DETECTION AND ENUMERATION OF …

1 NATIONAL STANDARD METHOD. DETECTION AND ENUMERATION . OF. ENTEROBACTERIACEAE. F 18. Issued by Standards Unit, Evaluations and Standards Laboratory Centre for Infections DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE. Issue no: 1 Issue date: Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the Regional Food, Water and Environmental Coordinators Forum Page 1 of 13. Reference no: F 18i1. This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency Email: 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.

2 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.

3 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 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.

4 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 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 Contributions to the development of the documents can be made by contacting Please note the references are now formatted using Reference Manager software.

5 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). DETECTION and ENUMERATION of Enterobacteriaceae. National Standard Method F 18 Issue 1. DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE. Issue no: 1 Issue date: Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the Regional Food, Water and Environmental Coordinators Forum Page 2 of 13. Reference no: F 18i1. This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency Email: INDEX. STATUS OF NATIONAL STANDARD METHODS .. 2. 3. AMENDMENT PROCEDURE .. 4. 5. BACKGROUND .. 5. 6. DEFINITIONS .. 6. SAFETY 6. EQUIPMENT .. 6. CULTURE MEDIA AND REAGENTS .. 7. SAMPLE PREPARATION OF THE SAMPLE.

6 8. PRE-ENRICHMENT .. 9. ISOLATION .. 9. CONFIRMATORY TESTS .. 9. CALCULATION OF 10. REPORTING RESULTS .. 10. DETECTION ..10. ENUMERATION .. 10. APPENDIX 1: DETERMINATION OF MOST PROBABLE NUMBER .. 11. APPENDIX 2: FLOWCHART SHOWING THE DETECTION AND ENUMERATION OF. ENTEROBACTERIACEAE .. 12. REFERENCES .. 13. DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE. Issue no: 1 Issue date: Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the Regional Food, Water and Environmental Coordinators Forum Page 3 of 13. Reference no: F 18i1. This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency Email: AMENDMENT PROCEDURE. Controlled document F 18. reference Controlled document title Standard Operating Procedure for DETECTION and ENUMERATION of Enterobacteriaceae Each National Standard Method has an individual record of amendments.

7 The current amendments are listed on this page. The amendment history is available from On issue of revised or new pages each controlled document should be updated by the copyholder in the laboratory. Amendment Issue no. Insert Page Section(s) involved Amendment Number/ Discarded Issue Date no. DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE. Issue no: 1 Issue date: Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the Regional Food, Water and Environmental Coordinators Forum Page 4 of 13. Reference no: F 18i1. This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency Email: STANDARD OPERATING PROCEDURE FOR THE. DETECTION AND ENUMERATION OF. ENTEROBACTERIACEAE. INTRODUCTION. Scope The method described is applicable to the DETECTION and ENUMERATION of Enterobacteriaceae in all food types, including milk and dairy products.

8 Background Regulation (EC) 852/20042 on the hygiene of foodstuffs and regulation (EC) 853/ 20043 laying down specific rules for food of animal origin will be enacted in English law on 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 Enterobacteriaceae, to be applied at the end of the manufacturing process of egg products, milk and other dairy products, dried infant formulae and dried dietary foods for special medical purposes, and to meat carcases after dressing but before chilling. The legislation also specifies the methods to be used, which for pasteurised milk, liquid dairy products, milk and whey powder, infant formulae and dried dietary foods is the horizontal method ISO 21528-16, a Most Probable Number method.

9 The method described below is based on ISO 21528-1. It allows DETECTION of Enterobacteriaceae and ENUMERATION by the most probable number (MPN) technique after incubation at 30 C (for milk and dairy products) or 37 C (for other food products). 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. DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE. Issue no: 1 Issue date: Issued by Standards Unit, Evaluations and Standards Laboratory in conjunction with the Regional Food, Water and Environmental Coordinators Forum Page 5 of 13. Reference no: F 18i1. This SOP should be used in conjunction with the series of SOPs from the Health Protection Agency Email: PRINCIPLE. The ENUMERATION of Enterobacteriaceae by the MPN technique involves six stages: Inoculation of three tubes of buffered peptone water per dilution of the test sample, using those dilutions appropriate to obtaining the required DETECTION parameters for that product Incubation of those tubes at 30 C 1 C or 37 C 1 C for 24 hours Subculture to Enterobacteriaceae Enrichment (EE) broth with incubation at 30 C.

10 1 C or 37 C 1 C for 24 hours Subculture of each tube to Violet Red Bile Glucose Agar (VRBGA) and incubation at 30 C or 37 C for 24 hours Confirmation of Enterobacteriaceae presence from tubes producing red-purple colonies on VRBGA by glucose fermentation and oxidase tests Determination of the MPN index from the number of positive tubes of selected dilutions using an MPN table and calculation of the Enterobacteriaceae count per gram or millilitre of sample DEFINITIONS. For the purpose of this method, the following definitions apply: Enterobacteriaceae Bacteria which at 30 C 1 C or 37 C 1 C are capable of forming characteristic colonies on violet red bile glucose agar (VRBGA) and that ferment glucose and show a negative oxidase reaction under the test conditions specified. DETECTION of Enterobacteriaceae Determination of the presence or absence of these microorganisms in a defined weight or volume of food.