SIST-TP CEN/TR 15214-2:2006
(Main)Characterization of sludges - Detection and enumeration of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes - Part 2: Miniaturised method (Most Probable Number) by inoculation in liquid medium
Characterization of sludges - Detection and enumeration of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes - Part 2: Miniaturised method (Most Probable Number) by inoculation in liquid medium
This part of the CEN Technical Report describes a miniaturized most probable number (MPN) method for the quantitative detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It is suitable to evaluate the log reduction of E. coli through treatment as well as the quality of the end product.
This method is convenient for material with dry residue of more than 10 %. For materials with dry residue less than 20 %, the procedure specified in CEN/TR 15214-1 will be used.
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen - Teil 2: Miniaturisiertes Verfahren durch Animpfen in Flüssigmedium (MPN-Verfahren)
Dieses Dokument legt ein miniaturisiertes Verfahren der wahrscheinlichsten Keimzahl (MPN-Verfahren) für den quantitativen Nachweis von Escherichia coli in Schlämmen, Böden, Bodenverbessererungsmitteln, Kultursubstraten und Bioabfall fest. Es eignet sich für die Bewertung der logarithmischen Abnahme von E. coli durch Behandlung sowie der Qualität des Endproduktes.
Das vorliegende Verfahren ist für Materialien mit einem Trockenrückstand von mehr als 10 % geeignet. Bei Materialien mit einem Trockenrückstand von weniger als 20 % wird das in CEN/TR 15214-1 festgelegte Verfahren angewendet.
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 2 : Méthode miniaturisée (nombre le plus probable) par ensemencement en milieu liquide
La présente partie du Rapport Technique CEN décrit une méthode miniaturisée (nombre le plus probable – NPP) de détection quantitative d’Escherichia coli dans les boues, les sols, les amendements du sol, les supports de culture et les biodéchets. Cette méthode convient pour évaluer la réduction log d'Escherichia coli pendant le traitement, ainsi que la qualité du produit fini.
Cette méthode s’applique aux matériaux ayant une teneur en matiere seche supérieure a 10 %. Le mode opératoire spécifié dans le CEN/TR 15214-2 sera utilisé lorsque les matériaux ont une teneur en matiere seche inférieure a 20 %.
Karakterizacija blata – Ugotavljanje prisotnosti in števila Echerichia coli v blatu, zemljini, izboljševalcih tal, rastnih substratih in bio-odpadkih - 2. del: Miniaturizirana metoda (najverjetnejše število – MPN) s cepljenjem v tekoči medij
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TP CEN/TR 15214-2:2006
01-julij-2006
.DUDNWHUL]DFLMDEODWD±8JRWDYOMDQMHSULVRWQRVWLLQãWHYLOD(FKHULFKLDFROLYEODWX
]HPOMLQLL]EROMãHYDOFLKWDOUDVWQLKVXEVWUDWLKLQELRRGSDGNLKGHO
0LQLDWXUL]LUDQDPHWRGD QDMYHUMHWQHMãHãWHYLOR±031 VFHSOMHQMHPYWHNRþLPHGLM
Characterization of sludges - Detection and enumeration of Escherichia coli in sludges,
soils, soil improvers, growing media and biowastes - Part 2: Miniaturised method (Most
Probable Number) by inoculation in liquid medium
Charakterisierung von Schlämmen - Quantitativer Nachweis von Escherichia coli in
Schlämmen, Böden, Bodenverbesserungsmitteln, Kultursubstraten sowie Bioabfällen -
Teil 2: Miniaturisiertes Verfahren durch Animpfen 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 2 : Méthode miniaturisée (nombre le plus probable) par ensemencement en milieu
liquide
Ta slovenski standard je istoveten z: CEN/TR 15214-2:2006
ICS:
13.030.20
13.080.30
65.080
SIST-TP CEN/TR 15214-2:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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TECHNICAL REPORT
CEN/TR 15214-2
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 2: Miniaturised method (Most Probable
Number) by inoculation 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 2 : Düngemitteln und Bodenverbesserern, Kultursubstraten
Méthode miniaturisée (nombre le plus probable) par sowie Bioabfällen - Teil 2: Miniaturisiertes Verfahren durch
ensemencement en milieu liquide Animpfen in 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 15214-2:2006: E
worldwide for CEN national Members.
