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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: 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 13
Reference no: F 18i1
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). Detection and Enumeration of Enterobacteriaceae. National
Standard Method F 18 Issue 1. http://www.hpa-standardmethods.org.uk/pdf_sops.asp.

INDEX
STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2

INDEX...................................................................................................................................................... 3
AMENDMENT PROCEDURE ................................................................................................................. 4
SCOPE................................................................................................................................................... 5
BACKGROUND ........................................................................................................................................ 5
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
6.1 PREPARATION OF THE SAMPLE ..................................................................................................... 8
6.2 PRE-ENRICHMENT ....................................................................................................................... 9
6.3 ENRICHMENT...............................................................................................................................9
6.4 ISOLATION ................................................................................................................................... 9
6.5 CONFIRMATORY TESTS ................................................................................................................ 9
7.0 CALCULATION OF RESULTS.................................................................................................. 10
8.0 REPORTING RESULTS ............................................................................................................ 10
8.1 DETECTION ...............................................................................................................................10
8.2 ENUMERATION .......................................................................................................................... 10
APPENDIX 1: DETERMINATION OF MOST PROBABLE NUMBER ................................................. 11
APPENDIX 2: FLOWCHART SHOWING THE DETECTION AND ENUMERATION OF
ENTEROBACTERIACEAE ................................................................................................................... 12
REFERENCES ...................................................................................................................................... 13

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. 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 Issue no. Insert Page Section(s) involved Amendment

Number/ Discarded Issue
Date no.

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.
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 6.1.06 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
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. 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.

1.0 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 ±
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
2.0 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.
Enumeration of Enterobacteriaceae
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 Enterobacteriaceae
family 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 Enterobacteriaceae 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 30°C ± 1°C or 37°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)
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 1.0 g
Sodium chloride 8.5 g
Water 1L
pH 7.0 ± 0.2 at 25°C
Buffered peptone water (BPW)

Single Double
strength strength
Peptone 10.0 g 20.0 g
Sodium chloride 5.0 g 10.0 g
Disodium hydrogen 9.0 g 18.0 g
phosphate dodecahydrate
Potassium dihydrogen 1.5 g 3.0 g
phosphate
Water 1L 1L
pH 7.2 ± 0.2 at 25°C
Dispense 10 mL volumes of single and double strength broth into tubes or bottles for
enumeration or 90 mL volumes of single strength broth for detection
Enterobacteriaceae Enrichment broth (EE)

Peptone 10.0 g
Glucose 5.0 g
Disodium hydrogen phosphate 6.45 g
Potassium dihydrogen phosphate 2.0 g
Ox bile 20.0 g
Brilliant green 0.0135 g
Water 1L
pH 7.2 ± 0.2 at 25°C
Dispense in 10 mL volumes in tubes or bottles

Violet Red Bile Glucose Agar (VRBGA)
Yeast extract 3.0 g
Peptone 7.0 g
Bile salts No.3 1.5 g
Glucose 10.0 g
Neutral red 3 mg
Crystal violet 2 mg
Agar 12 g
Water 1L
pH 7.4 ± 0.2 at 25°C
Glucose agar
Tryptone 10.0 g
Yeast extract 1.5 g
Glucose 10.0 g
Bromocresol purple 15 mg
Agar 12.0 g
Water 1L
pH 7.0 ± 0.2 at 25°C
Dispense in 10 mL amounts in test tubes or 15 ml amounts in universal containers.
Nutrient agar
Meat extract 10.0 g
Peptone 10.0 g
Agar 15.0 g
Water 1L
pH 7.5 ± 0.2 at 25°C
Plate Count agar

Yeast extract 2.5 g
Enzymatic digest of casein 5.0 g
Glucose 1.0 g
Agar 12.0 g
Water 1L
pH 7.0 ± 0.2 at 25°C
Oxidase reagent (prepare fresh as required or use commercially available equivalent)
6.0 SAMPLE PROCESSING

6.1 Preparation of the sample
6.1.1 Detection
Weigh x g (usually 10 g) of sample, add 9x mL (usually 90 mL) of single strength buffered
peptone water and homogenise. Incubate as described in section 6.2 and continue as
described in subsequent sections.

