SIST-TP CEN/TR 15214-3:2006

• Abstract:

  This part of the CEN Technical Report specifies a most probable number (MPN) method for the quantitative detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It allows further differentiation within the test than part 2 of this standard. It is suitable to evaluate the log reduction of E.coli through treatment as well as the quality of the end product.; The method is for material with dry residues of more than 10 %.; For materials with dry residues less than 10 %, the procedure given in CEN/TR 15214-1 should be used.

• Status: Published
• Publication Date: 30-Jun-2006
• ICS: 13.030.20 - Liquid wastes. Sludge
• ICS: 13.080.30 - Biological properties of soils
• ICS: 65.080 - Fertilizers
• Technical Committee: KAT - Soil quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Jul-2006
• Due Date: 01-Jul-2006
• Completion Date: 01-Jul-2006
• Directive: 2013-01-3557 - Uredba o predelavi biološko razgradljivih odpadkov in uporabi komposta ali digestata
• Ref Project: CEN/TR 15214-3:2006 - Characterization of sludges - Detection and enumeration of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes - Part 3: Macromethod (Most Probable Number) in liquid medium

• Refers: SIST EN 12880:2001 - Characterization of sludges - Determination of dry residue and water content
• Effective Date: 28-Jan-2026

SIST-TP CEN/TR 15214-3:2006 is a CEN Technical Report on detecting and enumerating Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It specifies a Most Probable Number (MPN) macromethod in liquid medium for materials with more than 10 % dry residue, so laboratories can assess faecal contamination, treatment log reduction and end-product quality.

What does SIST-TP CEN/TR 15214-3:2006 specify?

SIST-TP CEN/TR 15214-3:2006 specifies a quantitative MPN method for E. coli in sludge-related and organic soil materials. The method is intended for samples with dry residues above 10 %; for lower dry residues, CEN/TR 15214-1 is used instead.

The report is organized into the usual technical-report structure: scope, normative references, terms and definitions, principle, apparatus, sampling and hazards, reagents, procedure, expression of results, test report and performance data. Annex A gives method performance information, and the bibliography lists supporting references.

Annex	What it covers
Annex A (informative)	Performance of the method from comparative validation

What are the key requirements of SIST-TP CEN/TR 15214-3:2006?

SIST-TP CEN/TR 15214-3:2006 is built around a serial dilution MPN workflow, but the practical value is in how the sample is prepared, incubated and interpreted.

Sample preparation and applicability

Clause 1 limits the method to materials with more than 10 % dry residue. Clause 8.1 requires 20 g of wet sample in 180 ml of sterile 0.9 % NaCl, then shaking at a minimum of 150 rpm for 30 min to 20 h at 5 ± 3 °C. In practice, that means the lab must choose a mixing time that matches the sample type, with longer shaking for inhomogeneous materials.

Detection principle

Clause 4 uses dilution in 0.9 % NaCl, transfer of 1 ml portions into three tubes of MUG lauryl sulfate broth, incubation at 44 ± 1 °C for 40 ± 4 h, then reading gas, indole and fluorescence. This matters because the result depends on combining three reaction signals rather than counting colonies on a plate.

Safety and handling

Clause 6 treats these samples as potentially hazardous and gas-forming, with risks from pathogens, flammable gases and bursting containers. The report calls for gloves, face and eye protection, body protection and flame-proof equipment nearby. In practice, the method is not only a microbiology procedure - it also requires controlled handling and storage at 5 ± 3 °C.

Reagents and equipment

Clause 5 and Clause 7 require sterilized glassware, incubators at 36 ± 2 °C and 44 ± 1 °C, autoclaving, UV observation at 366 nm, MUG lauryl sulfate broth, sterile 0.9 % NaCl and Kovacs reagent. The media are prepared from suitable-grade chemicals or equivalent dehydrated products, which matters for reproducibility between laboratories.

Result calculation

Clause 8.3 requires dry residue to be determined in parallel with the microbiological analysis using EN 12880. Clause 9 then expresses the count as MPN per ml of primary suspension, per gram wet weight, and, where needed, per dry weight. This is important for comparing treated materials and reporting results in the form most useful to the buyer or process owner.