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CEN/TR 15214-2:2006 (E)
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
6.1 Introduction.7
6.2 General .7
6.3 Storage .7
6.4 Handling.7
7 Reagents, diluents and culture media.7
7.1 Tryptone salt diluent.8
7.2 Special diluent (SD) .8
7.3 Demineralized or distilled water .8
7.4 Culture medium: MUG/EC medium.8
8 Procedure .9
8.1 Sample preparation.9
8.2 Analysis .9
9 Expression of results.10
9.1 Determination of the characteristic number .10
9.2 Calculation of the MPN and its confidence interval .11
10 Test report .12
11 Performance data.12
Annex A (informative) MPN Statistical Table.13
6 dilutions (1/20 to 1/2000000) / 16 wells seeded per dilution .13
Annex B (informative) Synthetic sea salt.22
Annex C (informative) Performance data of the method .24
Annex D (normative) Quality criteria for the manufacturing of the medium in microtitre plates
(E.coli).27
Bibliography.28
2
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CEN/TR 15214-2:2006 (E)
Foreword
This Technical Report (CEN/TR 15214-2:2006) 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 standard.
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.
3
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CEN/TR 15214-2:2006 (E)
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 may cause outbreaks
of infection due to the production of contaminated food and animal feedstocks and may also be transmitted to
wild animals, consequently, there is a need to monitor rates to land.
Escherichia coli is a non-pathogenic, Gram negative bacterium with an exclusive faecal origin. Consequently,
it can be used as an indicator of faecal contamination. It can also be used to monitor the effectiveness of
disinfection treatments but it is comparatively sensitive (to heat, high pH) and cannot therefore reflect the
behaviour of all pathogens in sewage sludge. Suitable quality control procedures, at least those described in
ISO 8199, have to be applied.
WARNING — "Waste and sludge samples may contain hazardous and inflammable substances. They
may contain pathogens and be liable to biological action. Consequently it is recommended that these
samples should be handled with special care. The gases which may 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".
4
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CEN/TR 15214-2:2006 (E)
1 Scope
This part of the CEN Technical Report describes a miniaturized most probable number (MPN) method for the
quantitative detection of Escherichia coli in sludges, soils, soil improvers, growing media and biowastes. It is
suitable to evaluate the log reduction of E. coli through treatment as well as the quality of the end product.
This method is convenient for material with dry residue of more than 10 %. For materials with dry residue less
than 20 %, the procedure specified in CEN/TR 15214-1 will 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
EN ISO 9308-3, Water quality - Detection and enumeration of Escherichia coli and coliform bacteria in surface
and waste water - Part 3: Miniaturized method (Most Probable Number) by inoculation in liquid medium (ISO
9308-3:1998)
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 belongs to the family of Enterobacteriaceae, is 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
β-glucuronidase-positive micro-organism able to hydrolyse 4-.methylumbelliferyl-β-D-glucuronide (MUG) when
growing at an incubation temperature of 44 °C in the specified liquid medium
3.3
dry residue
the dry mass portion of the material obtained after the specified drying process. It is expressed as percent or
in grams per kilogram
[EN 12880:2000, 3.1].
5
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CEN/TR 15214-2:2006 (E)
4 Principle
This method is based on EN ISO 9308-3: “Water quality - Detection and enumeration of Escherichia coli and
coliform bacteria in surface and waste water – Part 3: Miniaturized method (Most Probable Number) by
inoculation in liquid medium”. The following text describes sample preparation in order to prepare a liquid
suspension, after which the analysis is performed following EN ISO 9308-3, reaching Most Probable Number
results in 100 ml. The number of E. coli is then calculated to express the number of E. coli per gram dry
residue of sludge. In order to perform the analysis of E. coli in sludges, soils, soil improvers, growing media
and biowastes, this document describes the whole procedure corresponding to the one of EN ISO 9308-3.
The detection and enumeration of E. coli from biological wastes and soils requires the following stages:
a) sample preparation: suspension of sludge in tryptone salt diluent;
b) inoculation of the diluted sample in a row of microtitre plate wells containing dehydrated culture medium;
c) examination of the microtitre plates under ultraviolet light at 366 nm in the dark after an incubation period
of 36 h minimum and 72 h maximum at (44 ± 1) °C. The presence of E. coli is indicated by a blue
fluorescence resulting from hydrolysis of MUG;
d) results are given as most probable number per 10 g sludge dry residue.
5 Apparatus
With the exception of equipment supplied sterile, the glassware shall be sterilized in accordance with the
instructions given in ISO 8199.
