parahaemolyticus possess thermostable direct haemolysin (TDH) and/or thermostable direct hemolysin-related hemolysin (TRH). TDH positive strains manifest Kanagawa phenomenon (KP). This characteristic is traditionally utilized in the identification of enterotoxigenic strains of V. parahaemolyticus. Strains possessing TRH do not share the haemolytic characteristics of TDH positive isolates and no conventional identification assay has been reported for TRH identification. Pathogenic strains in the environment are a minority and differentiation between enteropathogenic and total V. parahaemolyticus presence is therefore useful.

New Website of ISO/TC 34/SC 9 Available

This new webpage contains various sections to cover news (hot topic), on-going projects, guidelines and relevant information to contact committee manager as well as chairperson. Relevant additional information is given, for example, for the validation and verification of methods including some background information and links to a document for the transition period for the implementation of (EN) ISO 16140-3.

Another section is dedicated to (EN) ISO 11133 on culture media including a table containing a complete list of culture media and reagents used in ISO food and water microbiology standards with the names of the control strains which should be used for the performance testing.

There is also a part on (EN) ISO 19036 “Estimation of measurement uncertainty for quantitative determinations” which gives answers to the questions on this complex topic in the presentation of the approach of the recently revised standard ISO 19036:2019. A link to an Excel tool enables the user to implement the calculations of this standard is also provided.

(EN) ISO 11133:2014/AMD 2:2020

Microbiology of food, animal feed and water — Preparation, production, storage and performance testing of culture media — Amendment 2

This second amendment to the already published (EN) ISO 11133:2014 gives additional information by specifying control strains for the performance testing of confirmation and characterization media, reagents, dyes, stains and materials described in standards for the microbiological examination of samples from the food chain and water. The strains chosen have been selected preferentially those already cited in (EN) ISO 11133:2014. If a suitable strains was not available from this source, a strains from the catalogue of organisms compiled by the World Data Centre for Microorganisms (WDCM) has been selected. In most cases, more than one strain has been listed for both positive and negative reactions. The user may choose any of the strains cited for positive and negative reactions.

(EN) ISO 6579-1:2017 / AMD1:2020-03

Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp. — Amendment 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC

Amendmend 1:2020 includes an extension of the temperature range for incubation of the selective media from 37 °C ± 1 °´C to 34 °C to 38 °C without further tolerance (e.g. for MKTTn broth, XLD agar, SC medium, BS agar, confirmation media and reagents). This temperature range was already included in (EN) ISO 6579-1:2017 for incubation of non-selective media.

Status of Annex D “Detection of Salmonella enterica subspecies enterica serovars Typhi and Paratyphi” has been changed from “normative” to “informative”.

Composition of MSRV (Modified semi-solid Rappaport-Vassiliadis) agar and SC (Selenite cysteine) medium have been corrected for their preparation from single ingredients.

(EN) ISO 6887-5:2020-05

Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 5: Specific rules for the preparation of milk and milk products

This document specifies rules for the preparation of samples of milk and milk products and their suspensions for microbiological examination when the samples require a different preparation from the general methods specified in ISO 6887-1. It excludes the preparation of samples for both enumeration and detection test methods where preparation details are specified in the relevant International Standards.

Besides a list of diluents with their composition and performance testing, it describes general and specific procedures for preparation of the initial suspension and further decimal dilutions, e.g. for milk and liquid milk products, dehydrated milk products, cheese and cheese products, casein products, butter, milk-based products with low pH (e.g. yoghurts, probiotic milk products) and dehydrated milk-based infant foods with or without probiotics.

(EN) ISO 6887-5:2020 is intended to be used in conjunction with (EN) ISO 6887-1:2017 “Microbiology of the food chain — Preparation of test samples, initial suspension and decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial suspension and decimal dilutions”.

(EN) ISO 7932:2004 / Amd1:2020-05

Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of presumptive Bacillus cereus — Colony-count technique at 30 °C — Amendment 1: Inclusion of optional tests

Amendmend 1:2020 includes optional tests intended for complemenatry investigations (i.e. epidemiological) on isolated Bacillus cereus group strains obtained from the procedure as described in (EN) ISO 7932:2004.

In this amendmed, the term “B. cereus group” is used instead of “presumptive B. cereus” following the EFSA optinion published in 2016.

New Annex C describes a validated PCR method that targets both cytK gene variants (cytK-1 or cytK-2 gene variants of the gene encoding Cytotoxin K) and, if present, indicates which of the two forms is present. It also allows confirmation of isolates as B. cytotoxicus.

New Annex D describes a rapid and validated PCR method that targets the ces gene. A cereulide peptide synthetase (ces) is involved in the non-ribosomal syntehsis of cereulide. This cereulide, when poduced in food, can cause an emetic food poisoning syndrome.

The motility test described in Annex E allows for screening for presumptive B. anthracis among isolated B. cereus group. This test has strong limitations and it is intended to assist in differentiating B. anthracis from other B. cereus group members. The correct identification of B. anthracis strains is very complex and needs additional tests which are outside the scope of this document.

Microscopic examination of the parasporal crystal from Bacillus thuringiensis is described in Annex F. B. thuringiensis, one of the B. cereus group species, can be distinguished from the other species of this group by the microsocopic examination of the parasporal crystal formation. This method was evaluated through an interlaboratory study and performance characteristics are included in Annex F.