CEN/TR 15215-3:2006
(Main)Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 3: Presence/absence method by liquid enrichment in peptone-novobiocin medium followed by Rapport-Vassiliadis
Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 3: Presence/absence method by liquid enrichment in peptone-novobiocin medium followed by Rapport-Vassiliadis
This part of the CEN Technical Report specifies a presence/absence procedure to detect Salmonella spp using a four-stage presence/absence method in up to 50g (wet weight) sample.
The method has a limit of detection of approximately 10 cfu/50 g wet weight sludge.
NOTE The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen - Teil 3: Verfahren der Flüssiganreicherung in Peptonwasser mit Novobiocin in Kombination mit Rappaport-Vassiliadis-Medium zum qualitativen Nachweis des Vorkommens
In diesem Dokument wird Verfahren (Nachweisbarkeit oder keine Nachweisbarkeit) zum qualitativen Nachweis von Salmonella spp. festgelegt, unter Anwendung eines vierstufigen Verfahrens für bis zu 50 g Feuchtmasse.
Das Verfahren hat eine Nachweisgrenze von etwa 10 KBE/50 g Feuchtschlammmasse.
ANMERKUNG Der vorgesehene Anwendungsbereich umfasst die Untersuchung von unbehandelten und behandelten Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten und Bioabfall umfasst.
Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les boues, les sols, les amendements du sol, les supports de culture et les biodéchets - Partie 3: Présence/absence par enrichissement en milieu liquide peptone-novobiocine puis sur milieu Rapport-Vassiliadis
La présente partie du Rapport Technique CEN définit une méthode permettant la détection des Salmonella, et ce au moyen d’un mode opératoire par présence/absence en quatre étapes, mené sur un échantillon atteignant 50 g (masse humide).
La méthode a une limite de détection d’environ 10 ufc/50 g de masse de boue humide.
NOTE L’objectif est de couvrir le domaine des boues traitées et non traitées, sols, amendements du sol, supports de culture et biodéchets.
Karakterizacija blata – Ugotavljanje prisotnosti in števila Salmonela sp. v blatu, zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 3. del: Metoda s tekočo obogatitvijo v pepton-novobiocin mediju, ki mu sledi metoda po Vassiliadisu za kvalitativno določevanje prisotnosti
General Information
Overview
CEN/TR 15215-3:2006 defines a presence/absence method for detecting Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes. This CEN Technical Report specifies a four-stage liquid-enrichment procedure using Buffered Peptone Water (peptone‑novobiocin) as primary enrichment followed by Rappaport‑Vassiliadis (RV) selective enrichment. The procedure is designed for samples up to 50 g (wet weight) and has a reported limit of detection of approximately 10 cfu/50 g.
Key topics and technical requirements
Sample size and handling
- Process up to 50 g sample in a 1:10 ratio to primary enrichment (50 g → 450 ml Buffered Peptone Water + 40 mg/l Novobiocin).
- Collect at least 100 g wet weight for transport; chill samples to (5 ± 3) °C and deliver within 24 hours. Storage up to 36 hours at (5 ± 3) °C is recommended.
- Follow biohazard safety precautions; samples may ferment, generate gases and contain pathogens.
Four-stage detection principle
- Primary enrichment in peptone‑novobiocin medium (22 ± 2 h, 36 ± 2 °C, shaking ~150 rpm).
- Secondary (selective) enrichment: inoculate 0.1 ml into two RV tubes (10 ml each), incubate one at 36 ± 2 °C and another at 42 ± 1 °C for 22 ± 2 h (static).
- Plating: streak 10 µl from RV tubes onto XLD and BPLA agars; incubate (36 ± 2 °C, 22 ± 2 h). Subculture typical colonies onto nutrient agar for pure cultures.
- Confirmation: biochemical and/or serological identification (e.g., API 20E, O and H antigen tests).
Media and apparatus
- Buffered Peptone Water with Novobiocin; Rappaport‑Vassiliadis medium; XLD, BPLA and nutrient agar formulations specified.
- Standard microbiological equipment, incubators (36 ± 2 °C and 42 ± 1 °C), autoclave, membrane filters and aseptic tools.
Applications
- Routine monitoring of wastewater sludges, composts, soil amendments, growing media and biowaste for regulatory compliance and biosafety.
- Screening upstream of quantitative methods when presence/absence information is sufficient.
- Useful in environmental microbiology, sludge characterization, agricultural risk assessment and food‑safety surveillance where land application of organic wastes is concerned.
Who should use this standard
- Environmental and public‑health laboratories performing pathogen testing.
- Wastewater treatment facilities, sludge testing labs, agricultural labs and consultants assessing biosolids safety.
