prEN 18278
(Main)Soil improvers and growing media - Detection of Salmonella spp.
Soil improvers and growing media - Detection of Salmonella spp.
This document specifies a method for the detection of Salmonella spp. in soil improvers, soil improver blends, growing media and growing media blends. This document applies to material in solid form (including pre-shaped growing media) and liquid form.
This document specifies a technique for the detection of Salmonella spp. on two solid media with different enzymatic reactions, and it specifies a technique for confirmation of Salmonella spp. when presumptive colonies are detected.
NOTE: This method has been validated in an interlaboratory study with specific products that were present on the market during the study (Annex C). Products can change over time regarding their microbial composition and other characteristics, and the formulation of the products can also change in the future
Bodenverbesserungsmittel und Kultursubstrate - Bestimmung von Salmonellen spp.
Amendements du sol et supports de culture - Détection des salmonella
Izboljševalci tal in rastni substrati - Ugotavljanje prisotnosti salmonele (Salmonella spp.)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-januar-2026
Izboljševalci tal in rastni substrati - Ugotavljanje prisotnosti salmonele (Salmonella
spp.)
Soil improvers and growing media - Detection of Salmonella spp.
Bodenverbesserungsmittel und Kultursubstrate - Bestimmung von Salmonellen spp.
Amendements du sol et supports de culture - Détection des salmonella
Ta slovenski standard je istoveten z: prEN 18278
ICS:
65.080 Gnojila Fertilizers
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2026
ICS 65.080
English Version
Soil improvers and growing media - Detection of
Salmonella spp.
Amendements du sol et supports de culture - Détection Bodenverbesserungsmittel und Kultursubstrate -
des salmonella Bestimmung von Salmonellen spp.
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 223.
If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.
This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.
Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 18278:2026 E
worldwide for CEN national Members.
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 6
4 Principle . 7
5 Culture media and reagents . 7
6 Equipment and consumables . 8
7 Sampling . 8
8 Preparation of the test sample . 8
9 Procedure . 8
9.1 General. 8
9.2 Preparation of the initial suspension . 8
9.3 Non-selective pre-enrichment . 9
9.4 Selective enrichment . 9
9.5 Inoculation and incubation on two selective media . 9
9.6 Confirmation . 9
9.6.1 General. 9
9.6.2 Selection of colonies for confirmation . 10
9.6.3 Biochemical testing . 10
9.6.4 Serological testing . 12
9.6.5 Serotyping . 13
10 Expression of results . 13
11 Validation of the method . 13
11.1 Validation in partial accordance with EN ISO 16140-2 . 13
11.2 Performance characteristics. 13
11.2.1 Sensitivity . 13
11.2.2 Specificity . 13
11.2.3 LOD . 13
12 Test report . 14
13 Quality assurance . 14
Annex A (normative) Flow diagram of the procedure . 15
Annex B (normative) Composition and preparation of culture media and reagents . 16
Annex C (informative) Performance characteristics of the method . 24
Annex D (informative) Examples of selective media with different biochemical reactions
characteristic for Salmonella spp . 27
Annex E (informative) Additional examples and manufacturers of selective media with two
different biochemical reactions characteristic for Salmonella spp . 30
Bibliography . 31
European foreword
This document (prEN 18278:2026) 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 detect Salmonella spp. in soil improvers and growing media in order
to be able to control certain imposed hygienic requirements. The method described in this document is based
on EN ISO 6579-1.
Salmonella spp. are rod-shaped Gram-negative bacteria of faecal/intestinal origin within the family
Enterobacteriaceae. The presence of Salmonella spp. in soil improvers and growing media constitutes a
hazard of infection to those handling these materials. Furthermore, 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 Salmonella spp. 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
detection of Salmonella spp. 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”
1 Scope
This document specifies a method for the detection of Salmonella spp. in soil improvers and growing media.
It 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.
