Detection and Enumeration of Bacteria in Swabs and Other Environmental Samples PDF

Detection and enumeration of bacteria
in swabs and other environmental

samples
Microbiology Services
Food Water and Environmental
Microbiology
Standard Method
FNES4 [E1]
Detection and enumeration of bacteria in swabs and other environmental samples
About Public Health England

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Issued by
PHE Microbiology Services
Food, Water & Environmental Microbiology
Methods Working Group
Version number 2 Effective Date 17.12.13
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Page 2 of 21
Contents
About Public Health England 2
Contents 3
Status of Microbiology Services Food, Water and Environmental Microbiology Methods 4
Amendment history 5
Introduction 6
Scope 6
Background 6
1.0 Principle 7
2.0 Definitions 8
3.0 Safety considerations 8
3.1General safety considerations 8
3.2Specific safety considerations 8
3.3Laboratory containment 8
4.0 Equipment 9
5.0 Media,reagents and issue of sampling equipment 9
5.1Commercially prepared dacron swabs with neutralising buffer 9
5.2Commercially prepared sponge swabs with neutralising buffer 9
5.3Commercially prepared viscose tipped swabs with Amies charcoal medium 10
5.4Sampling bags for collection of other environmental samples 10
5.5Media and Reagents 10
6.0 Sample processing 11
6.1Sample preparation and dilutions 11
6.2Inoculation and incubation 13
7.0 Quality control 14
8.0 Calculation of results 14
9.0 Reporting of results 15
9.1Enumeration 15
9.2Detection 16
10.0 Reference facilities and referral of cultures 16
11.0 Acknowledgements and contacts 16
References 18
Appendix 1: Swabbing for pathogens and indicator organisms using dacron tipped swabs.
19
Appendix 2: Sponge swabs instructions for use 20
Appendix 3: Detection and enumeration of bacteria in swabs and other environmental
samples 21
Page 3 of 21
Status of Microbiology Services Food,

Water and Environmental Microbiology
Methods
These methods are well referenced and represent a good minimum standard for
food, water and environmental microbiology. However, in using Standard Methods,
laboratories should take account of local requirements and it may be necessary to
undertake additional investigations.
The performance of a standard method 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, Public
Health England (PHE) 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. PHE should at all times be acknowledged.
Citation for this document:

Public Health England. Detection and Enumeration of Bacteria in Swabs and other
Environmental Samples. Microbiology Services. Food, Water & Environmental
Microbiology Standard Method E1; Version 2.
Page 4 of 21
Amendment history
Controlled document FNES4 (E1)

reference
Controlled document title Standard Method for Detection and Enumeration of Bacteria in
Swabs and other Environmental Samples.
The amendment history is shown below. On issue of revised or new documents each controlled document
should be updated by the copyholder in the laboratory.
Amendment Version no. Insert Page Section(s) involved Amendment

Number/ Discarded version
Date no.
2/17.12.13 1.0 2 All All Updated to reflect move
to PHE and document
renumbered as FNES4
following the
implementation of Q-
pulse for Document
Management
5
Introduction Clarification of the swab
types that can be used
for testing
6 1.0 Principle Clarification of the swab
types that can be used
for testing
7 3.0 H&S Statement related to use
considerations of coloured text to
highlight hazards and
controls.
9 5.6 Media & Information note added
reagents regarding the use of
MRD
10 6.0 Sample Statement added to
processing clarify reporting of
multiple pathogens for
single samples.
Approximate
measurement removed
and replaced with
gravimetric dilution.
13 9.0 Reporting Comment added with
regards to reporting
when entire sample has
not been tested.
Page 5 of 21
Introduction
Scope
This method is applicable to the procedures used for examination of environmental samples
including swabs from carcasses in meat processing plants, swabs of food preparation
surfaces and other environmental samples such as cloths collected from the food
manufacturing environment and bottle rinses.
This support method must be used in conjunction with accredited methods for the detection
of bacteria in foods and includes the use of three different types of swab.
Background
The significance of food contact surfaces in food safety is highlighted in the Food Safety
Act 19901 which states under section 29 (c) that authorised officers can take a sample
from any food source, or a sample of any contact material, which is found by him on or in
any such premises. This implies that environmental samples are to be considered as part
of legislation. EU Legislation also states that Sampling of the production and processing
environment can be a useful tool to identify and prevent the presence of pathogenic micro-
organisms in foodstuffs2, and cleanliness standards have also been laid down for meat
premises in European Law3. A recommendation to undertake environmental monitoring of
the food preparation environment during the investigation of poor microbiological results is
also given in the HPA Guidelines for Assessing the Microbiological Safety of Ready to Eat
Foods Placed on the Market4.
The environment can be contaminated with a variety of micro-organisms derived from