- Regulatory bodies and quality assurance teams that need standardized presence/absence detection for Salmonella in solid organic matrices.
Related standards
- CEN/TR 15215-1 (membrane filtration method) and CEN/TR 15215-2 (liquid enrichment with MPN determination)
- EN 12880 (dry residue and water content of sludges)
- ISO 8199 (general guidance for microbial enumeration and quality control)
This standard provides clear procedural steps and media specifications for reliable screening of Salmonella in solid and semi-solid organic matrices, balancing sensitivity (LOD ≈ 10 cfu/50 g) with practical laboratory workflows.
Standards Content (Sample)
SLOVENSKI STANDARD
01-julij-2006
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]HPOMLQLL]EROMãHYDOFLKWDOUDVWQLKVXEVWUDWLKLQELRRGSDGNLKGHO 0HWRGDV
WHNRþRRERJDWLWYLMRYSHSWRQQRYRELRFLQPHGLMXNLPXVOHGLPHWRGDSR
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Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges,
soils, soil improvers, growing media and biowastes - Part 3: Presence/absence method
by liquid enrichment in peptone-novobiocin medium followed by Rapport-Vassiliadis
Charakterisierung von Schlämmen - Quantitativer Nachweis von Salmonella spp. in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 3: Verfahren der Flüssiganreicherung in Peptonwasser mit Novobiocin in
Kombination mit Rappaport-Vassiliadis-Medium zum qualitativen Nachweis des
Vorkommens
Caractérisation des boues - Détection et dénombrement de Salmonella spp. dans les
boues, les sols, les amendements du sol, les supports de culture et les biodéchets -
Partie 3: Présence/absence par enrichissement en milieu liquide peptone-novobiocine
puis sur milieu Rapport-Vassiliadis
Ta slovenski standard je istoveten z: CEN/TR 15215-3:2006
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 15215-3
RAPPORT TECHNIQUE
TECHNISCHER BERICHT
January 2006
ICS 07.100.99
English Version
Characterization of sludges - Detection and enumeration of
Salmonella spp. in sludges, soils, soil improvers, growing media
and biowastes - Part 3: Presence/absence method by liquid
enrichment in peptone-novobiocin medium followed by Rapport-
Vassiliadis
Détection et dénombrement de Salmonella spp. dans les Quantitativer Nachweis von Salmonella spp. in
boues, les sols, les engrais, les amendements organiques Schlämmen, Böden, Düngemitteln und Bodenverbesserern,
et les biodéchets - Partie 3: Présence/absence par Kultursubstraten sowie Bioabfällen - Teil 3: Verfahren der
enrichissement en milieu liquide peptone-novobiocine puis Flüssiganreicherung in Peptonwasser mit Novobiocin
sur milieu Rapport-Vassiliadis gefolgt durch Rapport-Vassiliadis zum qualitativen
Nachweis des Vorkommens
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 15215-3:2006: 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.6
5 Apparatus .6
6 Sampling and hazards .7
7 Reagents, diluents and culture media.7
8 Procedure .10
9 Expression of results.11
10 Test report .11
11 Performance data.11
Annex A (informative) Performance data from laboratory tests .12
Annex B (informative) Performance data with field samples .14
Bibliography.15
Foreword
This Technical Report (CEN/TR 15215-3:2006) has been prepared by Technical Committee CEN/TC 308
“Characterisation of sludges”, the secretariat of which is held by AFNOR.
This Technical Report does not replace any existing CEN method
This standard is divided into three parts:
- part 1 gives a membrane filtration method
- part 2 is a liquid enrichment method and determination by MPN and
- part 3 is a presence / absence method by liquid enrichment.
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 pathogen-contaminated materials in agriculture can cause outbreaks of
infection due to the production of contaminated food or animal feedstocks and may also be transmitted to wild
animals, consequently, there is a need to monitor rates to land. See CEN/TR 15215-2.
Examination for Salmonellae should only be carried out in laboratories competent for carrying out work
involving pathogens. 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 presence/absence procedure to detect Salmonella spp
using a four-stage presence/absence method in up to 50g (wet weight) sample.
The method has a limit of detection of approximately 10 cfu/50 g wet weight sludge.
