CEN/TR 15215-3:2006

SLOVENSKI STANDARD
01-julij-2006
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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 quality
and chemicals of recognised analytical grade, or a dehydrated diluent or complete medium prepared following
the manufacturer’s instructions. Prepare them with fit for purpose demineralised or distilled water free from
substances capable of inhibiting growth under the test conditions. (ISO 8199). If the media are not used
immediately, preserve them in the dark at (5 ± 3) °C for up to one month in conditions avoiding any alterations
in their composition.
NOTE The use of chemicals of other grades is permissible provided that they are shown to be of equivalent
performance in the test.
7.1 Buffered Peptone Water supplemented with 40mg/l Novobiocin
Buffered Peptone Water supplemented with 40 mg/l Novobiocin in 450 ml portions, prepared as follows:
 dissolve 1 g Novobiocin in 10 ml sterile distilled water and filter through membrane filter (pore size 0,2 µm,
(5.14)).
 transfer 180 µl of this solution into 450 ml (40 µg/ml) sterilized buffered peptone water (prepared
according to the manufacturer’s instructions) immediately before use, mix by gentle shaking.
7.2 Rappaport-Vassiliadis Medium
Prepared according to the manufacturer or:
Dissolve the following in 1000 ml water in a 2000 ml flask, while heating in a boiling wate
...