Most of the Salmonella serovars are detected with the method described in this document. For the detection
of some specific Salmonella serovars (e.g. Salmonella Typhi and Salmonella Paratyphi), additional cultivation
steps can be necessary.
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).
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).
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.
EN 12579, Soil improvers and growing media - Sampling
prEN 13040-2, Soil improvers and growing media — Part 2: Sample preparation for microbiological
examination
prEN 17732, Soil improvers and growing media — Terminology
EN ISO 7218, Microbiology of the food chain - General requirements and guidance for microbiological
examinations (ISO 7218)
3 Terms and definitions
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/
Under preparation. Stage at time of publication: prEN 13040-2:2025.
Under preparation. Stage at time of publication: prEN 17732:2025.
3.1
Salmonella spp
bacteria which form typical colonies on the solid selective media described in this document and which
display the morphological, physiological and biochemical characteristics described when the analysis is
carried out in accordance with this document
3.2
detection of Salmonella spp
analysis for the presence or absence of Salmonella spp. (3.1) in a particular mass or volume of product when
tests are carried out in accordance with this document
4 Principle
The detection of Salmonella spp. requires four successive steps as specified in Annex A. The four steps are:
— non-selective pre-enrichment;
— selective enrichment;
— inoculation and incubation on two selective media;
— confirmation of presumptive colonies with a serological or biochemical test.
The initial suspension on buffered peptone water (BPW) is incubated at 36 °C for 22 h for a non-selective
pre-enrichment. From the non-selective pre-enrichment obtained, Rappaport-Vassiliadis Soya (RVS) broth
is inoculated and incubated at 41,5°C for 24 h. From the selective enrichment obtained, two different
selective solid media based on different enzymatic reactions are inoculated and incubated at 36 °C for 24 h
(or according to the manufacturer’s instructions if explicitly recommended). After incubation, and depending
on the media chosen, it is determined if presumptive Salmonella spp. are present on the plates. Colonies of
presumptive Salmonella spp. are subcultured on a non-selective agar (if needed). The identity of presumptive
Salmonella spp. colonies is confirmed by means of appropriate serological or biochemical tests.
NOTE 1 Salmonella spp. can be present in small numbers and are often accompanied by considerably larger numbers
of other bacteria of the same family (Enterobacteriaceae) or of other families. Enrichment is used to allow the detection
of low numbers of Salmonella spp. or stressed Salmonella spp.
NOTE 2 Stressed microorganisms are defined here as those present in the environment that can be injured or that
can have developed in harsh environments. Such microorganisms can be difficult to detect because they struggle to
grow on selective media. However, under suitable conditions, they can repair the cellular damages and recover their
normal properties.
Validated alternative methods to detect Salmonella spp. based on molecular biology may be used if giving the
same results as those given in this document (or e.g. comparable to EN ISO 7218) and if they are validated
according to ISO 16140.
5 Culture media and reagents
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.
6 Equipment and consumables
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 Drying cabinet or oven, capable of operating between 25 °C and 50 °C.
6.3 Incubator(s), capable of operating in the ranges 36 °C ± 2 °C and 41,5 °C ± 1 °C.
6.4 Water bath, capable of operating at 36 °C ± 2 °C or at 47 °C to 50 °C.
6.5 Cooling unit, adjustable at 5 °C ± 3 °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 pH-meter, with a reading to the nearest 0,1 pH unit at 20 °C to 25 °C.
6.8 Sterile tubes, bottles or flasks with caps, of appropriate capacity.
6.9 Sterile filter, with a pore size of 0,22 μm.
6.10 Sterile Petri dishes with a diameter of approximately 90 mm or a diameter of approximately 140 mm
(optional).
7 Sampling
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.
8 Preparation of the test sample
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.
9 Procedure
9.1 General
The procedure as given in Annex A shall be followed.
9.2 Preparation of the initial suspension
The initial suspension shall be prepared according to prEN 13040-2 , based on a test portion of 25 g or 25 ml
adding buffered peptone water (BPW) as diluent to yield a minimum ten-fold dilution (mass or volume).
