HORIZONTAL Factsheet
E-COLI
version: 2/16/2016
SUBJECT
E-COLI
HORIZONTAL number
Existing standards used as basis
3
Sludge, soil, soil improvers, growing media, biowaste
…..
PrEN 15214-1, PrEN 15214-2, PrEN 15214-3,
Standardisation Committees
CEN TC308/WG1/TG5
Work package
3
Project
STREP FP6 HORIZONTAL-HYG
Expert
Prof. K. Clive Thompson
Organisation
ALcontrol Laboratories
Country
UK
Email
Status
clive.thompson@alcontrol.co.uk
Matrices
Desk study First
Comments Phase I
Final version
Phase II Ruggedness
Hygienic parameters
September 2003
October 2003
February 2004
Ongoing work
www.ecn.nl/horizontal
EIONET
Completion due by 31-05-06
Second consultation
Completion due by 30-09-06
Validation
Completion due by 30-08-07
Final consultation
Completion due by 31-10-07
Transfer to BT/TF151
Foreseen by 30-11-07
Summary of E-COLI procedure
The procedure related to detection, quantification and enumeration of E. coli in sludges, soils,
soil improvers, growing media and biowastes can be divided into three methods. These are
split into Parts 1, 2 and 3.
Part 1: Detection and enumeration of Escherichia coli in sludges, soils, soil improvers,
growing media and biowastes – Membrane filtration method for quantification. The scope of
the method specifies a membrane filtration procedure for the quantitative detection, by culture
of individual colonies on chromogenic agar media. This method is not suitable for materials
whose treatment will significantly reduce bacterial levels to less than 10 viable E. coli per g
wet weight such as lime addition, drying or pasteurisation. It is suitable to evaluate the log
reduction of E. coli throughout treatment, as well as the quality of the end product. This
method is for materials with dry residues <10 - 20% (w/v).
The principle of the method comprises of four stages; sample preparation and centrifugation;
pre-filtration; dilution and finally membrane filtration. The sample preparation stage involves
the crude homogenisation of the as received sample using simple laboratory techniques and
then taking a10g aliquot and adding 90ml of Maximal Recovery Diluent (MRD), this subsample is then homogenised and the pH is adjusted to pH 7.0± 1, before undergoing
centrifugation at 200-300g for 2 minutes. After centrifugation, the supernatant is decanted
from the tubes and pre-filtered. During this pre- filtration step, the sample is passed through a
coarse glass fibre filter disc (nominal pore size 2.7µ) to remove any fine debris present in the
sample. The procedure has been shown not to significantly reduce the recovery efficiency of
E. coli.
Following sample preparation, the sample is diluted. The number of dilutions to subsequently
HORIZONTAL Factsheet
E-COLI
version: 2/16/2016
filter will vary according to the presumed level of contamination of the material to be tested.
Typically, the sample solution should be serially diluted 10-1 – 10-3 using MRD, this will
permit enumeration of up to 104 E. coli per g wet weight sludge. Higher bacteria levels
(untreated sludge) will require additional dilutions of the filtrate to 10-7. The diluted sludge
sample is subsequently filtered, the membrane is recovered aseptically and incubated on
membrane lactose glucuronide agar, initially at 30 ± 1°C for 4 ± 0.5h to aid resuscitation of
stressed E. coli and then the temperature is increased to 44 ± 1°C for 14 ± 2h. The presence of
E. coli is indicated by green colonies resulting from the hydrolysis of BCIG. The method has
a limit of detection of approximately 10 cfu g-1 wet weight sludge, dependant on the solids
content which at high concentrations (>20% (w/v)) will restrict filtration of the sample
volume through the membrane, if not first diluted sufficiently. Samples can be confirmed
through a biochemical test (API20E) if required.
Part 2: Detection and enumeration of Escherichia coli from sludges, soils, soil improvers,
growing media and biowastes- Miniaturised method (MPN) in liquid medium.
The scope of the method is to detect E. coli in accordance with the requirements of the
European Sewage Sludge Draft Regulation.
The principle of the miniaturised method can be split into four stages; sample preparation;
inoculation; reading the microtitre plate and finally calculation of the result.
The as received sample is crudely homogenised using simple laboratory techniques and is
then prepared by suspending a sample aliquot equivalent to 10g (dry matter) with tryptone
diluent in a final volume of 100ml. This is then homogenised for a period of 1 minute in a
mixer and then subsequently analysed without delay.
