CEN/TC 223

Date: 2025-09-17

prEN 18280:2026

Document CEN/TC 223/WG 5/N 475

Secretariat: NEN

Soil improvers and growing media — Enumeration of Escherichia coli

Einführendes Element — Haupt-Element — Ergänzendes Element

Élément introductif — Élément central — Élément complémentaire

ICS:

Contents Page

European foreword 3

Introduction 4

1 Scope 5

2 Normative references 5

3 Terms and definitions 5

4 Principle 6

5 Culture media and reagents 6

6 Equipment and consumables 6

7 Sampling 7

8 Preparation of the test sample 7

9 Procedure 7

10 Expression of results 9

11 Validation of the method 10

12 Test report 10

13 Quality assurance 10

Annex A (normative) 11

Annex B (normative) Composition and preparation of culture media and reagents 12

B.1 General 12

B.2 Tryptone-bile-X-glucuronide (TBX) agar 12

B.3 MacConkey Agar No. 3 (optional) 13

B.4 Tryptone/tryptophan medium (optional) 13

B.5 Kovacs reagent (optional) 14

Annex C (informative) Performance characteristics of the method 15

C.1 Interlaboratory study 15

C.2 Statistical results for the enumeration of E. coli 15

Bibliography 18

European foreword

This document (prEN 18280:2026) has been prepared by Technical Committee CEN/TC 223 “Soil improvers and growing media”, the secretariat of which is held by NEN.

This document is currently submitted to the CEN Enquiry.

This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association.

Introduction

This document has been developed to enumerate E. coli in soil improvers and growing media in order to be able to control certain hygienic requirements. The method described in this document is based on EN ISO 16649‑2 for the enumeration of β-D-glucuronidase-positive E. coli.

The species Escherichia coli is a Gram-negative bacterium of faecal origin that consists of a large number of different strains. Consequently, it can be used as an indicator of faecal contamination and as a parameter to evaluate the sanitation process during the manufacturing process of soil improvers or growing media. The presence or absence of E. coli does not reflect the presence or absence of other pathogens in the material tested.

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for the enumeration of E. coli are only undertaken in properly equipped laboratories, under the control of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials. Persons using this document should be familiar with normal laboratory practice. This document does not purport to address all of the safety aspects, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices.

This document specifies a method for the enumeration of Escherichia coli (E. coli) in soil improvers and growing media. This document is applicable to material in solid form (including pre-shaped growing media) and liquid form.

This document is applicable to fertilizing product blends, where a blend is a mix of two or more fertilizing products belonging to the categories of fertilizers, liming material, soil improvers, growing media, inhibitors and plant biostimulants, and where soil improvers and/or growing media comprise the highest percentage in the blend by mass or volume, or in the case of liquid form by dry mass. If soil improvers and/or growing media do not comprise the highest percentage in the blend, the European Standard for the highest percentage in the blend applies. In case a blend is composed of fertilising products in equal quantity, the user of the standard decides which standard to apply.

NOTE 1 A soil improver or a growing medium consists of a single bulky (volume-building) component or a mix of bulky (volume-building) components (for example peat, wood fibres, coconut coir, compost, expanded perlite).

Strains of E. coli which do not grow at 44 °C ± 1 °C and, in particular, those that are β-D-glucuronidase-negative, such as E. coli O157, will not be detected. Some strains of Shigella spp. and Salmonella spp. within the family Enterobacteriaceae can also show β-D-glucuronidase activity at 44 °C ± 1 °C.

NOTE 2 This method has been validated in an interlaboratory study with specific products that were present on the market during the study (Annex C).

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.

EN 12579, Soil improvers and growing media - Sampling

prEN 13040‑2,^[1] Soil improvers and growing media — Sample preparation — Part 2: Sample preparation for microbiological examination

prEN 17732,^[2] Soil improvers and growing media — Terminology

EN ISO 7218, Microbiology of the food chain - General requirements and guidance for microbiological examinations (ISO 7218)

For the purposes of this document, the terms and definitions given in prEN 17732, prEN 13040‑2 and the following apply.