various sources. Estimation of the overall numbers of bacteria (Aerobic Colony Count, E.coli
and Enterobacteriaceae) present can provide useful information when assessing general
hygiene. Environmental monitoring can also be used as part of routine inspections of food
premises or in an investigation of a suspected food poisoning outbreak where surfaces are
thought to be likely vehicles of cross-contamination or if no food is available for examination.
Determination of the number of aerobic viable micro-organisms and the number of

Enterobacteriaceae on a specified area of a surface can provide an indication of the
cleanliness and allows monitoring of cleaning procedures over time.
Detection of Listeria species in a food processing premises can be used as an indicator of

poor cleaning. The presence of foodborne pathogens such as Listeria monocytogenes,
Salmonella, Campylobacter and E.coli O157 in a ready to eat food preparation area or retail
setting is of particular concern.
The main value of determining the colony counts on a surface is to assess the cleanliness of
that surface. If a swab is taken to demonstrate that procedures used for cleaning are
effective, swabbing should be performed after the surface has been cleaned. If it is
suspected that cleaning is not being done or the methods used for cleaning are not effective
then swabbing can be done as part of a routine hygiene inspection without prior cleaning. A
Page 6 of 21
high count can be an early warning of inadequate cleaning practices in food premises and
may highlight the need for further investigation but careful consideration must be given if the
surface is in use at the time of sampling. Cleaning of a surface prior to swabbing during
outbreak investigations should be avoided to maximise the possibility of detecting a
pathogen.
The method of choice for examination of surfaces is swabbing of a known area (10-100cm2)
using a sterile swab that has been moistened in 10mL of neutralising diluent. This semi-
quantitative approach enables enumeration of the micro-organisms per cm2 and can facilitate
interpretation of the results. When qualitative or detection tests only are to be performed the
area swabbed need not be known but this may limit the laboratorys ability to interpret the
significance of the results obtained. It is assumed that the sample has been taken by a
competent sampling officer who has been trained to carry out this procedure.
Microbiological examination of cleaning cloths can also be useful both in monitoring

general hygiene and in the investigation of outbreaks, since these cloths are frequently
used to wipe food preparation surfaces, and therefore have the potential to spread
microbial contamination over the surfaces. Since many cloths are stored wet and may
contain food debris, they have the potential to support the growth of high numbers of
bacteria if not sanitised appropriately. Both enumeration of hygiene indicators and
detection of pathogens can be performed on these samples by rinsing the entire cloth in
diluent or a growth medium.
1.0 Principle
Three types of commercially available swabs are issued to customers by PHE
laboratories. Two contain a measured volume of commercially prepared neutralising buffer
or neutralising diluent while the third has Amies charcoal media. Neutralising buffer/diluent
is used to neutralise the effect of disinfectants that have been used to clean surfaces,
charcoal containing media has a similar effect. Surfaces are generally swabbed by the
customer (with the exception of complex investigations where laboratory staff may be
asked to assist with sampling). The laboratory provides the customer with instructions and
offers training for use of the swabs but the effectiveness of the swabbing procedure used
is often outside the control of the laboratory.
When enumeration tests are required a template enclosing a known surface area (eg10cm
x10cm) is used. Swabs in diluent are refrigerated at 2-8oC and are submitted to the
laboratory as soon as possible to ensure that they are examined on the day of collection or
at least within 24 hours of collection. The swabs are vortex mixed or homogenised to aid
release of organisms into the diluent. The swab diluent and serial dilutions if necessary are
subjected to the same methods as used for examination of food homogenates.
The target count for enumeration tests is in the range of 0 to 300 colony forming units (CFU)
per plate. Samples likely to have counts that exceed the countable limit per plate (method
dependent) must be diluted.
Page 7 of 21
Detection of pathogens from a known surface area can be performed by addition of
enrichment media to the swab sample. In outbreak situation a large random area can also
be sampled to maximise the likelihood of detection.
Enumeration and detection test can be performed on cloths and other environmental
samples such as bottle rinses by adding measured volumes of diluent or enrichment
media.
2.0 Definitions
For definitions of target organisms refer to the corresponding PHE standard methods for
foods for the bacteria of interest.
3.0 Safety considerations

