Iso 16649-3 e Coli NMP

NATIONAL STANDARD METHOD
ENUMERATION OF GLUCURONIDASE POSITIVE

ESCHERICHIA COLI MOST
PROBABLE NUMBER METHOD
F 22
Issued by Standards Unit, Evaluations and Standards Laboratory
Centre of Infections
ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

Issue no: 1 Issue date: 14.12.05 Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the
Regional Food, Water and Environmental Coordinators Forum Page 1 of 12
Reference no: F 22i1
This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency
www.evaluations-standards.org.uk
Email: standards@hpa.org.uk
STATUS OF NATIONAL STANDARD METHODS

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information obtained in different laboratories. This in turn facilitates standardisation of surveillance
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in their healthcare services. The methods are well referenced and represent a good minimum
standard for clinical and public health microbiology. However, in using National Standard Methods,
laboratories should take account of local requirements and may need to undertake additional
investigations. The methods also provide a reference point for method development.
National Standard Methods are developed, reviewed and updated through an open and wide
consultation process where the views of all participants are considered and the resulting documents
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member. The current list of participating organisations can be obtained by emailing
standards@hpa.org.uk.
The performance of standard methods depends on the quality of reagents, equipment, commercial
and in-house test procedures. Laboratories should ensure that these have been validated and shown
to be fit for purpose. Internal and external quality assurance procedures should also be in place.
Whereas every care has been taken in the preparation of this publication, the Health Protection
Agency or any supporting organisation cannot be responsible for the accuracy of any statement or
representation made or the consequences arising from the use of or alteration to any information
contained in it. These procedures are intended solely as a general resource for practising
professionals in the field, operating in the UK, and specialist advice should be obtained where
necessary. If you make any changes to this publication, it must be made clear where changes have
been made to the original document. The Health Protection Agency (HPA) should at all times be
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The HPA is an independent organisation dedicated to protecting peoples health. It brings together the
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Suggested citation for this document:

Health Protection Agency (2005). Enumeration of -glucuronidase positive Escherichia coli Most
Probable Number method National Standard Method F 22 Issue 1. http://www.hpastandardmethods.org.uk/pdf_sops.asp.

INDEX
STATUS OF NATIONAL STANDARD METHODS ........................................................................................ 2
INDEX .................................................................................................................................................................... 3
AMENDMENT PROCEDURE ............................................................................................................................ 4
1.0
PRINCIPLE ............................................................................................................................................... 6
2.0
DEFINITIONS ........................................................................................................................................... 6
3.0
SAFETY CONSIDERATIONS................................................................................................................ 6
4.0
EQUIPMENT............................................................................................................................................. 6
5.0
CULTURE MEDIA AND REAGENTS................................................................................................... 7
6.0
SAMPLE PROCESSING ........................................................................................................................ 8
7.0
CALCULATION OF RESULTS.............................................................................................................. 9
8.0
REPORTING RESULTS ......................................................................................................................... 9
APPENDIX 1: DETERMINATION OF MOST PROBABLE NUMBER ...................................................... 10

APPENDIX 2: FLOWCHART SHOWING THE ENUMERATION OF GLUCURONIDASE POSITIVE
ESCHERICHIA COLI ........................................................................................................................................ 11
REFERENCES ................................................................................................................................................... 12

AMENDMENT PROCEDURE
Controlled document
reference
Controlled document title
F 22
Standard Operating Procedure for Enumeration of -glucuronidase
positive Escherichia coli Most Probable Number method
Each National Standard Method has an individual record of amendments. The current amendments
are listed on this page. The amendment history is available from standards@hpa.org.uk.
On issue of revised or new pages each controlled document should be updated by the copyholder in
the laboratory.
Amendment
Number/
Date
Issue no.
Discarded
Insert
Issue
no.
Page
Section(s) involved
Amendment