ISO and IEC maintain terminology 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

Escherichia coli

bacteria which form typical blue colonies on tryptone-bile-X-glucuronide (TBX) agar (β-D-glucuronidase-positive) at 44 °C ± 1 °C under the conditions specified in this method

3.2

enumeration of Escherichia coli

determination of the number of colony-forming units (CFU) of β-D-glucuronidase-positive Escherichia coli per gram or per millilitre of product or per sample device, when the analysis and calculation is carried out in accordance with this document

The initial suspension and/or aliquots of appropriate dilutions are used to inoculate the selective medium tryptone-bile-glucuronide (TBX) agar through pour plating, which is incubated for 21 h ± 3 h at 44 °C. After inoculation and incubation on TBX agar, β-D-glucuronidase-positive E. coli are enumerated and, if necessary, confirmed by a positive indole reaction.

Follow current laboratory practices in accordance with standards comparable to EN ISO 7218. The composition of culture media and reagents and their preparation are specified in Annex B. For performance testing of culture media, it is advised to follow the procedures of EN ISO 11133.

Disposable equipment is an acceptable alternative to reusable glassware if it has suitable specifications. The usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following shall be used.

6.1 Apparatus for dry sterilization (oven) and/or wet sterilization (autoclave).

6.2 Incubator(s), capable of maintaining a temperature of 36 °C ± 2 °C, 44 °C ± 1 °C and 44°C to 47°C.

6.3 Water bath, capable of maintaining a temperature between 44 °C and 47 °C.

6.4 Cooling unit, adjustable to 5 °C ± 3 °C.

6.5 pH-meter, capable of reading to the nearest 0,1 pH unit at 20 °C to 25 °C.

6.6 Sterile loops, of 10 μl volume (approximate diameter 3 mm) and of 1 µl volume, inoculating needles or inoculating wires.

6.7 Sterile tubes, bottles or flasks with caps, of appropriate capacity.

6.8 Sterile Petri dishes, with a diameter of approximately 90 mm or a diameter of approximately 140 mm (optional).

6.9 Graduated pipettes or automatic pipettes and sterile tips, with wide opening if necessary, of nominal capacities of 1 ml and 10 ml.

Sampling is not part of the method specified in this document. Follow EN 12579 dealing with soil improvers and growing media. It is important that the laboratory receives a sample that is representative of the product under consideration. The sample should not have been damaged or changed during transport or storage.

Prepare the test sample from the laboratory sample in accordance with prEN 13040‑2. If there is no specific International Standard available, it is recommended that the parties concerned come to an agreement on this subject.

The procedure as given in Annex A shall be followed.

The time elapsing between the start of the preparation of the initial suspension and the moment when the plates are inoculated shall not exceed 45 min.

To check sterility, a control pour plate shall be prepared.

Preparation of the initial suspension and further decimal dilutions shall be performed according to prEN 13040‑22.

Aliquots of the initial suspension should only be used for cultivation if no toxic or inhibiting substances are expected in the sample. For blended products with high amounts of minerals, it is recommended to use aliquots from the first dilution (10⁻²) and higher. Liquid materials may be used undiluted if they do not contain any toxic or inhibiting substances.

For enumeration, one plate per dilution shall be used with at least two successive dilutions. To improve the reliability of the results, two plates per dilution are recommended.

If only one dilution is used, then two plates of this dilution shall be used to improve reliability of the results.

For laboratories that do not operate under quality assurance principles, two plates per dilution shall be used to improve reliability of the results.

Using a sterile pipette or a micropipette (6.9), 1 ml of the test sample (if liquid) or 1 ml of the appropriate dilution shall be transferred to a sterile Petri dish (6.8).

Where greater dilutions are made and low counts are expected, the number of inoculated plates for enumeration tests should be increased to ensure that an inoculation volume corresponding to at least 0,1 g of the sample is analysed. If the colony counts are expected to be close to a given limit value, it is recommended to increase the accuracy of the method by adding 10 ml distributed into one pour-plated Petri dish with a diameter of 140 mm, or at least three pour-plated Petri dishes with a diameter of 90 mm.