Usual microbiological laboratory equipment and in particular:
5.1 Apparatus for sterilisation by dry heat (oven) or steam (autoclave).
5.2 Thermostatic incubator regulated at (44 ± 1) °C.
5.3 Tunnel drier or vertical laminar airflow cabinet (preferably class II).
R
5.4 Homogeniser (e.g. Stomacher , Seward Laboratories or equivalent).
5.5 Sterile homogeniser bags, 250 ml volume, with or without integrated mesh to exclude large particulate
R
matter (e.g. Stomacher , Seward Laboratories 6041, 6041/STR or equivalent).
5.6 Ultraviolet observation chamber (Wood’s Lamp , 366 nm).
WARNING — UV light causes irritation of eye and skin. Use protective glasses and gloves.
5.7 Portable refractometer (optional).
5.8 pH meter with an accuracy of ± 0.1.
5.9 Sterile test tubes of 25 ml volume, or flasks with similar capacity.
5.10 Sterile flasks, of nominal capacities e.g. 250 ml.
5.11 Sterile graduated pipettes, glass or disposable plastic ware, capable of dispersing 2 ml and 18 ml.
5.12 Adjustable or pre-set 8-channel multi-pipette or any other suitable system used for measuring
and distributing 200 µl per well.
6
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CEN/TR 15214-2:2006 (E)
5.13 Sterile tips for multi-pipette.
5.14 Equipment for membrane filtration according to ISO 8199, including membrane filters with a
nominal pore size of 0,2 µm, for sterilization of liquid media.
5.15 Sterile microtitre plates – 96 wells, 350 µl, flat-bottomed, non-fluorescent.
5.16 Sterile adhesive covering strips for sealing microtitre plates.
5.17 Sterile Petri dishes, 90 mm in diameter.
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, preferably
chilled at (5 ± 3) °C.
6.2 General
Samples are liable to ferment, particularly if untreated, and can 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.
Bursting glass bottles containing sludge can produce micro-organism contaminated shrapnel. Plastic bottles
can also burst and produce a hazardous spray and aerosol.
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, face
and eye protection, and sufficient body protection to guard against bottles bursting. The gas evolved is usually
flammable so all equipment used 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 demineralised or distilled water free from substances
capable of inhibiting growth under the test conditions. 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 providing that they are shown to be of equivalent
performance in the test.
7
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CEN/TR 15214-2:2006 (E)
7.1 Tryptone salt diluent
Per litre:
a) 1 g of casein peptone;
b) 8,5 g of sodium chloride.
Sterilise the solution in a steam steriliser (5.1) at (121 ± 3) °C for (15 ± 1) min.
7.2 Special diluent (SD)
Synthetic sea salt 22,5 g
Bromophenol blue solution (optional) 10 ml
Distilled water 1 000 ml
Sterilize in the autoclave (5.1) at (121 ± 3) °C for (15 ± 1) min.
The bromophenol blue solution is prepared by adding 0,04 g in 100 ml of 50 % ethanol. It is only used to
colour the SD blue and avoid confusing with demineralised or distilled water.
NOTE 1 A typical analysis of a commercially available and suitable synthetic sea salt is given in informative Annex B.
NOTE 2 SD ready to use is commercially available by several suppliers.
7.3 Demineralized or distilled water
Demineralized or distilled water free from substances inhibiting growth under test conditions.
Sterilize in the autoclave at (121 ± 3) °C for (15 ± 1) min.
7.4 Culture medium: MUG/EC medium
Composition:
Tryptone 40 g
Salicin 1 g
Triton x 100 1 g
MUG* (4-methyl-umbelliferyl β-D-glucuronide) 100 mg
Demineralized or distilled water 1 000 ml
* the fluorogenic constitutent MUG is dissolved in 2 ml of N-N-dimethylformamide.
WARNING — N-N dimethylformamide is toxic. Harmful by inhalation, in contact with skin and is
swallowed. May cause cancer. Use in a chemical fume hood.
Successively add Tryptone, Salicin and Triton to one litre of water, while maintaining a gentle heat and
magnetic stirring, then bring to the boil until completely dissolved. Allow to cool and add the MUG solution.
Adjust the pH to (6,9 ± 0,2).
8
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CEN/TR 15214-2:2006 (E)
Sterilize by filtration with membranes of average pore size 0,2 µm (5.14).
Distribute in 96-well microtitre plates (5.15) with a volume of 100 µl of media in each well (minimum capacity
350 µl) and dehydrate immediately in a tunnel drier or laminar airflow cabinet (5.3).