STANDARD OPERATING PROCEDURE ENUMERATION

OF -GLUCURONIDASE POSITIVE ESCHERICHIA COLI
MOST PROBABLE NUMBER METHOD
INTRODUCTION
Scope
The method described is applicable to the detection and enumeration of Escherichia coli in all food types,
including milk and dairy products.
Background
Regulation (EC) 852/20042 on the hygiene of foodstuffs and regulation (EC) 853/ 20043 will be enacted in
English law on 1.1.06 as the Food Hygiene Regulations 20054. Associated legislation5 specifies
microbiological criteria and methods for testing for a number of food commodities. Included in the
hygiene criteria are specifications for Escherichia coli, to be applied at the end of the manufacturing
process of shelled and shucked products of cooked crustaceans and molluscan shellfish, pre-cut fruit and
vegetables, unpasteurised fruit and vegetable juices and minced meat, meat products and mechanically
separated meat. For cheeses made from milk that has undergone heat treatment, a hygiene criterion is
applied at the beginning of the ripening period for cheeses unable to support the growth of E.coli and at
the end of the ripening period if able to support the growth of E.coli, ie: at the time of manufacturing when
the E.coli count is expected to be highest. The legislation also specifies the methods to be used, which
for shelled and shucked products of cooked crustaceans and molluscan shellfish is the horizontal method
ISO TS 16649-36, a Most Probable Number method. There is also a food safety criterion for Escherichia
coli applied to live bivalve molluscs, echinoderms, tunicates and gastropods, for which ISO TS 16649-3 is
specified, and this is addressed by Standard Method F16 Enumeration of Escherichia coli in Raw
Molluscs by the Multiple Tube Technique.
The method described below is based on ISO TS 16649-3. It allows detection of Escherichia coli and
enumeration by the most probable number (MPN) technique after incubation at 37C. It is particularly
suitable for use when levels are likely to be low (less than 100/g or 10/mL), and for recovery from
products (dried, frozen etc) when the target organism is likely to be stressed. Although other parts of ISO
16649 are specified in the legislation for examining the remainder of the food commodities listed above,
part 3 is also suitable due to the nature of the samples and the use of the following method is
recommended for routine purposes.

1.0
PRINCIPLE
The enumeration of Escherichia coli by the MPN technique involves four stages:
2.0
Inoculation of three tubes of minerals modified glutamate medium per dilution of the
test sample, using those dilutions appropriate to obtaining the required detection
parameters for that product
Incubation of those tubes at 37C 1C for 24 hours
Subculture to TBX agar with incubation at 44C 1C for 20 - 24 hours
Determination of the MPN index from the number of positive tubes of selected
dilutions using an MPN table and calculation of the Escherichia coli count per gram
or millilitre of sample
DEFINITIONS
For the purpose of this method, the following definitions apply:
Escherichia coli
Bacteria that are capable of forming producing acid from lactose and cleave 5-bromo-4chloro-3-indolyl--D glucuronide (BCIG) under the test conditions specified
Enumeration of Escherichia coli
Determination of the most probable number per gram or millilitre of these microorganisms.
3.0
SAFETY CONSIDERATIONS
Normal microbiology precautions apply. In addition, members of the Escherichia coli
species may be pathogenic to man and therefore isolation and identification must be
performed by trained laboratory personnel in a properly equipped laboratory and under the
supervision of a trained microbiologist. Care must be taken in the disposal and sterilisation
of all test materials. Procedures involving subculturing from pre-enrichment and enrichment
broths and handling of Escherichia coli cultures must be performed in a designated area of
the laboratory.
4.0
EQUIPMENT
Usual laboratory equipment and in addition:
Top pan balance capable of weighing 0.1 g
Gravimetric diluter (optional)
Stomacher
Vortex mixer
Incubator at 37C 1C and 44C 1C
Automatic pipettors and associated sterile pipette tips capable of delivering up to 10 mL and
1 mL volumes (optional)
Pipettes (total delivery) 10 mL and 1 mL graduated in 0.1 mL volumes (optional)
Stomacher bags (sterile)

