Updates on ISO and Other Standards for Microbiological Analysis of the Food Chain and Water

ISO standards are reference methods implemented in the field of microbiological analysis of the food chain from primary production stage to food and feed products including food production and handling and in the field of water quality. IDF and ISO jointly publish standards for the dairy sector and IFU publishes own methods for the analysis of fruit and vegetable juices.

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

NEW (EN) ISO 15213:2023

Microbiology of the food chain — Horizontal method for the detection and enumeration of Clostridium spp. — Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique

The International Organization for Standardization (ISO) is publishing the three-part EN ISO 15213 series. The series specifies the enumeration and detection of sulfite-reducing Clostridium spp., including C. perfringens, in a wide range of foods that includes pet food and animal feed, samples from the primary production stage and environmental samples in food and feed production and handling.

• Part 1: Enumeration of sulfite-reducing Clostridium spp. by colony-count technique. It has replaced ISO 15213:2003
• Part 2: Enumeration of Clostridium perfringens by colony-count technique. It is set to replace EN ISO 7937:2004
• Part 3: Detection of Clostridium perfringens. This part will be newly published

NEW (EN) ISO 7704:2023

Water quality — Requirements for the performance testing of membrane filters used for direct enumeration of microorganisms by culture methods

The International Organization for Standardization (ISO) has recently published the EN ISO 7704:2023 standard, which specifies the requirements for performance testing of membrane filters used for the retention and subsequent direct enumeration of microorganisms by culture methods. It applies to the analysis of different water samples, including:

• Drinking water, bottled water and other water samples with expected low numbers of microorganisms.
• Water with expected higher numbers of microorganisms, for example, surface water or process water

This document applies to manufacturers producing membrane filters and microbiological laboratories using membrane filters for their own testing or providing these to other end users.

(EN) 20976-2:2022

Microbiology of the food chain — Requirements and guidelines for conducting challenge tests of food and feed products — Part 2: Challenge tests to study inactivation potential and kinetic parameters

Under the general principles of the Codex Alimentarius on food hygiene, it is the responsibility of the food business operators (FBOs) to control microbiological hazards in food and manage microbial risks. Therefore, FBOs implement validated control measures within the hazard analysis and critical control point (HACCP) system and conduct studies in order to investigate compliance with the food safety criteria throughout the food chain.

In the framework of microbial risk assessment (MRA), several complementary approaches are developed to estimate risks posed by pathogens or spoilage microorganisms in the food chain. MRA is adopted by regulators under the auspices of the international agency for setting food standards. Challenge testing is one of the recognized approaches used to validate control measures within the HACCP system, as well as to assess microbiological safety and quality of food, food production, food storage conditions, and food preparation recommendations dedicated to consumers.

Therefore, this document provides technical rules, calculations, and approaches to investigate the ability of an inoculated microorganism of concern to grow, survive or inactivated in the raw materials, intermediate or end products under reasonably foreseeable food processes, storage and use conditions. The objective and the scope of the study are to determine the experimental design and the selection of the study conditions and to assess the extent of microbial inactivation. Regulatory authorities can have different recommendations, and these differences have been included as much as possible. It is, however, possible that specific requirements need to be incorporated to get regulatory approval for the challenge test.

As the growth and inactivation studies are different, the ISO 20976 series consists of two parts, under the general title Microbiology of the food chain — Requirements and guidelines for conducting challenge tests of food and feed products:

• Part 1: Challenge tests to study the growth potential, lag time and maximum growth rate.
• Part 2: Challenge tests to study inactivation potential and kinetic parameters.

The use of the ISO 20976 series involves expertise in relevant areas such as food microbiology, food science, food processing and statistics. The statistical expertise encompasses an understanding of sampling theory and design of experiments, statistical analysis of microbiological data, and an overview of scientifically recognized and available mathematical concepts used in predictive modeling.

For practical reasons, the term “food” includes feed.

This document specifies the protocols for conducting microbiological challenge tests for inactivation studies on vegetative bacteria and bacterial spores in the raw materials and ingredients, intermediate or end products.

The use of this document can be extended to yeasts that do not form mycelium.

IFU METHOD OF ANALYSIS NO. 2 (2022)

Method on the Detection and Enumeration of Acid-Tolerant Spoilage Microorganisms of Fruits and Related Products

It is known that no single plating medium can be perfect for determining the Total Count of microorganisms present in all products. The purpose of a suitable culture medium is to enable the growth of all the microorganisms having the potential to spoil finished products, the so-called potential spoilage microorganisms. Largely, due to the low pH of fruit products, the growth of most microorganisms is inhibited and limited mostly to the Acid-Tolerant types mentioned below.

1. Lactic Acid Bacteria, predominantly the genera Lactobacillus and Leuconostoc, can be isolated from low Brix level products (such as juices) and during the earliest stages of the juice concentration process.
2. Yeasts mostly exhibit osmoduric characteristics and may be encountered as the main potential spoilage microorganisms of products with Brix levels of more than 32° Bx and up to 65° Bx.
3. Molds should be regarded mainly as secondary contaminants, due either to rotten raw material or improper storage and re-infection during processing.
4. Some other Acid-Tolerant Types, such as Acetic Acid Bacteria and or Gluconobacter, may be part of the spoilage flora, especially in soft drinks.