3.1 General safety considerations
Normal laboratory precautions apply 5-6.

All laboratory activities associated with this Standard Method must be risk assessed locally
to identify hazards 7-8. Appropriate controls must be in place to reduce the risk to staff or
other groups. Staff must be trained to perform the activities described and must be
provided with any personal protective equipment (PPE) specified in this method. Review of
this method must also include a review of local risk assessments to ensure that controls
are appropriate and effective.
Information Note: Throughout this method hazards are identified using red text. Where
a means of controlling a hazard has been identified this is shown in green text.
3.2 Specific safety considerations
Refer to individual food methods for specific safety considerations
3.3 Laboratory containment
Swabs, cloths or other environmental samples that are considered to be high risk for the
isolation of VTEC O157, must be handled in a CL3 Laboratory.
Page 8 of 21
4.0 Equipment
Top pan balance capable of weighing to 0.1g
Stomacher
Vortex mixer
Stomacher bags (sterile)
Other equipment as detailed in PHE, MS, FW&E Standard Methods
5.0 Media,reagents and issue of

sampling equipment
5.1 Commercially prepared dacron swabs with neutralising buffer
These are stored at ambient temperature and must be used before the displayed expiry
date. Instructions for use and a template are issued to the customer (see Appendix 1).
10mL neutralising buffer with dacron tipped swab (egTechnical Service

Consultants Ltd, product code TS5-42)
Sterile templates (eg 10cm x 10cm plastic templates, Technical Service
Consultants Ltd, product code TS15-T)
5.2 Commercially prepared sponge swabs with neutralising buffer
These are stored at ambient or under refrigeration (2 25C) and must be used before the
displayed expiry date. They should not be used if they appear to have dried out during
storage (the chance of swabs drying out during their shelf-life may be reduced by storing
between 2 and 8C). Instructions for use with a template are issued to the customer (see
Appendix 2).
SpongeSicle swabs or sponge swabs containing 10mL neutralising buffer (eg

3M, product code SSL-10NB)
Sterile templates (eg 10cm x 10cm plastic templates, Technical Service
Consultants Ltd, product code TS15-T)
Page 9 of 21
5.3 Commercially prepared viscose tipped swabs with Amies charcoal

medium
These swabs are used for the detection of a single pathogen only. They are stored at
ambient or under refrigeration (2 25C) and must be used before the displayed expiry
date.
Amies charcoal swabs (eg Technical Service Consultants Ltd, product TS-5
18)
5.4 Sampling bags for collection of other environmental samples
Other environmental samples including cloths or utensils can be collected in sampling

bags with wire closures or in a food grade bag. Bottles to be rinsed should be capped (if
possible) and placed in a food grade bag. Food grade bags are not provided by the
laboratory.
5.5 Media and Reagents
Equivalent commercial dehydrated media may be used; follow the manufacturers

instructions.
Peptone Saline Diluent (PSD)
Peptone 1.0 g
Sodium chloride 8.5 g
Water 1L
pH 7.0 0.2 at 250C
Information note: This media is often referred to as Maximum Recovery Diluent

(MRD) in the laboratory
Buffered peptone water (ISO formulation)
Enzymatic digest of casein 10.0 g

Disodium hydrogen phosphate dodecahydrate 9.0 g
or anhydrous disodium hydrogen phosphate 3.5 g
Potassium dihydrogen phosphate 1.5 g
Water 1L
pH 7.0 0.2 at 25C
Page 10 of 21
Neutralising Diluent (ISO 18593-3)
Sorbitan monooleate (Polysorbate 80) 30.0 g

Saponin 30.0 g
Sodium thiosulphate 5.0 g
Lecithin 3.0 g
Peptone 1.0 g
L-histidine 1.0 g
Water 1L
pH 7.0 0.2 at 250C
Refer to individual food test methods for culture media and reagents.
6.0 Sample processing