The manufacturing of the medium shall meet the quality criteria given in Annex D.
NOTE Some microtitre plates containing the MUG/EC medium already distributed are commercially available.
8 Procedure
8.1 Sample preparation
8.1.1 Determination of the dry residue content
The dry residue of the sample is measured using the method described in EN 12880.
8.1.2 Suspension preparation
Weigh a representative sub-sample of 10 g (dry residue) into a homogeniser bag (5.5) with an integrated
mesh if large debris is to be excluded.
Add an appropriate volume of tryptone salt diluent (7.1) so that the final volume is 100 ml. Place the
homogeniser bag (5.5) in a homogeniser (5.4) and homogenize for 1 min.
Process without delay.
NOTE For disinfectant (e.g. lime, peracetic acid) and treated sludges, a suitable neutralisation procedure must be
used (see for example EN 1040:1997).
8.2 Analysis
8.2.1 Preparation of dilutions
Prepare serial dilutions in special diluent (SD, 7.2) from 1/20 to 1/2000000:
Vigorously stir the primary suspension (8.1.2) in order to obtain a homogeneous suspension and, using a
sterile pipette, immediately transfer 18 ml of this homogenised suspension to a first tube (5.9) containing 18 ml
of special diluent (1/2 dilution).
Using a fresh pipette (5.11), transfer 2 ml of this first dilution (homogenised) to a second 18 ml special diluent
tube (1/20 dilution).
From this second tube (dilution 1/20 carefully homogenised) proceed to another 1/10 dilution giving the
following dilution (1/200), while adding 2 ml in 18 ml of special diluent.
Continue as above until all the dilutions to 1/2000000 have been prepared.
NOTE Appropriate precautions should be taken as aerosols may be created by the diluting and pipetting.
9
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CEN/TR 15214-2:2006 (E)
8.2.2 Inoculation and incubation of microtitre plates
Inoculate a microtitre plate (5.15) containing the MUG/EC medium in each well (7.4) while distributing each
dilution from 1/20 to 1/2 000 000 in 16 wells each (dilution ½ is only used for the preparation of the serial
dilutions).
Transfer the contents of the first dilution tube (1/20) to an empty sterile Petri dish of 90 mm diameter (5.17).
Using a multi-channel pipette (5.12) with 8 sterile tips (5.13), distribute 200 µl per wells into 16 wells of the
microtitre plate (5.15) corresponding to this first dilution (use the 2 first columns on the left side of the
microtitre plate).
For subsequent dilutions (1/200, 1/2000, etc.), operate in an identical manner, changing the Petri dish and the
row of 8 sterile tips between each dilution, and using for each successive dilution the 2 following 8 wells
columns of the microtitre plate. The two last 8 wells columns on the right of the microtitre plate should
correspond to the 1/2000000 dilution.
Alternatively, any other suitable system (5.12) may be used to distribute 200 µl of each dilution per well.
WARNING — Beware of contamination via an overflow from one well to another.
Once the microtitre plate is inoculated, cover with the disposable adhesive tape (5.16) provided for this
purpose.
Incubate the microtitre plate in an incubator (5.2) at (44 ± 1) °C for a minimum of 36 hours, and a maximum of
72 hours.
NOTE The microtitre plates should be handled with care, without tilting.
8.2.3 Reading
Place each microtitre plate with the adhesive on, in the UV observation chamber (5.6).
Consider all blue fluorescent wells as being positive.
9 Expression of results
9.1 Determination of the characteristic number
For each of the 6 inoculated dilutions, note the number of positive wells.
EXAMPLE
1/20 16+ out of 16
1/200 16+ out of 16
1/2 000 12+ out of 16
1/20 000 5+ out of 16
1/200 000 0+ out of 16
1/2 000 000 0+ out of 16
10
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CEN/TR 15214-2:2006 (E)
Identify the corresponding characteristic number (CN) corresponding to the number of positive wells of the 3
last dilutions giving a number of positive wells > 0, according to the instructions given in ISO 8199 for NPP
calculation.
In the example, the CN will be 16/12/5 for dilutions 1/200;1/2 000;1/20 000.
9.2 Calculation of the MPN and its confidence interval
The Most Probable Number (MPN) is a statistical estimation of the density of micro-organisms, assumed to
correspond to a Poisson distribution in the volumes inoculated. Confidence intervals are attached to this MPN.