6.1.2 Enumeration
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 Pre-enrichment
If a low level of detection is required (<10/g or mL), add 10 mL of test sample if liquid, or 10
mL of the 10-1 suspension, to each of three tubes containing double strength buffered
peptone water (BPW). Add 1 mL of the test sample if liquid, or 1 mL of the 10-1 suspension,
to each of three tubes containing single strength BPW. Add 1 mL of each further dilution,
as required, to each of three tubes containing single strength BPW. Carefully mix the
inoculum and the medium. Incubate all inoculated tubes at 30°C (if milk or dairy product) or
37°C (other food products) for 24 ± 2 hours.
Positive control: Inoculate a tube of single strength medium with Escherichia coli NCTC
9001.
Negative control: Inoculate a tube of single strength medium with Enterococcus faecalis
NCTC 775.
6.3 Enrichment
At the end of incubation transfer 1 mL from each tube to 10 mL of Enterobacteriaceae
Enrichment broth (EE). Incubate EE tubes at 30°C (milk and dairy products) or 37°C (other
food products) for 24 ± 2 hours.
6.4 Isolation
At the end of incubation subculture each tube to a third section of a VRBGA plate. Incubate
the plates at 30°C (milk and dairy products) or 37°C (other food products) for 24 ± 2 hours.
Examine the plates for the presence of purple-red colonies with a diameter of 0.5 mm or
greater, with or without a red zone of precipitated bile. Record the results.
6.5 Confirmatory tests

From each section of VRBGA showing presence of typical colonies select a well isolated
suspect Enterobacteriaceae colony to perform further tests.
Fermentation test
Prior to use, steam or boil the glucose agar for 10 minutes and allow to set. Inoculate by a
deep stab inoculation of tubes of glucose agar, then use the same inoculum to streak onto a
non-selective medium such as Nutrient Agar (NA) or Plate Count Agar (PCA). Place the
media in an incubator at 37°C for 24 + 2 hours. Enterobacteriaceae produce a yellow colour
throughout the glucose medium. Also examine the plate count agar to check for purity.
Oxidase test
Use the growth on PCA to perform an oxidase test according to Standard Method BSOP TP
26. Development of a purple colour within 10 seconds denotes a positive reaction.
Members of the Enterobacteriaceae are oxidase negative.

7.0 CALCULATION OF RESULTS
Count the number of tubes containing confirmed Enterobacteriaceae 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 (eg:
milk and dairy products), divide the MPN index by 10. If 10 mL of the undiluted liquid
sample has been used, divide the MPN index by 100. 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

