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NATIONAL STANDARD METHOD

ENUMERATION OF βGLUCURONIDASE POSITIVE ESCHERICHIA COLI – MOST PROBABLE NUMBER METHOD

F 22 Issued by Standards Unit, Evaluations and Standards Laboratory Centre of Infections

ENUMERATION OF β 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: [email protected]

STATUS OF NATIONAL STANDARD METHODS National Standard Methods, which include standard operating procedures (SOPs), algorithms and guidance notes, promote high quality practices and help to assure the comparability of diagnostic information obtained in different laboratories. This in turn facilitates standardisation of surveillance underpinned by research, development and audit and promotes public health and patient confidence 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 reflect the majority agreement of contributors. Representatives of several professional organisations, including those whose logos appear on the front cover, are members of the working groups which develop National Standard Methods. Inclusion of an organisation’s logo on the front cover implies support for the objectives and process of preparing standard methods. The representatives participate in the development of the National Standard Methods but their views are not necessarily those of the entire organisation of which they are a member. The current list of participating organisations can be obtained by emailing [email protected]. 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 acknowledged. The HPA is an independent organisation dedicated to protecting people’s health. It brings together the expertise formerly in a number of official organisations. More information about the HPA can be found at www.hpa.org.uk. The HPA aims to be a fully Caldicott compliant organisation. It seeks to take every possible precaution to prevent unauthorised disclosure of patient details and to ensure that patient-related records are kept under secure conditions1. More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the development of the documents can be made by contacting [email protected]. Please note the references are now formatted using Reference Manager software. If you alter or delete text without Reference Manager installed on your computer, the references will not be updated automatically.

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

ENUMERATION OF β 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 2 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: [email protected]

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 Β GLUCURONIDASE POSITIVE ESCHERICHIA COLI ........................................................................................................................................ 11 REFERENCES ................................................................................................................................................... 12

ENUMERATION OF β 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 3 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: [email protected]

AMENDMENT PROCEDURE Controlled document reference Controlled document title

F 22 Standard Operating Procedure for Enumeration of β-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 [email protected]. 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

ENUMERATION OF β 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 4 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: [email protected]

STANDARD OPERATING PROCEDURE ENUMERATION OF β-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 37°C. 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.

ENUMERATION OF β 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 5 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: [email protected]

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 37°C ± 1°C for 24 hours

•

Subculture to TBX agar with incubation at 44°C ± 1°C 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-β-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 37°C ± 1°C and 44°C ± 1°C 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)

ENUMERATION OF β 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 6 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: [email protected]

5.0

CULTURE MEDIA AND REAGENTS Equivalent commercial dehydrated media may be used; follow the manufacturer’s instructions. Peptone saline diluent (Maximum recovery diluent) Peptone Sodium chloride Water pH 7.0 ± 0.2 at 25°C

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 25°C

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 25°C

20.0 g 1L

Dipotassium hydrogen phosphate diluent Dipotassium hydrogen phosphate Water pH 7.5± 0.2 at 25°C

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

ENUMERATION OF β 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 7 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: [email protected]

Ammonium chloride Water pH 6.7 ± 0.1 at 25°C

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 25°C

6.0

SAMPLE PROCESSING

6.1

Preparation of the sample

20.0 g 1.5 g 144 µmol 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 37°C for 24 ± 2 hours. Positive control: Inoculate a tube of single strength medium with Escherichia coli NCTC 13216 (weak β-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 44°C for 20 - 24 hours. Examine the plates for the presence of blue or blue-green colonies, indicating the presence of β – glucuronidase positive Escherichia coli. Record the results.

ENUMERATION OF β 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 8 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: [email protected]

7.0

CALCULATION OF RESULTS Count the number of tubes containing confirmed β-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.

ENUMERATION OF β 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 9 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: [email protected]

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 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.

ENUMERATION OF β 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 10 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: [email protected]

APPENDIX 2: FLOWCHART SHOWING THE ENUMERATION OF Β 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)

Ø 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

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

Ø Subculture all tubes showing any trace of acid to a 1/3rd segment of TBX and incubate at 44°C ± 1°C for 20 - 24 h

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

Ø Count tubes which yield blue or blue-green colonies growth as positive for Escherichia coli

Ø Calculate the count per g or mL and report

ENUMERATION OF β 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 11 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: [email protected]

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.

ENUMERATION OF β 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 12 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: [email protected]