The MPN corresponding to the identified CN can be obtained from the MPN table given in Annex A
i
(informative). The confidence limits at 95% are attached to this MPN .
i
Proceed then as follows:
identify the intermediary MPN (MPN ) from the CN directly in the table in Annex A;
i
the final MPN per 1 ml of suspension (8.1.2), taking into account the dilution steps corresponding to the
CN, is calculated from MPN as follows:
i
* If CN correspond to dilutions 1/20, 1/200 and 1/2 000, MPN/ml suspension = MPN
i
* If CN correspond to dilutions 1/200, 1/2 000 and 1/20 000, MPN/ml suspension = 10 x MPN
i
* If CN correspond to dilutions 1/2 000, 1/20 000 and 1/200 000, MPN/ml suspension = 100 x MPN
i
* If CN correspond to dilutions 1/20 000, 1/200 000 and 1/2 000 000, MPN/ml suspension = 1 000 x MPN
i
The result per g Dry residue is then calculated as follows:
E. coli MPN/g Dry residue = [MPN x 100 ml of total suspension]/10 g Dry residue initially subsampled in 100
ml.
In the example of § 9.1:
If CN = 16/12/5 for dilutions 1/200, 1/2 000 and 1/20 000, the MPN statistical table in Annex A gives
i
1 758.2/ml, with a Lower limit of 1 018.2/ml and a upper limit of 3 036.2/ml.
The MPN is then 17 582/ml suspension (Lower limit = 10 182/ml; Upper limit = 30 362/ml).
The result per g Dry residue is then:
E. coli = 175 820 MPN/g Dry residue (Lower limit = 101 820; Upper limit = 303 620).
If none of the wells is positive, express the result in the following form:
< n/ml, n being the MPN for 1 positive well under the dilution conditions employed.
Calculate the MPN result per ml of suspension (8.1.2) as indicated above.
A free MPN software adapted to this standard and giving a final result/g Dry residue, is also available to the
address aglae@nordnet.fr.
11
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CEN/TR 15214-2:2006 (E)
10 Test report
The test report shall contain the following information:
a) reference to this Technical Report;
b) all information necessary for complete identification of the sample;
c) details of sample pre-treatment, if carried out;
d) results of the determination according to Clause 9.
Any detail not specified in this European Standard and any other factor which may have affected the results.
11 Performance data
Information concerning the repeatability and reproducibility of the procedure, obtained using interlaboratory
tests, is given in Annex C (informative).
12
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CEN/TR 15214-2:2006 (E)
Annex A
(informative)
MPN Statistical Table
For Microtitre plate 96 wells
6 dilutions (1/20 to 1/2000000) / 16 wells seeded per dilution
Table A.1
Characteristic Number MPNi in Lower Upper Characteristic Number MPNi in Lower Upper
1 ml limit limit 1 ml limit limit
0/16 0/16 1/16 5,60 0,80 41,00 2/16 1/16 0/16 18,00 5,70 56,40
0/16 0/16 2/16 11,30 2,70 46,70 2/16 1/16 1/16 24,00 8,80 65,50
2/16 1/16 2/16 30,00 12,10 74,60
0/16 1/16 0/16 5,60 0,80 41,00
0/16 1/16 1/16 11,30 2,70 46,70 2/16 2/16 0/16 24,10 8,80 65,60