The diluent used will depend on the tests to be performed. If enumeration tests only are
being performed on a swab Peptone Saline Diluent (PSD) can be used as the diluent. If
the swab will be subjected to detection of Salmonella species, in addition to enumeration
tests BPW can be used. Detection of pathogens other than Salmonella from swabs
requires the addition of selective enrichment media. Due to this only one pathogen can be
sought per swab. Cloths or other environmental samples for detection of a single pathogen
are prepared by adding 500mL of the appropriate enrichment medium. If a cloth or other
environmental sample is being investigated for more than one pathogen by enrichment or
if enumeration tests are being performed in addition to pathogen testing neutralising
diluent should be added, the sample homogenised and the resulting liquid aliquoted to
perform these tests. The resulting suspension can be mixed with the enrichment media for
the pathogens of interest. If this is performed a comment must be added to the report to
reflect that the entire sample was not tested (see section 9).
6.1 Sample preparation and dilutions
6.1.1 Preparation of dacron swabs
The sample comprises a swab in a tube of 10 mL neutralising buffer.
Preparation for enumeration tests

Transfer the neutralising buffer and swab tip to a sterile universal container. Vortex mix the
universal container for a total of one minute. All vortexed universals must be allowed to
stand for 5 minutes to allow settling of aerosols. This is equivalent to a 100 dilution and
gives a lower limit of detection of 10 CFU per swab if 1mL is plated. Further dilutions can be
performed if required.
Page 11 of 21
Preparation for detection tests

Transfer the contents of the universal (including the swab tip) to a sterile bag with wire
closures. Add the appropriate enrichment medium for the test required to gravimetrically
prepare a 10-1 dilution.
6.1.2 Preparation of sponge swabs
The sample comprises of a sponge moistened in 10mL of neutralising buffer inside a

plastic bag with wire closures. The handle should have been aseptically removed at the
time of use. If all, or a portion of the handle is still in place this should be aseptically
removed by pushing it up through the bag and snapping it off. It must be noted on the
report that the handle was not fully removed.
Preparation for enumeration tests

Add 90 mL of diluent to the bag containing the sponge swab to prepare a 10 -1 dilution.
Place the plastic bag with sponge swab inside a stomacher and homogenise for between
30 seconds and 3 minutes. The homogenisation time required will depend on the
manufacturer instructions.
This gives rise to a lower limit of detection of 100 CFU per swab if 1mL is plated. Lower limits
of detection can be achieved by reducing the amount of diluent added eg 10 mL can be
added equivalent to a 1 in 2 dilution giving a limit of detection of 20 CFU per swab.

Add 90mL of the required enrichment medium to the swab bag and homogenise for
between 30 seconds and 3 minutes in a stomacher. The homogenisation time required will
depend on the manufacturer instructions.
6.1.3 Preparation of charcoal swabs
The sample comprises a swab in a tube of Amies Charcoal medium.

Transfer the contents of the tube (including the swab tip) to a sterile bag with wire
closures. Add the appropriate enrichment medium for the test required to cover the swab
and prepare a a 10-1 dilution gravimetrically and homogenise for between 30 seconds and 3
minutes in a stomacher. The homogenisation time required will depend on the manufacturer
instructions of the equipment used.
6.1.4 Preparation of cloths and other environmental samples

The cloth samples that will be submitted are likely to vary considerably in their size and
degree of moisture content. It may also be the case that the cloth contains disinfectant and
it is important that an attempt to dilute this disinfectant is made. It is therefore
recommended that 500mL of diluent is added to the cloth regardless of size and moisture
content. The diluent added will depend on the tests to be performed. It is recommended
that if enumeration tests only are being performed neutralising diluent is used. The sample
should be homogenised for between 30 seconds and 3 minutes in a stomacher. The
homogenisation time required will depend on the manufacturer instructions and the sample.
Where this is not possible the sample should be manipulated manually for at least 2
Page 12 of 21
minutes. This resulting suspension is considered to be the neat or 100 dilution. Where
enumeration is being performed in addition to the detection of pathogens neutralising
diluent should be used and 25mL, aliquoted to 225mL of the appropriate enrichment
media.
Other environmental samples including scrubbing brushes, nozzles and utensils are
placed in a sampling bag. When enumeration tests are being performed at least 100mL of
the appropriate diluent is added. These samples must be manipulated manually for at least
2 minutes, again the resulting suspension is considered to be the neat or 100 sample.
When carrying out a pathogen detection test, sufficient enrichment media must be added
to fully immerse the sample.
6.1.5 Preparation of bottle rinses
The bottle submitted for rinsing is removed from the sample bag aseptically. If the bottle is
intended for single use, add 20mL of PSD. If the bottle is re-usable and has been washed
add 20mL of neutralising diluent. The internal surfaces of the bottle are thoroughly rinsed
by rotating the bottle gently 12 times in one direction. The bottle is allowed to stand for 15-
30 minutes. Rinse the internal surfaces of the bottle again by rotating the bottle gently 12
times in one direction9. Using a sterile pipette transfer the bottle rinse fluid to a sterile
universal container. This is the neat or 100 sample. When carrying out pathogen detection
tests, add 100 mL of enrichment media to the bottle, rinse the bottle as described above
and transfer the broth to a closure bag or sterile container.
6.2 Inoculation and incubation
6.2.1 Enumeration methods
For all samples, dilutions should be selected that will give colony counts within the
appropriate range for the test being performed.
Proceed using PHE MS FW&E Standard methods for Foods.
Using a separate sterile pipette for each dilution, aseptically transfer 0.5mL or 1mL
volumes (pour plates methods only) of the initial dilution (10 0 or 10-1) of the sample to the
appropriate medium. If necessary repeat the procedure with further decimal dilutions
prepared in PSD.
6.2.2 Detection methods
For all samples gravimetrically prepare a 10-1 dilution in the appropriate enrichment
medium. Enumeration tests can also be performed when samples have been diluted in
BPW. If multiple pathogen tests are to be performed sample homogenates can be
aliquoted to the appropriated enrichment media to achieve a 10-1 dilution.
Proceed using PHE MS FW&E Standard methods for Foods.