The microtitre plate is inoculated with 2ml of primary suspension mixed with 18ml of special
diluent. The dilutions are prepared from this solution from a starting concentration of 10-1
down to a final dilution of 10-6. Once the microtitre plate is inoculated it is incubated for
between 36-72 h at 44 ±0.5 °C
The presence of E. coli under Ultra Violet light at 366nm is indicated by a blue florescence
resulting from hydrolysis of MUG. The results are expressed as Most Probable Number per g
dry weight sludge.
Part 3: Detection and enumeration of Escherichia coli from sludges, soils, soil improvers,
growing media and biowastes – macromethod MPN in liquid medium.
The scope of the method described in this standard to detect Escherichia coli in sewage
sludge in accordance with the requirements of the European Sewage Sludge Draft Regulation.
The principle of the macromethod MPN in liquid medium can be split into four parts; sample
preparation; dilution; inoculation and finally interpretation and reading.
The preparation of the primary suspension is achieved by placing 20 g (wet weight) of the
crudely homogenised as received sample (sludge/compost etc.) obtained by using simple
laboratory techniques, into 180 ml 0.9% m/V sterile NaCl solution. This is then shaken at a
minimum of 150 rpm for up to 20 h at 6 ± 2°C.
For relatively homogeneous samples (e.g. mesophilic digested sludges), only a 30 min
shaking is necessary for the homogenisation step. A 20 hour shaking is only necessary for dry
hydrophobic and/or inhomogeneous samples.
For disinfectant (e.g. lime, peracetic acid) treated sludges, a suitable pre-treatment for
neutralising the disinfecting agent and suitable pH adjustment is required.
The samples are prepared for analysis using a serial tenfold dilution (1 ml of primary
suspension + 9 ml 0.9% m/V sterile NaCl solution) up to 10-7. From each dilution step is
transferred 1 ml per tube; into 3 tubes each containing 5 ml MUG Fluorocult lauryl sulphate
broth.
The samples are then incubated for 40 ± 4 h at 44 ± 1°C. The confirmation of E. coli is
HORIZONTAL Factsheet
E-COLI
version: 2/16/2016
achieved through the adding 0.5 ml NaOH 1N to each tube, and observing for fluorescence
with 366 nm UV-light irradiation.
The result per gram of sludge is then calculated according to E. coli / g (wet weight) = 10 x E.
/ ml of Primary Suspension.
Placement of E-COLI method in overall structure HORIZONTAL
SLUDGE, SOIL, TREATED BIOWASTE AND RELATED
WASTES
SAMPLING
IN-SITU ,TRANSPORT, STORAGE
SAMPLE PRETREATMENT - INORG
INORGANIC
CONSTITUENTS
PHYSICAL
PROPERTIES
SAMPLE PRETREATMENT - ORG & BIOL
LEACHING
ORGANIC
PARAMETERS
BIOLOGICAL
PARAMETERS
SAMPLE PRETREATMENT
- HYG
HYGIENIC
PARAMETERS
PAH
pH
Bulk density
Characterisation
leaching tests
Impurities (e.g
glass, plastic)
E. Coli
Stability (bio)
Salmonella
Germinating
weed seeds
Clostridium
Phytotoxicity
Enterococci
AOX
Electrical
conductivity
Dry matter
Compliance
leaching tests
PCB
Organic matter*
Solidity
LAS, NP,NPE
Nutrients N, P,
K*
Thixotropic
behaviour
DEPH, DBP
Trace elements
solid *
Trace elements
determination*
Piling
behaviour
Helminth ova
BFR
Flowability
Rapid methods
PCCD/PCDF
Soluble P
Pharmaceuticals
Virusses and
bacteriophages
Plant
Pathogens
Position of E-COLI method in the sequence of steps from sampling to reporting
Sampling Taking of
plan
sample
Transport & Preparation
storage
of test
portion
Extraction/
Leaching
Pre-treatment
Detection &
Summary
Quantification, reports
Enumeration
1)Sample conservation, storage and transport are issues that generally are covered in as the final step in sampling standards
2) Preparation of the test portion from a laboratory sample is sometimes part of the next step (digestion or analysis preparation) or can
be covered in a separate standard
3) Summary of test results is a concise summary of the relevant data for the end-user, which can be merged in an overall test report
covering all aspects relevant for the given situation.
4) After defining sampling strategy based on the question to be answered either one of the sampling standards will generally apply. All
the following steps will be the same for either route.
5) It is at present not clear where extraction of organic parameters begins and pre-treatment ends. It seems that pre-treatment now includes
the extraction step.
Major issues of discussion
HORIZONTAL Factsheet
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E-COLI
version: 2/16/2016