prEN 13040-2

SLOVENSKI STANDARD
01-julij-2024
Izboljševalci tal in rastni substrati - Priprava vzorcev - 2. del: Priprava vzorcev za
mikrobiološke preskuse
Soil improvers and growing media - Sample preparation - Part 2: Sample preparation for
microbiological examination
Bodenverbesserungsmittel und Kultursubstrate - Probenvorbereitung - Teil 2:
Probenvorbereitung für mikrobiologische Untersuchungen
Amendements du sol et supports de culture - Préparation des échantillons - Partie 2 :
Préparation des échantillons pour l’examen microbiologique
Ta slovenski standard je istoveten z: prEN 13040-2
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
June 2024
ICS 65.080
English Version
Soil improvers and growing media - Sample preparation -
Part 2: Sample preparation for microbiological
examination
Bodenverbesserungsmittel und Kultursubstrate -
Probenvorbereitung - Teil 2: Probenvorbereitung für
mikrobiologische Untersuchungen
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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13040-2:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 6
5 Reagents . 6
6 Apparatus and equipment . 7
7 Transportation and storage of samples . 7
8 Procedure . 8
9 Further dilutions . 9
Annex A (normative) Culture media and reagents . 11
A.1 General. 11
A.2 Phosphate buffer . 11
A.3 Double buffered phosphate buffer . 12
Annex B (informative) Examples of product types with their proposed corresponding procedural
steps for sample preparation . 13
Bibliography . 18

European foreword
This document (prEN 13040-2:2024) 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 the CEN Enquiry.
This document has been prepared under a standardization request addressed to CEN by the European
Commission.
Introduction
This document describes the general requirements for the preparation of samples and initial suspensions
prior to microbiological examination of soil improvers and growing media.
Any special diluents or practices required for particular materials or microorganisms in specific standard
methods take priority over the general requirements listed in this document.
This document is one of the series of standards as listed below:
— prEN 13040-1, Soil improvers and growing media — Sample preparation — Part 1:
Sample preparation for chemical and physical tests, determination of dry matter content, moisture
content and laboratory bulk density;
— prEN 13040-2, Soil improvers and growing media — Sample preparation — Part 2:
Sample preparation for microbiological examination.
WARNING — In order to safeguard the health of laboratory personnel, it is essential that the procedures
described in this document are only undertaken in properly equipped laboratories, under the supervision
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 the general requirements for the preparation of samples and initial suspensions
prior to microbiological examinations of soil improvers and growing media. This method is intended
especially for sample preparation prior to microbiological examinations of e.g. E. coli, Salmonella spp. and
Enterococcaceae.
Because of the large variety of soil improvers and growing media, this method might not be appropriate
in every detail for certain materials. This method might not be appropriate in every detail for certain
products. In this case, different methods which are specific to these products can be used if necessary, for
justified technical reasons.
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
CEN/TS 17732, Soil improvers and growing media — Terminology
ISO 7218, Microbiology of the food chain — General requirements and guidance for microbiological
examinations
EN ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media (ISO 11133)
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 12579, CEN/TS 17732 and the
following apply.
3.1
laboratory sample
in relation to chemical and physical testing, a final sample intended for laboratory testing and in relation
to microbiological testing, each separate segment sample intended for laboratory testing
3.2
test portion
quantity of material drawn from the test sample (or from the laboratory sample if both are the same) and
on which the tests and observations are actually carried out
Note 1 to entry: Sometimes a preparation of the laboratory sample (3.1) s required before the test portion is taken
out, but this is uncommon in microbiological examinations.
3.3
test sample
sample prepared from the laboratory sample (3.1) and from which test portions (3.2) will be taken
Note 1 to entry: Preparation of the laboratory sample (3.1) before taking out the test portion (3.2) is rarely used in
microbiological examinations.
In preparation.
3.4
initial suspension
primary dilution obtained after the test portion (3.2) has been treated with, normally, a nine-fold quantity
of diluent
Note 1 to entry: A closer ratio between the diluent and the quantity of material is often not recommended because
of possible inhibiting influences of the matrix.
3.5
further dilution
suspension or solution obtained by mixing a measured volume of the initial suspension (3.4) with an x-
fold volume of diluent and by repeating this operation with further dilutions until a dilution series is
obtained that is suitable for the inoculation of culture media
Note 1 to entry: Normally ten-fold dilutions are used to yield a decimal dilution series, but other ratios may be
required for specific purposes.
4 Principle
Sample preparation prior to microbiologal examination consists of two consecutive steps, for which the
second step depends on the intent of the subsequent microbiological examination, meaning enumeration
or detection.
1) Take the test portion (3.2) out of the laboratory sample (3.1):
2) Preparation:
a) For enumeration
Preparation of an initial suspension in a way to obtain an as uniform as possible distribution of
microorganisms contained in the test portion. Preparation, if necessary, of further dilutions in order
to reduce the number of microorganisms per unit volume to allow counting of colonies after
incubation.
b) For detection
Preparation of a first enrichment in a way to obtain an as uniform as possible distribution of
microorganisms contained in the test portion.
To restrict the enumeration range to a given optimal interval, or if high numbers of microorganisms are
expected, only those (decimal) dilutions that are necessary may be inoculated to perform the
enumeration comparable to calculation in e.g. ISO 7218 .
5 Reagents
Follow current laboratory practices in accordance with standards comparable to ISO 7218 . The
composition of culture media and reagents and their preparation are specified in Annex A. For uniformity
of results, in the preparation of media use either dehydrated complete medium or use constituents of
uniform quality and chemicals of recognized analytical grade. For performance testing of culture media,
follow the procedures in accordance with standards comparable to EN ISO 11133 and Annex A.
6 Apparatus and equipment
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) or wet sterilization (autoclave).
6.2 Equipment for size reduction; apparatuses (aseptic) may include:
— scissors;
— tweezers;
— straight scalpels;
— knives;
— hammers (used with a sterile bag around the sample);
— saws;
— spatulas;
— mortar and pestle.
6.3 Homogenization devices; apparatuses (aseptic) may include:
— a magnetic stirrer;
— orbital shaker, with bottles or plastic bags (glass beads may be used in the case of viscous or thick
materials);
— peristaltic blender (paddle blender), with sterile bags, preferable with a filter;
— vortex mixer (for dilutions).
6.4 Other laboratory equipment may include:
— pipettes or pipettor and sterile tips, to dispense 0,1 ml to 1 ml with tips (with large opening if
necessary);
— scales and gravimetric dilution controllers, capable of weighing to 1 % of the mass;
— sterile bowls, wide-mouth containers or plastic bags.
7 Transportation and storage of samples
The laboratory sample shall be transported and stored without compaction or any other treatment which
may irreversibly alter its moisture content, particle size, packing characteristics or any feature which
affects density.
The laboratory samples, as submitted to the laboratory, shall be stored so that they shall not undergo any
further decomposition, physical damage, hydration or dehydration. Recommended storage should be at
5 °C ± 3 °C, but not frozen.
oSIST prEN 13040-2:2
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