Page 13 of 21
7.0 Quality control

Quality control of media and internal quality assurance checks should be performed
according to in-house procedures. See PHE MS FW&E Standard methods for Foods for
each method.
8.0 Calculation of results

For enumeration tests counts should be calculated, where possible, using dilutions giving
15 to 150 or 300 colonies on the plate. The upper countable limit used is method
dependent; please refer to individual food methods.
Calculate count per swab or cloth as follows:
Count = _____C_____ x n3
v (n1 + 0.1n2)d
where : -
C is the sum of colonies on all plates counted;

v is the volume applied to each plate;
n1 is the number of plates counted at the first dilution;
n2 is the number of plates counted at the second dilution;
n3 is the original volume of neat suspension (ie 10 for swab, 500 or 100 for other
samples);
d is the dilution from which the first count was obtained eg10-2 is 0.01;
If the swab is from a measured area the count can be divided by the area swabbed in cm2.
eg Sponge swab, area swabbed 100cm2, tested for Enterobacteriaceae.
45 colonies confirmed at 10-2 dilution.
Count = _____45_____ x 10
1(1 + 0)0.01
=4.5x104
This count is the count per swab so to calculate the result per cm 2 it is necessary to divide
this by the area swabbed which in this case is 100.
Result per cm2 is 4.5x102 CFU per cm2.

Page 14 of 21
eg Dacron swab, area swabbed 100 cm2 tested for E.coli.
Example: 15 colonies on plate 1 and 18 colonies on plate 2 at 10 0 dilution (neat).
Count = _____33_____ x 10
0.5 (2 + 0)1
=3.3x102
This count is the count per swab so to calculate the result per cm 2 it is necessary to divide
this by the area swabbed which in this case is 100.
Result per cm2 is 3.3 CFU per cm2
9.0 Reporting of results

The test report must specify the method used, all details necessary for complete identification
of the sample and details of any incidents that may have influenced the result.
Take as the result the count per swab, per cloth, per item or per cm2 expressed as a whole
number to two significant figures (if less than 100).
If count is 100 or more, report counts with one figure before and one figure after the
decimal point in the form of:
a x 10b CFU per swab, cloth, item, or cm2
where a is never less than 1.0 or greater than 9.9 and b represents the appropriate power
of ten. Round counts up if the last figure is 5 or more, and down if the last figure is 4 or
less.
eg : 1920 CFU per swab = 1.9 x 103 CFU per swab