NOTE The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
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
Salmonella spp.
member of the family of Enterobacteriaceae, these are Gram-negative, non-sporulating, rod-shaped bacteria,
most of which are motile. They can be distinguished from other genera of the Enterobacteriaceae family by
biochemical methods and serologically identified by their somatic or flagellar antigens (O and H-antigens)
3.2
method definition
Salmonella spp. capable of being enriched in peptone water supplemented with Novobiocin and growth in RV
medium
3.3
cfu, colony forming unit
growth of individual bacterial cells into visible colonies on agar media, including on membrane filters
overlaying the agar media
3.4
vegetative bacteria
those bacteria which are capable of normal growth in broth or on agar media without pre-culture resuscitation
3.5
sub-lethally damaged bacteria
those bacteria which have been stressed but not killed in treatment processes or storage
3.6
resuscitation
stimulation to vegetative growth of sub-lethally damaged bacteria previously incapable of growth on agar
media
3.8
presumptive positives
isolates which are believed to be Salmonella spp., but not yet confirmed
3.9
dry residue
the dry mass portion of the sludge obtained after the specified drying process. It is expressed as percent or in
grams per kilogram
[EN 12880:2000, 3.1]
4 Principle
This is a presence/absence method including recovery of sub-lethally damaged Salmonella spp. Designed to
process samples of up to 50 g wet weight. If lower sample quantities are processed, the relationship between
the amount of sample and primary recovery medium shall be maintained
The detection of Salmonella spp. requires four stages.
a) culturing of bacteria in a primary selective medium;
b) enrichment in a secondary selective medium which inhibits the growth of other micro-organisms but
promotes that of Salmonellae (selective enrichment);
c) preparation of pure cultures by inoculation of two different solid media with subcultures;
d) biochemical and serological identification
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 example 125 ml, 200 ml, 500 ml and 2 000 ml.
5.2 Thermostatic incubators regulated at (36 ± 2) °C (gyratory shaking) and (42 ± 1) °C (static).
5.3 Autoclave (Steam sterilizer).
5.4 Refrigerator.
5.5 Sterile plastics culture dishes, with lid of about 90 mm in diameter.
5.6 Sterile graduated pipettes, of nominal capacities 1 and 10 ml.
5.7 Inoculating loop (e.g. platinum-iridium wire), of diameter approximately 3 mm.
5.8 Apparatus for shaking the culture tubes.
5.9 Culture tubes, 25 ml capacity, or equivalent containers.
5.10 Vortex mixer suitable for 25 ml capacity culture tubes or equivalent containers.
5.11 Laboratory spatula.
5.12 pH meter, with temperature compensation and pH measuring cell.
5.13 Membrane filtration equipment.
5.14 Filter membrane, for media sterilisation (0,2 µm cellulose nitrate 47 mm diameter).
5.15 Adjustable micropipettor up to 200 µl capacity.
5.16 Boiling water bath.
6 Sampling and hazards
6.1 Introduction
Take samples of at least 100 g wet weight and deliver them to the laboratory as quickly as possible
(within 24 hours). In order to prevent propagation or inactivation of Salmonella during transport to the
laboratory and subsequent storage, the necessary precautions depending upon the matrix shall be taken.
NOTE Generally chilling the sample to (5 ± 3) °C is recommended.
6.2 General
Samples are liable to ferment particularly if not treated, and may contain pathogenic micro-organisms. It is
essential to keep them away from any food or drink, and to protect any cuts. When transporting and handling
samples, it is essential that national and international regulations relating to biohazardous samples are
followed.
See also the Warning note in the introduction.
6.3 Storage
It is not advisable to store samples in the open laboratory. If samples are to be stored, store them at (5 ± 3) °C
for a maximum period of 36 hours.
6.4 Handling
Cleanliness when working is essential. When handling sludge samples, it is necessary to wear gloves, a face
and eye protection, sufficient body protection to guard against bottles bursting. The gas evolved is usually
flammable, so all equipment in the vicinity shall be flame proof to avoid any source of ignition.
See also the Warning note in the introduction.
7 Reagents, diluents and culture media
To ensure reproducible results, prepare culture media and diluents using either constituents of uniform qua
...
Frequently Asked Questions
CEN/TR 15215-3:2006 is a technical report published by the European Committee for Standardization (CEN). Its full title is "Characterization of sludges - Detection and enumeration of Salmonella spp. in sludges, soils, soil improvers, growing media and biowastes - Part 3: Presence/absence method by liquid enrichment in peptone-novobiocin medium followed by Rapport-Vassiliadis". This standard covers: This part of the CEN Technical Report specifies a presence/absence procedure to detect Salmonella spp using a four-stage presence/absence method in up to 50g (wet weight) sample. The method has a limit of detection of approximately 10 cfu/50 g wet weight sludge. NOTE The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
This part of the CEN Technical Report specifies a presence/absence procedure to detect Salmonella spp using a four-stage presence/absence method in up to 50g (wet weight) sample. The method has a limit of detection of approximately 10 cfu/50 g wet weight sludge. NOTE The objective is to cover untreated and treated sludges, soils, soil improvers, growing media and biowastes.