0/16 1/16 2/16 16,90 5,20 54,90 2/16 2/16 1/16 30,10 12,10 74,70
2/16 2/16 2/16 36,20 15,60 83,90
0/16 2/16 0/16 11,30 2,70 46,80
0/16 2/16 1/16 17,00 5,30 54,90 2/16 3/16 0/16 30,20 12,20 74,80
2/16 3/16 1/16 36,30 15,70 84,00
0/16 3/16 0/16 17,00 5,30 55,00
0/16 3/16 1/16 22,70 8,10 63,50 2/16 4/16 0/16 36,40 15,70 84,20
2/16 4/16 1/16 42,50 19,30 93,40
0/16 4/16 0/16 22,80 8,20 63,60
2/16 5/16 0/16 42,60 19,40 93,60
1/16 0/16 0/16 5,80 0,80 41,00
1/16 0/16 1/16 11,60 2,90 47,10 3/16 0/16 0/16 18,5 6 57,2
1/16 0/16 2/16 17,40 5,50 55,50 3/16 0/16 1/16 24,7 9,2 66,5
3/16 0/16 2/16 30,9 12,6 75,9
1/16 1/16 0/16 11,70 2,90 47,20 3/16 0/16 3/16 37,1 16,2 85,4
1/16 1/16 1/16 17,50 5,50 55,60
1/16 1/16 2/16 23,30 8,40 64,40 3/16 1/16 0/16 24,8 9,2 66,6
3/16 1/16 1/16 31 12,6 76,1
1/16 2/16 0/16 17,50 5,50 55,70 3/16 1/16 1/16 37,3 16,2 85,5
1/16 2/16 1/16 23,30 8,50 64,50
1/16 2/16 2/16 29,20 11,60 73,30 3/16 2/16 0/16 31,1 12,7 76,2
3/16 2/16 1/16 37,4 16,3 85,7
1/16 3/16 0/16 23,40 8,50 64,60 3/16 2/16 2/16 43,6 20 95,2
1/16 3/16 1/16 29,30 11,70 73,40
3/16 3/16 0/16 37,5 16,4 85,8
1/16 4/16 0/16 29,40 11,70 73,60 3/16 3/16 1/16 43,8 20,1 95,3
1/16 4/16 1/16 35,20 15,10 82,50 3/16 3/16 2/16 50,1 23,9 104,9
1/16 5/16 0/16 35,30 15,10 82,60 3/16 4/16 0/16 43,9 20,2 95,5
3/16 4/16 1/16 50,2 24 105,1
2/16 0/16 0/16 12,00 3,00 47,60
2/16 0/16 1/16 17,90 5,70 56,30 3/16 5/16 0/16 50,4 24,1 105,3
2/16 0/16 2/16 23,90 8,80 65,40 3/16 5/16 1/16 56,7 28 114,9
3/16 6/16 0/16 56,9 28,1 115,1
13
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CEN/TR 15214-2:2006 (E)
Characteristic Number MPNi in Lower Upper Characteristic Number MPNi in Lower Upper
1 ml limit limit 1 ml limit limit
4/16 0/16 0/16 25,5 9,6 67,8 5/16 4/16 0/16 60,8 30,5 121
4/16 0/16 1/16 32 13,2 77,5 5/16 4/16 1/16 67,7 34,9 131,4
4/16 0/16 2/16 38,4 16,9 87,3 5/16 4/16 2/16 74,6 39,2 141,9
4/16 0/16 3/16 44,9 20,8 97
5/16 5/16 0/16 67,9 35 131,7
4/16 1/16 0/16 32,1 13,2 77,6 5/16 5/16 1/16 74,9 39,4 142,3
4/16 1/16 1/16 38,5 17 87,4
4/16 1/16 2/16 45 20,8 97,2 5/16 6/16 0/16 75,1 39,6 142,6
4/16 1/16 3/16 51,5 24,8 107,1 5/16 6/16 1/16 82,1 44 153,3
4/16 2/16 0/16 38,6 17 87,6 5/16 7/16 0/16 82,4 44,2 153,7
4/16 2/16 1/16 45,1 20,9 97,4
4/16 2/16 2/16 51,7 24,9 107,3 6/16 0/16 0/16 41,2 18,6 91,5
6/16 0/16 1/16 48,2 22,8 102,1
4/16 3/16 0/16 45,3 21 97,6 6/16 0/16 2/16 55,3 27,1 112,7
4/16 3/16 1/16 51,8 25 107,5 6/16 0/16 3/16 62,2 31,5 123,3
4/16 3/16 2/16 58,4 29,1 117,4
6/16 1/16 0/16 48,4 22,9 102,3
4/16 4/16 0/16 52 25,1 107,7 6/16 1/16 1/16 55,4 27,2 112,9
4/16 4/16 1/16 58,6 29,2 117,7 6/16 1/16 2/16 62,2 31,6 123,6
4/16 4/16 2/16 65,2 33,3 127,7 6/16 1/16 3/16 69,7 36,1 134,4
4/16 5/16 0/16 58,8 29,3 118 6/16 2/16 0/16 55,6 27,3 113,2
4/16 5/16 1/16 65,4 33,4 128 6/16 2/16 1/16 62,8 31,8 124
6/16 2/16 2/16 69,9 36,3 134,8
4/16 6/16 0/16 65,6 33,6 128,3 6/16 2/16 3/16 77,1 40,8 145,7
5/16 0/16 0/16 33,1 13,8 79,2 6/16 3/16 0/16 63 31,9 124,3
5/16
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