9.3 Non-selective pre-enrichment
The initial suspension (9.2) shall be incubated at 36 °C (6.3) for 22 h ± 2 h. The pre-enrichment culture may
be stored at 5 °C (6.5) for 72 h maximum after the incubation.
9.4 Selective enrichment
After the incubation, the RVS broth (B.2) shall be inoculated with 0,1 ml of the pre-enriched initial suspension
(9.3). If the RVS broth has been stored at 5 °C (6.5), it shall be brought to room temperature before
inoculation. It is recommended to minimize the transfer of particulate material from the pre-enrichment into
the selective enrichment medium.
The inoculated RVS broth shall be incubated at 41,5°C (6.3) for 24 h ± 3 h. The selective enrichment may be
stored at 5 °C (6.5) for 72 h maximum after the incubation.
9.5 Inoculation and incubation on two selective media
Two selective media based on different biochemical reactions characteristic for Salmonella spp. (Annex D)
shall be chosen by the testing laboratory. From the selective enrichment (9.4), the surface of two selective
media shall be inoculated by means of a 10 μl loop (6.6) or comparable, so that well-isolated colonies are
obtained after incubation.
The plates with the two selective media shall be allowed to reach room temperature if they were stored at
5 °C (6.5). If necessary, dry the surface of the plates before use.
To obtain well-isolated colonies, large-size Petri dishes (with a diameter of approximately 140 mm) or two
normal-size plates (with a diameter of approximately 90 mm) may be used (6.10).
The plates with the two selective media shall be incubated at 36 °C (6.3) for 24 h ± 3 h, or according to the
manufacturer’s instructions if explicitly recommended.
After incubation, both selective media shall be checked for the presence of colonies which, from their
characteristics, are considered to be presumptive Salmonella spp.
9.6 Confirmation
9.6.1 General
Biochemical or serological testing may be used to determine whether a presumptive colony belongs to the
genus Salmonella or not.
For serological testing, it is recommended to subculture this colony on a non-selective agar (B.4) at
36 °C (6.3) for 24 h ± 3 h to get a pure culture with enough material to work with. For biochemical testing, it
is recommended to use a selective medium (Annex D) for subculturing and to incubate it according to the
manufacturer’s instructions, to ensure that there is a pure culture with enough material to work with.
Any other method capable of unambiguously identifying Salmonella spp. than the one described in this
document [e.g. polymerase chain reaction (PCR) analyses or Matrix-Assisted Laser Desorption Ionization -
Time-of-Flight Mass Spectrometry (MALDI-TOF)] may be used for confirmation of Salmonella spp. if giving
the same results as the method specified in this document.
In cases where identification of Salmonella spp. is unclear, after choosing either biochemical testing (9.6.3)
or serological testing (9.6.4), the testing option that had not been chosen initially shall be tried. After that, if
the identification of Salmonella spp. is still unclear, other tests shall be carried out (e.g. PCR).
For the characterization of Salmonella spp. strains (optional), full serotyping is needed. Guidance for
serotyping is given in e.g. CEN ISO/TR 6579-3.
9.6.2 Selection of colonies for confirmation
Mark presumptive colonies on each plate (9.5). If well-isolated colonies are available on the selective
media (9.5), the biochemical or serological confirmation may be performed directly on a presumptive colony.
It is recommended to subculture this colony.
Select one presumptive colony for confirmation. If the result is negative, select a combination of up to four
presumptive colonies from both selective media (testing up to five colonies in total).
9.6.3 Biochemical testing
9.6.3.1 General
Inoculate the biochemical confirmation media with each of the cultures obtained from the colonies selected
in 9.6.2. For biochemical confirmation of Salmonella spp., at least the tests specified in 9.6.3.2 to 9.6.3.4 shall
be performed. The tests specified in 9.6.3.5 and 9.6.3.6 may also be performed when the results of the other
confirmation tests do not give a clear identification. The use of biochemical strips (9.6.3.7) replaces the tests
specified in 9.6.3.2 to 9.6.3.4.