Approximately 15 ml (for 90 mm Petri dishes) or 45 ml (for 140 mm Petri dishes) of TBX agar (B.2), cooled down to 44 °C to 47 °C in a water bath (6.3) or an incubator (6.2), shall be poured into each Petri dish containing the respective volume of the test sample or an appropriate dilution.

The inoculum shall be carefully mixed with the medium and the mixture shall be allowed to solidify, with the Petri dishes standing on a cool horizontal surface.

The time which elapses between the inoculation of the empty Petri dish and pouring of the medium shall not exceed 15 min.

The inoculated plates shall be incubated upside down in an incubator (6.2) set at 44 °C for 21 h ± 3 h.

After incubation, all plates showing less than 150 typical colonies and less than 300 total (typical and atypical) colonies shall be used for the enumeration. Atypical colonies shall not be considered for the enumeration.

When embedded in TBX agar, typical E. coli colonies have a diameter greater than 1 mm and appear dark to light blue as a result of the β-D-glucuronidase reaction.

On the surface of the TBX agar, typical colonies appear blue in the centre with a white edge.

Confirmation is required in case of doubt on whether E. coli colonies are present on the primary agar medium (TBX) [5, 6].

Confirmation is recommended if the calculated result will be above a given limit value (for example the limit value specified in Regulation (EU) 2019/1009 [4]).

Any other method capable of unambiguously identifying E. coli than the one described in this document [e.g. commercially available biochemical test kits, serological tests, polymerase chain reaction (PCR) analyses or Matrix-Assisted Laser Desorption Ionization - Time-of-Flight Mass Spectrometry (MALDI-TOF)] may be used for confirmation if giving the same results as the method specified in this document.

If typical β-D-glucuronidase-positive E. coli colonies have been confirmed, that shall be stated in the results.

If it is not possible to isolate a single colony due to high background flora, a pure culture shall be established by subculturing. With a sterile loop (6.6) streak plates from up to 5 typical colonies from each countable plate (9.4) shall be prepared on poured TBX agar (B.2) or MacConkey agar No. Three (B.3), yielding single colonies after incubation.

Any other medium giving a clear distinction between E. coli and other bacteria may be used.

TBX agar (B.2) shall be incubated at 44 °C for 21 h ± 3 h, or MacConkey agar No. Three (B.3) at 36 °C (6.2) for 21 h ± 3 h. On TBX agar, typical colonies show a blue centre with a white edge. On MacConkey agar No. 3, non-lactose fermenters form colourless colonies, coliform bacteria appear as intense violet-red colonies and typical E. coli colonies additionally show bile precipitation. Typical colonies may be subjected to a subsequent indole reaction (9.5.3).

The indole reaction, indole test or tryptophan test can be used to differentiate Salmonella and Enterobacter (indole-negative) from E. coli (indole-positive) [5, 6].

At least 5 typical single colonies shall be picked from each countable plate (9.4), or pure typical single colonies shall be picked from each isolation (9.5.2) with a sterile loop (6.6). For each suspected colony, 3 ml to 5 ml of tryptone/tryptophan medium (B.4) shall be inoculated. If less than 5 typical colonies are present, all colonies shall be tested.

The inoculated tryptone/tryptophan medium shall be incubated at 36 °C (6.2) for 21 h ± 3 h. After incubation, 1 ml of the Kovacs reagent (B.5) shall be added.

The formation of a red ring indicates a positive reaction. A yellow-brown ring indicates a negative reaction.

Commercially available kits for the detection of the indole reaction can be used if they show equivalent results to the method specified in this document.

The number of colony forming units (CFU) of E. coli per gram or per ml of test portion (N) shall be calculated using Formula (1). In special cases (e.g. no colonies detected, more than 150 typical colonies, etc.) results shall be expressed according to the instructions stated in EN ISO 7218.