5.0
CULTURE MEDIA AND REAGENTS

Equivalent commercial dehydrated media may be used; follow the manufacturers
instructions.
Peptone saline diluent (Maximum recovery diluent)
Peptone
Sodium chloride
Water
pH 7.0 0.2 at 25C
1.0 g
8.5 g
1L
Buffered peptone water (BPW)

Peptone
Sodium chloride
Disodium hydrogen
phosphate dodecahydrate
Potassium dihydrogen
phosphate
Water
pH 7.2 0.2 at 25C
10.0 g
5.0 g
9.0 g
1.5 g
1L
Sodium citrate diluent

Tri-sodium citrate dihydrate
Water
pH 7.5 0.2 at 25C
20.0 g
1L
Dipotassium hydrogen phosphate diluent

Dipotassium hydrogen
phosphate
Water
pH 7.5 0.2 at 25C
20.0 g
1L
Minerals modified glutamate medium (MMGM)
Lactose
Sodium glutamate
Sodium formate
L(+) Arginine hydrochloride
L(-) Aspartic acid
L(-) Cystine
Thiamine
Nicotininc acid
Pantothenic acid
Magnesium sulphate heptahydrate
Ammonium iron (III) citrate
Calcium chloride dihydrate
Dipotassium hydrogen phosphate
Single
strength
10.0 g
6.35 g
0.25 g
0.024 g
0.02 g
0.02 g
0.001 g
0.001 g
0.001 g
0.100 g
0.010 g
0.010 g
0.90 g
Double
strength
20.0 g
7.7 g
0.50 g
0.048 g
0.04 g
0.04 g
0.002 g
0.002 g
0.002 g
0.200 g
0.020 g
0.020 g
1.8 g

Ammonium chloride
Water
pH 6.7 0.1 at 25C
2.5 g
1L
5.0 g
1L
Tryptone Bile Glucuronide Agar (TBX)

Enzymatic digest of casein
Bile salts No 3
BCIG
Dimethyl sulphoxide (DMSO)
Agar
Water
pH 7.2 0.2 at 25C
6.0
SAMPLE PROCESSING
6.1
Preparation of the sample
20.0 g
1.5 g
144 mol
3 ml
9 g to 18 g
1L
Prepare the test portion, the 10-1 initial suspension and further decimal dilutions as
described in Standard Method D1: Milk and Dairy Products - Preparation of Samples and
Decimal Dilutions or Standard method F2: Food products Preparation of Samples and
Dilutions. Use a separate pipette for each dilution.
6.2
Procedure
If a low level of detection is required (eg for shelled and shucked cooked shellfish and
crustaceans) add 10 mL of the 10-1 suspension to each of three tubes containing double
strength MMGM, 1 mL of the 10-1 and 10-2 dilutions to each of three tubes of single strength
MMGM. For most other sample types add 1 mL of the 10-1, 10-2 and 10-3 dilutions to each
of three tubes of single strength MMGM. For cheese made from pasteurised milk also use
a 10-4 dilution. Carefully mix the inoculum and the medium. Incubate all inoculated tubes at
37C for 24 2 hours.
Positive control: Inoculate a tube of single strength medium with Escherichia coli NCTC
13216 (weak -glucuronidase producer).
Negative control: Inoculate a tube of single strength medium with Klebsiella aerogenes
NCTC 9528 (for TBX)
6.3
Isolation
At the end of incubation examine each tube for any trace of acid production, manifested as
a yellow coloration; record results. Subculture each tube showing acid production to a third
section of a TBX plate to obtain isolated colonies. Incubate the plates at 44C for 20 - 24
hours. Examine the plates for the presence of blue or blue-green colonies, indicating the
presence of glucuronidase positive Escherichia coli. Record the results.