9.6.3.2 Triple sugar/iron (TSI) agar
Colony material is spread on the slanted surface of the TSI agar (B.5) using an inoculation loop (6.6). The
agar butt is then inoculated with the same colony material by stabbing it to the bottom with a needle (6.6).
Incubate at 36 °C (6.3) for 24 h ± 3 h.
Interpret the changes in the medium as follows:
a) butt
— yellow: glucose positive (glucose fermentation);
— red or unchanged: glucose negative (no fermentation of glucose);
— black: formation of hydrogen sulphide;
— bubbles or cracks: gas formation from glucose;
b) slant surface
— yellow: lactose and/or sucrose positive (lactose and/or sucrose fermentation);
— red or unchanged: lactose and sucrose negative (no fermentation of lactose or sucrose).
The majority of the typical Salmonella cultures show alkaline (red) slants and acid (yellow) butts with gas
formation (bubbles) and (in about 90 % of the cases) formation of hydrogen sulfide (blackening of the agar).
When a lactose-positive Salmonella is isolated, the TSI slant is yellow. Thus, preliminary confirmation of
Salmonella cultures shall not be based on the results of the TSI agar test only (see 9.6.3.1).
NOTE Kligler-Hajna medium gives similar results as TSI agar.
9.6.3.3 Urea agar
Streak the urea agar (B.6) slant surface. Incubate at 36 °C (6.3) for up 24 h ± 3 h.
If the reaction is positive, urea is hydrolysed, liberating ammonia. This changes the colour of phenol red to
rose-pink and later to deep cerise. The reaction is often apparent after 2 h to 4 h.
Typical Salmonella cultures do not hydrolyse urea, so that the colour of the urea agar will remain unchanged.
9.6.3.4 L-Lysine decarboxylation medium (LDC)
Inoculate just below the surface of the liquid LDC medium (B.7). Incubate at 36 °C (6.3) for 24 h ± 3 h.
Turbidity and a purple colour after incubation indicate a positive reaction. A yellow colour indicates a
negative reaction.
The majority of the typical Salmonella cultures show a positive reaction in LDC.
9.6.3.5 Detection of β-galactosidase (optional)
The β-galactosidase test distinguishes Salmonella enterica subspecies arizonae and diarizonae and other
members of the Enterobacteriaceae (all give a positive reaction) from other subspecies of Salmonella enterica
(in general these give a negative reaction).
Several procedures to perform the β-galactosidase test exist. An example is given below.
Suspend a loopful of the suspected colony in a tube containing 0,25 ml of the saline solution (B.10).
Add one drop of toluene and shake the tube. Place the tube in a water bath set at 36 °C (6.4) and leave for
several minutes (approximately 5 min). Add 0,25 ml of the reagent for detection of β-galactosidase (B.8) and
mix.
Incubate the tube at 36 °C (6.3 or 6.4) and leave for up to 24 h.
A yellow colour indicates a positive reaction. The reaction is often apparent after 20 min.
If prepared paper discs are used for the detection of β-galactosidase, follow the manufacturer’s instructions.
9.6.3.6 Medium for indole reaction (optional)
The indole test may be used when there is a need to differentiate Salmonella (generally indole negative, see
Table 1) from Escherichia coli and Citrobacter (both indole positive), as these organisms can give typical
reactions on some of the Salmonella isolation media.
Inoculate a tube containing 5 ml of the tryptone/tryptophan medium (B.9.1) with the suspected colony.
Incubate at 36 °C (6.3) for 24 h ± 3 h. After incubation, add 1 ml of the Kovacs reagent (B.9.2).
The formation of a red ring (surface layer) indicates a positive reaction. A yellow-brown ring (surface layer)
indicates a negative reaction.
9.6.3.7 Biochemical s
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