CEN/TR 15215-3:2006 is classified under the following ICS (International Classification for Standards) categories: 07.100.99 - Other standards related to microbiology. The ICS classification helps identify the subject area and facilitates finding related standards.
You can purchase CEN/TR 15215-3:2006 directly from iTeh Standards. The document is available in PDF format and is delivered instantly after payment. Add the standard to your cart and complete the secure checkout process. iTeh Standards is an authorized distributor of CEN standards.
CEN/TR 15215-3:2006 표준은 슬러지, 토양, 토양 개선제, 성장 매체 및 바이오 폐기물에서 살모넬라(Salmonella spp.)를 발견하고 수량화하는 방법에 대한 중요한 자료를 제공합니다. 이 문서는 특히 50g(습중량)의 샘플을 사용하여 슬러지 내 살모넬라의 존재 여부를 확인하는 네 단계의 접근 방식을 규정합니다. 이 표준은 약 10 cfu/50g 습중량 슬러지를 검출할 수 있는 한계를 가지고 있어, 매우 민감한 검출 시스템을 제공하여 여러 종류의 샘플에서 슬러지를 효과적으로 평가할 수 있도록 돕습니다. 다양한 종류의 슬러지와 토양 샘플을 아우르는 이 표준은 오염 모니터링 및 환경 안전성 평가에 필수적인 요소로 작용합니다. 또한, 슬러지의 처리 여부에 관계없이 적용 가능하므로, 이 표준은 다양한 환경에서 일관된 결과를 제공하는 강점을 가집니다. CEN/TR 15215-3:2006 표준은 살모넬라와 같은 병원균의 안전성을 평가하는 데 중요한 역할을 하며, 이는 농업 및 환경 관리 분야에서의 중요성이 더욱 강조됩니다. 따라서 이 표준의 개발과 적용은 생태계 보존과 인간 건강 보호에 기여하고, 환경 모니터링 및 공공 건강 관리를 위한 지속 가능한 방법을 마련하는 데 도움을 줍니다.
CEN/TR 15215-3:2006は、スラッジ、土壌、土壌改良材、栽培メディアおよびバイオ廃棄物におけるSalmonella spp.の検出と定量に関する規格であり、その重要性と適用範囲が明確に示されています。この文書では、最大50g（湿重量）のサンプルを用いて、液体培養法による存在/不在の手法を詳細に定義しています。 この規格の強みは、サルモネラの存在を検出するための四段階の手法を採用している点にあります。これにより、サンプル中のSalmonella spp.の検出限界が約10 cfu/50 gの湿重量スラッジという高い感度を持ち、さまざまな条件下での適用が可能です。未処理および処理済みのスラッジ、土壌、土壌改良材、栽培メディアおよびバイオ廃棄物に焦点を当てることで、規格は多面的な用途に対して柔軟性を持っています。 さらに、この手法は、衛生基準の確保と食の安全性の向上に寄与することが期待されており、環境保護の観点からも重要です。この文書は、スラッジおよび関連する材料の管理、評価、および規制において不可欠なリソースとなるでしょう。
The CEN/TR 15215-3:2006 standard provides a comprehensive methodology for the characterization of sludges, specifically focusing on the detection and enumeration of Salmonella spp. in various substrates, including sludges, soils, soil improvers, growing media, and biowastes. The standard's scope is well-defined, detailing a four-stage presence/absence procedure that is designed to effectively determine the presence of Salmonella spp. using a sample weight of up to 50 grams. One of the key strengths of this standard is its clearly articulated limit of detection, which is approximately 10 cfu/50 g of wet weight sludge. This sensitivity is crucial for both regulatory compliance and public health safety, as it allows for the effective monitoring of potentially hazardous microorganisms in diverse environments. The method outlined in CEN/TR 15215-3:2006 is robust, as it covers both untreated and treated sludges, demonstrating its relevance in a variety of contexts within waste management and agricultural sectors. Moreover, the standardized approach ensures consistency and reliability across laboratories and field applications, which is critical for comparability of results in environmental monitoring. The emphasis on a liquid enrichment culture in peptone-novobiocin medium followed by the Rapport-Vassiliadis method further enhances the thoroughness of the detection process. This combination allows for an effective identification of Salmonella, ensuring that the characterization of sludges remains within the framework of safety and environmental protection. Overall, the CEN/TR 15215-3:2006 standard is a valuable resource for professionals in the field, as it guides the systematic detection of Salmonella spp., contributes to public health initiatives, and supports the continued improvement of waste treatment practices. Its relevance and strength in addressing both scientific and practical needs within the realm of sludge characterization cannot be overstated.
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