7.0
CALCULATION OF RESULTS
Count the number of tubes containing confirmed -glucuronidase positive Escherichia coli
at each dilution and use the table (Appendix 1) to obtain the MPN/g or mL. This table
assumes the use of 10-1, 10-2 and 10-3 dilutions. If 10 mL of the 10-1 dilution have been used
as the first dilution, divide the MPN index by 10. If more than three dilutions have been
used, select the three consecutive dilutions that give a category 1 MPN index and multiply
the MPN index by the appropriate power of 10. If no combination with category 1 is
available, use the combination with category 2; if more than one combination with category
2 is obtained, use the one with the highest number of positive tubes.
8.0
REPORTING RESULTS
Report the result as the most probable number of Escherichia coli per gram or mL. If the
result is less than 10, report to the nearest whole number. If the result lies between 10 and
99 report that number. If the result is 100 or more, report as a number between 1.0 and 9.9
multiplied by 10x, where x is the appropriate power of 10. If Escherichia coli was detected
but the count is less than one, report detected, less than 1 per g or mL. If no tubes are
positive, report the result as less than the lowest MPN value per gram or mL for the dilutions
used, or as not detected in 1g or 1 mL if the lowest MPN value is 0.3.

APPENDIX 1: DETERMINATION OF MOST PROBABLE

NUMBER
Determination Of Most Probable Number
Number of positive results
-1
-2
10 dilution 10
dilution
0
0
0
0
0
0
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
0
0
1
1
2
3
0
0
0
1
1
2
2
3
0
0
0
1
1
1
2
2
2
3
3
0
0
0
1
1
1
1
2
2
2
2
3
3
3
3
Confidence limits
-3
10 dilution
0
1
0
1
0
0
0
1
2
0
1
0
1
0
0
1
2
0
1
2
0
1
2
0
1
0
1
2
0
1
2
3
0
1
2
3
0
1
2
3
MPN per
g/ml
<3
3
3
6.1
6.2
9.4
3.6
7.2
11
7.4
11
11
15
16
9.2
14
20
15
20
27
21
28
35
29
36
23
38
64
43
75
120
160
93
150
210
290
240
460
1100
>1100
Category
> 95%
3
2
0
3
0
1
2
0
1
3
2
3
3
1
2
0
1
2
0
1
3
0
3
0
1
1
3
1
1
3
0
1
1
2
3
1
1
1
Lower
limit
0
0.1
0.1
1.2
1.2
3.5
0.2
1.2
4
1.3
4
4
5
5
1.5
4
5
4
5
9
5
9
9
9
9
5
9
16
9
17
30
30
18
30
30
90
40
90
200
> 99%
Upper
limit
9.4
9.5
10
17
17
35
17
17
35
20
35
35
38
38
35
35
38
38
38
94
40
94
94
94
94
94
104
181
181
199
360
380
360
380
400
990
990
1980
4000
Lower
limit
0
0
0
0.5
0.5
1.8
0.1
0.5
2
0.6
2
2
2
2
0.7
2
2
2
2
5
2
5
5
5
5
3
5
10
5
11
20
20
12
20
20
50
30
50
100
Upper
limit
14
14
16
25
25
46
25
25
46
27
46
46
52
52
46
46
52
52
52
142
56
142
142
142
142
142
157
250
250
270
440
520
430
520
560
1520
1520
2830
5700
Adapted from de Man JC, 1983. Eur J Appl Biotechnol. 17, 301-305
Category 1:
Results have the greatest chance of being obtained. There is only at most 5% chance of obtaining a result that is less
likely than the least likely one in this category
Category 2:
Results have less chance of being obtained than even the least likely one in category 1, but there is only at most 1%
chance of obtaining a result that is less likely than the least likely one in this category
Category 3:
Results have less chance of being obtained than even the least likely one in category 2, but there is only at most 0.1%
chance of obtaining a result that is less likely than the least likely one in this category
Category 0:
The result is one of those that have less chance of being obtained than even the least likely one in category 3. There is
only a chance of 0.1% of obtaining a result in this category without anything being wrong.