(1)

where

When confirmation is necessary, a given number A (generally 5 per plate) of β-D-glucuronidase-positive E. coli colonies shall be confirmed from each of the dishes retained for counting.

After confirmation, the number of colonies (a) complying with identification criteria shall be calculated for each of the dishes using Formula 2.

a = × 𝐶 (2)

where

And replace ΣC by Σa in Formula (1) to account for the ratio of verified colonies.

NOTE 1 Σa is the total number of colonies complying with identification criteria from all of the dishes selected.

NOTE 2 If Σa is < 10, the final result is expressed as an estimated value.

This standardized reference method was validated in accordance with ISO 5725‑5 [6]. All relevant data were reported in an interlaboratory study report [7].

The performance characteristics of the method (reproducibility, repeatability) were determined in an interlaboratory study. The method has been validated for four different matrices, as listed in Table C.1. All data are given in Annex C.

The test report shall specify at least the following information:

— the test method used, with reference to this document, i.e. prEN 18280:2025;

— the sampling method used (EN 12579) with an indication of all deviations from the sampling method, if known;

— the size of the test portion and/or the nature of the objects examined;

— all operating conditions not specified in this document, or regarded as optional or informative (including informative annexes), together with details of any incidents which can have influenced the test result(s);

— any deviations from this document, including all operating conditions not specified in this document, or regarded as optional, together with details of any incidents which may have influenced the result(s);

— all information necessary for the complete identification of the sample;

— the test result(s) obtained in 1 g or 1 ml of product;

— if repeatability has been checked, the final result obtained;

— the date(s) of the start and end of the test.

The laboratory should have a quality control system to ensure that the equipment, reagents and techniques are suitable for the method. The use of positive controls, negative controls and blanks are part of the method. Performance testing of culture media is described in EN ISO 11133.

1. 
   (normative)

Diagram of the procedure

Figure A.1 — Flow diagram of the method for the enumeration of Escherichia coli in soil improvers and growing media

1. 
   (normative)
   
   Composition and preparation of culture media and reagents
   1. General

The general specifications of EN ISO 11133 are applicable to the preparation and performance testing of the culture media described in this Annex. If culture media or reagents are prepared from dehydrated complete media/reagents or if ready-to-use media/reagents are used, follow the manufacturer’s instructions regarding preparation, storage conditions, expiry date and use. The shelf life of the media indicated in this Annex has been determined in some studies. The user shall verify the performance criteria of the media and/or reagents under their own storage conditions if it differs from the manufacturer’s instructions.

• 1. Tryptone-bile-X-glucuronide (TBX) agar
     1. Composition

• • 1. Preparation

Dissolve the BCIG in the dimethyl sulfoxide or in the diluent recommended by the manufacturer. Dissolve all components in water and heat to boiling.

If necessary, adjust the pH (6.5) to 7,2 ± 0,2 at 25 °C after sterilization.

Sterilize the medium in the autoclave (6.1) set at 121 °C ± 3 °C for 15 min. Immediately cool the medium in the water bath (6.3) at 44 °C to 47 °C up to the use in the pour plate method.

For the production of plates for the confirmation reaction, cool the medium to 44 °C to 47 °C in a water bath (6.3), mix and pour into sterile Petri dishes (6.8). Allow to solidify.

Immediately before use, dry the agar plates carefully (preferably with the lids off and the agar surface downwards) until the surface of the agar is dry.

Store the poured plates protected from drying at 5 °C (6.4) for up to 4 weeks.

• 1. MacConkey Agar No. 3 (optional)
     1. Composition

• • 1. Preparation

Dissolve all components in water and heat to boiling.

If necessary, adjust the pH (6.5) to 7,1 ± 0,2 at 25 °C after sterilization.

Sterilize the medium in the autoclave (6.1) set at 121 °C ± 3 °C for 15 min. Immediately cool the medium in the water bath (6.3) at 44 °C to 47 °C, mix and pour into sterile Petri dishes (6.8). Allow to solidify.

Immediately before use, dry the agar plates carefully (preferably with the lids off and the agar surface downwards) until the surface of the agar is dry.