Reporting and performance

Clause 10 requires the report to identify the standard used, the sample, any pre-treatment and the results, plus any relevant deviations. Clause 11 points to Annex A for repeatability and reproducibility information, which helps a laboratory judge whether the method fits quality control or validation work.

What terms does SIST-TP CEN/TR 15214-3:2006 define?

• Escherichia coli (E. coli) - A Gram-negative, non-sporulating, rod-shaped bacterium that grows at 44 °C; in this method it must also be β-glucuronidase-positive, produce indole from tryptophan and form gas from lactose.
• Method definition - The operational definition of E. coli used in this report, based on the specific biochemical reactions read in the test medium.
• Dry residue - The dry mass portion of the sample after the specified drying process, expressed as percent or grams per kilogram.
• Most Probable Number (MPN) - A statistical estimate of microbial concentration derived from the pattern of positive tubes across dilution steps.
• MUG - 4-methylumbelliferyl-β-D-glucuronide, the substrate used to detect fluorescence from β-glucuronidase activity.

Who uses SIST-TP CEN/TR 15214-3:2006?

SIST-TP CEN/TR 15214-3:2006 is used by microbiology laboratories, environmental testing labs and sludge treatment operators that need to measure E. coli in sludges, soils, soil improvers, growing media and biowastes. It is relevant for quality managers checking treatment performance, for operators monitoring storage or disinfection steps, and for buyers or regulators who need a documented hygiene result for an end product or land-applied material.

Which standards are used with SIST-TP CEN/TR 15214-3:2006?

• EN 12880:2000 - Provides the dry residue and water content method used when results are needed on a dry-weight basis.
• ISO 8199 - Gives general guidance for enumeration of microorganisms by culture and sterilization of glassware.
• CEN/TR 15214-1 - Supplies the procedure to use when materials have less than 10 % dry residue.
• CEN/TR 15214-2 - Gives the miniaturised semi-quantitative MPN method in the same series.

What does the SIST-TP CEN/TR 15214-3:2006 document contain?

The document gives the full laboratory workflow: sample suspension, serial dilution, inoculation of MUG lauryl sulfate broth, incubation, and reading of gas, fluorescence and indole reactions. It also describes the equipment, reagents and safety precautions needed to run the method consistently.

For result handling, it provides the MPN calculation approach, conversion to wet-weight and dry-weight expressions, and the content required in the test report. Annex A adds validation information from comparative testing on compost and sludge, including statistical treatment of the data for repeatability and reproducibility.

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Characterization of sludges - Detection and enumeration of Escherichia coli in sludges,
soils, soil improvers, growing media and biowastes - Part 3: Macromethod (Most
Probable Number) in liquid medium
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 3: Makroverfahren in Flüssigmedium (MPN-Verfahren)
Caractérisation des boues - Détection et dénombrement des Escherichia coli dans les
boues, les sols, les amendements du sol, les supports de culture et les biodéchets -
Partie 3 : Macrométhode (nombre le plus probable) par ensemencement en milieu
liquide
Ta slovenski standard je istoveten z: CEN/TR 15214-3:2006
ICS:
13.030.20
13.080.30
65.080
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL REPORT
CEN/TR 15214-3
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
January 2006
ICS 07.100.99
English Version
Characterization of sludges - Detection and enumeration of
Escherichia coli in sludges, soils, soil improvers, growing media
and biowastes - Part 3: Macromethod (Most Probable Number)
in liquid medium
Caractérisation des boues - Détection et dénombrement Charakterisierung von Schlämmen - Quantitativer
des Escherichia coli dans les boues, les sols, les engrais, Nachweis von Escherichia coli in Schlämmen, Böden,
les amendements organiques et les biodéchets - Partie 3 : Düngemitteln und Bodenverbesserern, Kultursubstraten
Macrométhode (nombre le plus probable) par sowie Bioabfällen - Teil 3: Makroverfahren in
ensemencement en milieu liquide Flüssigmedium (MPN-Verfahren)
This Technical Report was approved by CEN on 3 September 2005. It has been drawn up by the Technical Committee CEN/TC 308.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TR 15214-3:2006: E
worldwide for CEN national Members.