APPENDIX 2: FLOWCHART SHOWING THE

ENUMERATION OF GLUCURONIDASE POSITIVE
ESCHERICHIA COLI
Prepare 10-1, 10-2 and 10-3 dilutions of the sample as required in appropriate diluent
(adjust pH of 10-1 if necessary to 6.8 0.2)
For shelled & shucked cooked crustaceans and shellfish use three tubes of double strength MMGM
and 10 ml of 10-1, three tubes of single strength MMGM and 1 mL of 10-1 and 10-2 dilutions
For most other products use three tubes of MMGM with 1mL of 10-1, 10-2, 10-3 dilutions
For pasteurised cheese samples and pre-cut fruit and vegetables use three tubes of MMGM and 1 mL
of 10-1, 10-2, 10-3 and 10-4 dilutions
Place in an incubator at 37C 1C for 24 2 h
Subculture all tubes showing any trace of acid to a 1/3rd segment of TBX and incubate at 44C 1C for
20 - 24 h
Examine plates for presence of blue or blue-green colonies and record
Count tubes which yield blue or blue-green colonies growth as positive for Escherichia coli
Calculate the count per g or mL and report

REFERENCES
1. Department of Health NHS Executive: The Caldicott Committee. Report on the review of patientidentifiable information. London. December 1997.
2. The European Parliament and the Council of the European Union. Regulation (EC) No 852/2004 of
the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. Official
Journal
of
the
European
Union.
L226.
http://europa.eu.int/eurlex/pri/en/oj/dat/2004/l_226/l_22620040625en00030021.pdf. p. 3-21.
3. The European Parliament and the Council of the European Union. Regulation (EC) No 853/2004 of
the European Parliament and of the Council of 29 April 2004 laying down specific hygiene rules for
food and animal origin. Official Journal of the European Union. L226, 25.6.2004.
http://europa.eu.int/eur-lex/pri/en/oj/dat/2004/l_226/l_22620040625en00220082.pdf. p. 22-82.
4. The Food Hygiene (England) (No.2) Regulations 2005 Draft Statutory Instrument. England: HMSO;
2005.
5. SANCO 4198/2004 rev.19 (PLSPV/2001/4198/4198R19-EN.doc). Draft Commission Regulation on
Microbiological Criteria for Foodstuffs. 2005.
6. BS ISO 16649 Microbiology of food and animal feeding stuffs -- Horizontal method for the
enumeration of beta-glucuronidase-positive Escherichia coli -- Part 3: Most probable number
technique using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. London: British Standards Institution
(BSI); 2005.


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NATIONAL STANDARD METHOD

ENUMERATION OF GLUCURONIDASE POSITIVE

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

STATUS OF NATIONAL STANDARD METHODS

Suggested citation for this document:

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

CULTURE MEDIA AND REAGENTS................................................................................................... 7

SAMPLE PROCESSING ........................................................................................................................ 8

REPORTING RESULTS ......................................................................................................................... 9

APPENDIX 1: DETERMINATION OF MOST PROBABLE NUMBER ...................................................... 10

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

STANDARD OPERATING PROCEDURE ENUMERATION

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

Incubation of those tubes at 37C 1C for 24 hours

Subculture to TBX agar with incubation at 44C 1C for 20 - 24 hours

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

CULTURE MEDIA AND REAGENTS

Buffered peptone water (BPW)

Sodium citrate diluent

Dipotassium hydrogen phosphate diluent

Minerals modified glutamate medium (MMGM)

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

Tryptone Bile Glucuronide Agar (TBX)

Preparation of the sample

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

APPENDIX 1: DETERMINATION OF MOST PROBABLE

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

APPENDIX 2: FLOWCHART SHOWING THE

Place in an incubator at 37C 1C for 24 2 h

Examine plates for presence of blue or blue-green colonies and record

Calculate the count per g or mL and report

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

ENUMERATION OF GLUCURONIDASE POSITIVE ESCHERICHIA COLI MOST PROBABLE NUMBER METHOD

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