8.1 Detection
Report the result as Detected or Not detected in the weight examined.
8.2 Enumeration
Report the result as the most probable number of Enterobacteriaceae 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 Enterobacteriaceae are
detected but are less than 1, 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 1 g 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 Confidence limits
-1 -2 -3
10 dilution 10 10 dilution MPN per Category > 95% > 99%
dilution g/ml
Lower Upper Lower Upper
limit limit limit limit
0 0 0 <3 0 9.4 0 14
0 0 1 3 3 0.1 9.5 0 14
0 1 0 3 2 0.1 10 0 16
0 1 1 6.1 0 1.2 17 0.5 25
0 2 0 6.2 3 1.2 17 0.5 25
0 3 0 9.4 0 3.5 35 1.8 46
1 0 0 3.6 1 0.2 17 0.1 25
1 0 1 7.2 2 1.2 17 0.5 25
1 0 2 11 0 4 35 2 46
1 1 0 7.4 1 1.3 20 0.6 27
1 1 1 11 3 4 35 2 46
1 2 0 11 2 4 35 2 46
1 2 1 15 3 5 38 2 52
1 3 0 16 3 5 38 2 52
2 0 0 9.2 1 1.5 35 0.7 46
2 0 1 14 2 4 35 2 46
2 0 2 20 0 5 38 2 52
2 1 0 15 1 4 38 2 52
2 1 1 20 2 5 38 2 52
2 1 2 27 0 9 94 5 142
2 2 0 21 1 5 40 2 56
2 2 1 28 3 9 94 5 142
2 2 2 35 0 9 94 5 142
2 3 0 29 3 9 94 5 142
2 3 1 36 0 9 94 5 142
3 0 0 23 1 5 94 3 142
3 0 1 38 1 9 104 5 157
3 0 2 64 3 16 181 10 250
3 1 0 43 1 9 181 5 250
3 1 1 75 1 17 199 11 270
3 1 2 120 3 30 360 20 440
3 1 3 160 0 30 380 20 520
3 2 0 93 1 18 360 12 430
3 2 1 150 1 30 380 20 520
3 2 2 210 2 30 400 20 560
3 2 3 290 3 90 990 50 1520
3 3 0 240 1 40 990 30 1520
3 3 1 460 1 90 1980 50 2830
3 3 2 1100 1 200 4000 100 5700
3 3 3 >1100
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 DETECTION
AND ENUMERATION OF ENTEROBACTERIACEAE
For detection weigh x g or mL and add 9 x g of BPW; homogenise
Ø
For enumeration prepare 10-1, 10-2 and 10-3 dilutions of the sample as required in MRD
(adjust pH of 10-1 if necessary to 6.8 ± 0.2)
Ø
For milk and most dairy products use 3 tubes of double strength BPW with 10mL of 10-1 dilution and
three tubes of single strength BPW with 1mL of 10-1 and 10-2 dilutions (detection limit 0.3/g)
For frozen dairy products and most other foods use three tubes of single strength BPW with 1mL of
10-1, 10-2 and 10-3 dilutions (detection limit 3/g)
Ø
Place in an incubator at 30°C ± 1°C (milk and dairy products) or 37°C ± 1°C (other products) for 24 ± 2 h
Ø
Transfer 1 mL of all tubes to 10 mL of EE broth and place in an incubator at 30°C ± 1°C (milk and dairy
products) or 37°C ± 1°C (other products) for 24 ± 2 h
Ø
Subculture all tubes to a 1/3 segment of VRBGA and incubate at 30°C ± 1°C (milk and dairy products)
or 37°C ± 1°C (other products) for 24 ± 2 h
Ø
Subculture a typical colony from each tube yielding growth of presumptive Enterobacteriaceae on VRBGA
to glucose agar and NA or PCA. Incubate media at 37°C ± 1°C for 24 ± 2h
Ø
Examine glucose agar for the presence of fermentation; examine PCA for purity and oxidase reaction
Ø
Count tubes which yield oxidase negative cultures that show fermentative growth as positive for
Enterobacteriaceae
Ø
Calculate the count per g or mL and report

REFERENCES
1. Department of Health NHS Executive: The Caldicott Committee. Report on the review of patient-
identifiable 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/eur-
lex/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. http://www.food.gov.uk/multimedia/pdfs/fh2regs05draftsiengland.pdf
5. SANCO 4198/2004 rev.19 (PLSPV/2001/4198/4198R19-EN.doc). Draft Commission Regulation on

Microbiological Criteria for Foodstuffs. 2005.
http://www.food.gov.uk/multimedia/pdfs/microcriteria2005reg.pdf
6. BS ISO 21528: Microbiology of food and animal feeding stuffs - horizontal method for the detection
and enumeration of Enterobacteriaceae. Part 1: Detection and enumeration by MPN technique with
pre-enrichment. London: British Standards Institution (BSI); 2004.


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NATIONAL STANDARD METHOD

DETECTION AND ENUMERATION

Issued by Standards Unit, Evaluations and Standards Laboratory

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

More details can be found on the website at www.evaluations-standards.org.uk. Contributions to the

Suggested citation for this document:

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

STATUS OF NATIONAL STANDARD METHODS ................................................................................ 2

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

Amendment Issue no. Insert Page Section(s) involved Amendment

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

3.0 SAFETY CONSIDERATIONS

Top pan balance capable of weighing 0.1 g

5.0 CULTURE MEDIA AND REAGENTS

Peptone saline diluent (Maximum recovery diluent)

Buffered peptone water (BPW)

Enterobacteriaceae Enrichment broth (EE)

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

Plate Count agar

Oxidase reagent (prepare fresh as required or use commercially available equivalent)

6.0 SAMPLE PROCESSING

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

6.5 Confirmatory tests

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

8.0 REPORTING RESULTS

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

For detection weigh x g or mL and add 9 x g of BPW; homogenise

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

5. SANCO 4198/2004 rev.19 (PLSPV/2001/4198/4198R19-EN.doc). Draft Commission Regulation on

DETECTION AND ENUMERATION OF ENTEROBACTERIACEAE

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