Contents Page
Foreword .3
Introduction.4
1 Scope .5
2 Normative references .5
3 Terms and definitions.5
4 Principle.5
5 Apparatus .6
6 Sampling and hazards .6
7 Reagents, diluents and culture media.7
8 Procedure .8
9 Expression of the results .9
10 Test report .9
11 Performance data.10

Annex A (informative) Performance of method.11
Bibliography.12

Foreword
This Technical Report (CEN/TR 15214-3:2006) has been prepared by Technical Committee CEN/TC 308
“Characterization of sludges”, the secretariat of which is held by AFNOR.
This Technical Report does not replace any existing CEN method.
The standard is divided into three parts:
Part 1 gives a membrane filtration for quantification
Part 2 gives a miniaturised semi-quantitative MPN method and
Part 3 gives a semi-quantitative macro method
Introduction
Sludges, soils, soil improvers, growing media and biowastes can contain pathogenic micro-organisms such as
Salmonella spp. which occur mainly in the intestinal tract of humans and animals and are transmitted through
faecal contamination. The use of such contaminated materials in agriculture may cause outbreaks of infection
due to the production of contaminated food and animal feedstocks. It may also be transmitted to wild animals.
There is a need to monitor the efficacy of the storage and treatment processes to control pathogens such as
Salmonella spp., and application rates to land.
Escherichia coli is a non-pathogenic, Gram negative bacterium with faecal origin. Consequently, it can be
used as an indicator of faecal contamination. It can also be used to monitor the effectiveness of pasteurization
or disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the
behaviour of all pathogens in these materials. Suitable quality control procedures, at least those described in
ISO 8199, have to be applied.
WARNING — "Waste and sludge samples can contain hazardous and inflammable substances. They
can contain pathogens and be liable to biological action. Consequently, it is recommended that these
samples should be handled with special care. The gases which can be produced by microbiological
activity are potentially inflammable and will pressurise sealed bottles. Exploding bottles are likely to
result in infectious shrapnel and/or pathogenic aerosols. Glass bottles should be avoided wherever
possible. National regulations should be followed with respect to microbiological hazards associated
with this method".
1 Scope
This part of the CEN Technical Report specifies a most probable number (MPN) method for the quantitative
detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It allows further
differentiation within the test than part 2 of this standard. It is suitable to evaluate the log reduction of E.coli
through treatment as well as the quality of the end product.
The method is for material with dry residues of more than 10 %.
For materials with dry residues less than 10 %, the procedure given in CEN/TR 15214-1 should be used.
2 Normative references
The following referenced documents are indispensable for the application 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 12880:2000, Characterisation of Sludges - Determination of Dry Residue and Water Content.
ISO 8199, Water quality – General guide to the enumeration of micro-organisms by culture.
3 Terms and definitions
For the purposes of this Technical Report, the following terms and definitions apply.
3.1
Escherichia coli
Escherichia coli, which belongs to the family of Enterobacteriaceae, are Gram-negative, non-sporulating, rod-
shaped, lactose positive bacteria able to grow at 44 °C. Most E.coli strains are able to produce indole from
tryptophane and are β-glucuronidase-positive
3.2
method definition
for the purpose of the present method, the following E. coli definition shall apply: - β-glucuronidase-positive
and able to hydrolyse 4-methylumbelliferyl-B-D-glucoronide (MUG) when at an incubation temperature of
44 °C in the specified liquid medium. In addition, indole shall be produced from tryptophan and gas produced
from lactose
3.3
dry residue
the dry mass portion of the sample obtained after the specified drying process. It is expressed as percent or in
grams per kilogram
[EN 12880:2000, 3.1].
4 Principle
This method is based on that described to Schindler 1991 (Fluorcult™ or equivalent).
a) Suspension of the sample in 0.9 % m/V sodium chloride;
b) Serial dilutions of this suspension in the same diluent;
c) Transfer 1ml of these diluted suspensions into 3 tubes containing 9 ml MUG fluorocult lauryl sulfate broth;
d) Incubation at (44 ± 1) °C for (40 ± 4) h;
e) Detection of gas production, indole formation and fluorescence;
f) Quantification by the MPN technique.
5 Apparatus
With the exception of equipment supplied sterile, the glassware shall be sterilised in accordance with the
instructions given in ISO 8199.
Usual microbiological laboratory equipment, and in particular:
5.1 Wide-mouth glass flasks or beakers for exampl
...