Store the poured plates protected from drying at 5 °C (6.4) for up to 4 weeks.

• 1. Tryptone/tryptophan medium (optional)
     1. Composition

• • 1. Preparation

Dissolve the components in water and heat to boiling whilst stirring continuously to dissolve all ingredients completely.

If necessary, adjust the pH (6.5) to 7,5 ± 0,2 at 25 °C after sterilization.

Dispense 3 ml to 5 ml of the medium into each of several tubes (6.7).

Sterilize for 15 min in the autoclave (6.1) set at 121 °C ± 3 °C.

Store the sterilized medium at 5 °C (6.4) for up to 3 months.

• 1. Kovacs reagent (optional)
     1. Composition

• • 1. Preparation

Mix the components.

Store the complete reagent, in closed flasks in the dark, at 5 °C (6.4) for up to 6 months.

1. 
   (informative)
   
   Performance characteristics of the method
   1. Interlaboratory study

An interlaboratory study involving 11 laboratories in six countries was carried out. The following matrices were included in the study, ensuring representation of soil improvers and growing media available on the market at the time of the interlaboratory study: peat, biowaste compost, green compost, solid digestate (Table C.1). The study was organized in 2025 by BIPEA as part of Standardization Request M/564 (including amendments) aimed at providing European standardization deliverables in support of the European Fertilising Product Regulation (FPR; EU 2019/1009).

Table C.1 — Samples selected for the inter-laboratory study

• 1. Statistical results for the enumeration of E. coli

The values of the performance characteristics for each type of soil improver and/or growing media, derived from this interlaboratory study are shown in Table C.2, and were calculated in accordance with ISO 5725‑5 [6] and ISO 5725‑6 [8] with the application of the algorithms A and S detailed in ISO 13528 [9].

Table C.2 — Results of data analysis on enumeration of E. coli obtained from this interlaboratory study

Across matrices and pathogen levels, the repeatability standard deviation was estimated to be 0,067 log10 (CFU/g) and the reproducibility standard deviation was estimated to be 0,213 log10 (CFU/g).

The absolute difference between two independent single test results, obtained using the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time, will in no more than 5 % of the cases be greater than 0,188 log10 (CFU/g) across matrices and pathogen levels, or the repeatability limit r for the matrices and pathogen levels specified in Table C.2.

The absolute difference between two independent single test results, obtained using the same method on identical test material in different laboratories with different operators using different equipment, will in no more than 5 % of the cases be greater than 0,596 log10 (CFU/g) across matrices and pathogen levels, or the reproducibility limit R for the matrices and pathogen levels specified in Table C.2.

Bibliography

[1] EN ISO 11133, Microbiology of food, animal feed and water - Preparation, production, storage and performance testing of culture media (ISO 11133)

[2] ISO 16649‑2, Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli — Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide

[3] ISO 16140‑2, Microbiology of the food chain — Method validation — Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method

[4] Kilian M., Bulow P. Rapid diagnosis of Enterobacteriaceae. Detection of bacterial glycosidases. Acta Pathol Microbiol. Scand. Sect. B. 1976, 84 pp. 245–251

[5] Le Minor L., Buissière J., Novel G., Novel M. Relation entre le sérotype et l’activité β-glucuronidasique chez les Salmonella. Ann. Microbiol. (Paris). 1978 Aug–Sep, 129B (2) pp. 155–165

[6] Statistical ILS Report for WI 00223114 (2025), available as doc. N 1924 on the CEN Documents platform of CEN/TC 223 ‘Soil improvers and growing media’ (access can be obtained via national standardization bodies)

[7] ISO 5725‑6:1994, Accuracy (trueness and precision) of measurement methods and results – Part 6: Use in practice of accuracy values.

[8] ISO 13528:2022, Statistical methods for use in proficiency testing by interlaboratory comparison

1. Under preparation. Stage at time of publication: prEN 13040-2:2025. ↑

2. Under preparation. Stage at time of publication: prEN 17732:2025. ↑