The main changes, compared to IFU Method No. 2:1996 and IFU No.7:1998, are given below:

• The title of the method has been changed.
•  The optional usage of several media has been changed to the usage of one liquid (Malt extract broth) and one solid medium [Orange serum agar (OSA) medium].
• A new enumeration procedure by filtration technique is introduced for clear or membrane-filterable fruit juice products and ingredients with a low Brix index.
• A new detection procedure by enrichment technique is introduced for the detection of 1 cfu in 10 mL.
• A new confirmation procedure by growth in Tryptic soy broth (acidified) has been added.
• Performance testing for the quality assurance of the culture media has been added to Annex B.
• Performance characteristics for this method have been added to Annex C.
• Matrix-dependent special processes have been added to Annex D.

The main technical changes listed in the Foreword, introduced in this document compared to IFU Method No. 2:1996 and IFU No.7:1998, are considered major (as specified by ISO 17468). This document specifies methods for the detection and enumeration of potential spoilage microorganisms of fruits and related products of high acid and low pH.

This document applies to:

• Juice and juice-related products and their ingredients intended for human consumption
• Environmental samples including processed water in juice and juice-related production and handling
• Other beverages not containing juices and their ingredients including syrups.

This method is intended to detect and enumerate mesophilic acid-tolerant potential spoilage microorganisms at 30 °C, specific included groups may need deviating optimal growth conditions.

A list of all IFU Methods can be found on the IFU International Fruit and Vegetable Juice Association website (see www.ifu-fruitjuice.com).

IDF FACTSHEET 22/ 2022

Enumeration of butyric acid forming (cheese spoiling) clostridia – methodical considerations

Late blowing in cheese is a significant problem, especially in the manufacture of hard or semi-hard cheeses. Spoilage contributes to wastage in the food chain, decreases efficiency and generates severe economic losses in hard cheese manufacturing. Costs of damage associated with cheese-spoiling clostridia have been confirmed to be significant.

Only a few or even single clostridial spores per liter of raw milk may cause severe spoilage of hard cheese. For clostridial enumeration, various most probable number (MPN) procedures including different media formulations are currently in use. A comparison of the most common procedures is provided in Table 1 of the IDF Factsheet.

The IDF Factsheet can be found (for free) on the International Dairy Federation (IDF) website (see www.fil-idf.org).

(EN) ISO 23419:2022

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 guides 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 the microbiology of the food chain includes viruses and fungi, this document is only intended for bacteria and applies to all currently available next-generation DNA sequencing technologies.

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

1. Handling of bacterial cultures
2. Axenic genomic DNA isolation
3. Library preparation, sequencing, and assessment of raw DNA sequence read quality and storage
4. Bioinformatics analysis for determining genetic relatedness, genetic content and predicting phenotype, and bioinformatics pipeline validation
5. Metadata capture and sequence repository deposition
6. Validation of the end-to-end WGS workflow (fit for purpose for intended application).

This document applies 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

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 can grow and form colonies in a solid medium after aerobic incubation at 30 °C.

The method described in this document applies to:

• Products intended for human consumption
• Products intended for feeding animals (including pets)
• Environmental samples in food 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

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 can grow and form colonies on the surface of a solid medium after aerobic incubation at 30 °C.

The method described in this document applies to:

• Products intended for human consumption
• Products intended for feeding animals (including pets)
• Environmental samples in food 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 1000 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, and other foods that can contain heat-sensitive organisms)
• Products containing obligately aerobic bacteria 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 with intense color that prevents the recognition of colonies in a pour plate
• Products for which a distinction between different types of colonies 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

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 amplification 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 metrologically 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 detect 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, and 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
• Compliance testing has been added
• In Annex C, two procedures to set PCR-method-based specifications have been added

(EN) ISO 6888-1:2021 AND (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 1: Method using Baird-Parker agar medium

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 aerobic 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 apply to:

• Products intended for human consumption
• Products intended for animal feeding
• Environmental samples in food and feed production, handling
• Samples from the primary production stage

Because of the large variety of products in the food chain, however, these horizontal methods maybe not be 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 examine 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.

Important 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 minor. They have a minor impact on the performance characteristics of this method.

Results of the inter-laboratory 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 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 an alternative to the coagulase test for confirmation
• The flow diagram procedure in Annex A of parts 1 and 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/amendment 1: 2003, precision data; and for part 2 from ISO 6888-2:1999/amendment 1:2003 precision data) have been updated and added to Annex 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 for ISO/TC 34/SC 9

This new webpage contains various sections to cover news (hot topic), ongoing projects, guidelines and relevant information to contact the committee manager as well as the 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 that 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 are the ones that are cited in (EN) ISO 11133:2014. If a suitable strain was not available from this source, a strain 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

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

Amendment 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.

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

The 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

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., yogurts, 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 feeds — Horizontal method for the enumeration of presumptive Bacillus cereus — Colony-count technique at 30 °C — Amendment 1: Inclusion of optional tests.

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

In this amendment, the term “B. cereus group” is used instead of “presumptive B. cereus” following the EFSA opinion 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 synthesis of cereulide. This cereulide, when produced in food, can cause an emetic food poisoning syndrome.

The motility test described in Annex E allows for screening for presumptive B. anthracis among the 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 microscopic examination of the parasporal crystal formation. This method was evaluated through an inter-laboratory study and performance characteristics are included in Annex F.