235,000 CFU per swab = 2.4 x 105 CFU per swab
9.1 Enumeration
9.1.1 Area swabbed not specified
Report the count as CFU per swab.
9.1.2 Area specified
Report as CFU per cm2. Include a comment on the report (eg Results expressed per cm2
are calculated using the area tested as stated by the customer).
Page 15 of 21
9.1.3 Cloth or other environmental samples
Report the count as CFU per cloth or per sample.
9.1.4 Bottle rinses
Report the count as CFU per bottle.
9.2 Detection
If a single pathogen test has been carried out only ie no other tests performed on the sample
Report as:-
Not Detected per swab, per cloth or per item.
or
DETECTED per swab, per cloth or per item.
If more than one pathogen test has been carried out (cloths or other samples only) or
enumeration tests have been performed in addition to Salmonella (swabs) report as in swab
or in item with the comment Entire sample not tested
10.0 Reference facilities and referral

of cultures
Any pathogen detected in a swab or in other environmental samples, particularly during
the investigation of an outbreak must be referred to a reference laboratory for further
typing. Please refer to advice provided in individual food methods.
11.0 Acknowledgements and contacts

This Standard Method has been developed, reviewed and revised by Microbiology
Services, Food, Water and Environmental Microbiology Methods Working Group.
Page 16 of 21
The contributions of many individuals in Food, Water and Environmental
laboratories, reference laboratories and specialist organisations who have provided
information and comment during the development of this document are
acknowledged.
For further information please contact us at:
Public Health England

Microbiology Services
Food Water & Environmental Microbiology Laboratories
Central Office
Colindale
London
NW9 5EQ
E-mail: [email protected]
Page 17 of 21
RE
References
1. Food Safety Act 1990.
2. European Commission. 2005. Commission regulation (EC) No 2073/2005 of 15

November 2005 on microbiological criteria for foodstuffs. Official Journal of the
European Union L 338 22.12.2005:1-26.
3. European Commission. 2001. Commission decision 2001.471/EC of 8 June 2001

laying down rules for the regular checks on the general hygiene carried out by
operators in establishments according to Directive 64/433/EEC on health conditions
for the production and marketing of fresh meat and Directive 71/118/EC on health
problems affecting the production and placing on the market of fresh poultry meat.
L165 21.06.2001;48-53.
4. Health Protection Agency. 2009. Guidelines for assessing the microbiological safety
of ready-to-eat foods placed on the market. London: Health Protection Agency.
5. Health and Safety Executive. Biological Agents: Managing the risks in laboratories
and healthcare premises; 2005. http://www.hse.gov.uk/biosafety/biologagents.pdf
6. Control of Substances Hazardous to Health Regulations 2002. General COSHH.

Approved Code of Practice and Guidance, L5. Suffolk: HSE Books; 2002.
7. Health and Safety Executive. Five steps to risk assessment: a step by step guide to
safer and healthier workplace, IND(G) 163 (REVL). Suffolk: HSE Books; 2002.
8. Health and Safety Executive. A guide to risk assessment requirements: common

provisions in health and safety law, IND (G) 218 (L). Suffolk; HSE Books; 2002.
9. Roberts, D and Greenwood, M (Editors) Practical Food Microbiology, 3rd Edition,

2003
Page 18 of 21
Appendix 1: Swabbing for pathogens and

indicator organisms using dacron tipped
swabs.
This procedure can be used after cleaning to assess cleaning standards it can also be used if no cleaning protocol
is in place. During an outbreak investigation swab surfaces prior to cleaning when possible.
Consumables (supplied by the laboratory on request) Supplier/product code

1 x sterile swab with 10ml Neutralising Buffer TSC Ltd, TS5-42
2
1 x sterile 100cm template TSC Ltd, TS 15-T
1. Put on a clean overcoat and wash hands thoroughly prior to sampling. Sterile gloves are not necessary
unless the sample is likely to lead to legal proceedings.
2. Select the area to be investigated. If the surface is not flat and indicator organisms are being sought
estimate the area to be swabbed.
3. If the area is flat remove a template from its bag by the handle and hold it between the thumb and index
finger of one hand. Ensure that the template does not come into contact with any other object and place in
the selected position.
4. Open the swab pack (do not remove the sterile swab from the pack at this stage).
5. Remove the lid from the tube containing neutralising buffer.
6. Remove the swab from the packaging and insert it into the neutralising buffer to moisten it. Squeeze
excess liquid from the swab by pressing the cotton tip against the side of the tube.
7. Replace the lid on the neutralising buffer tube.
8. Using the moistened swab, swab the entire template area for 30 seconds as shown in Figure 1 using firm
even pressure and rotation of the swab.
9. Remove the cap of the tube containing neutralising buffer and insert the swab. The top of the swab then
becomes the lid of the tube. Push swab firmly in place until it is fully engaged-it should click.
10. The sample should be labelled appropriately and placed in a suitable cool box in an upright position and
transported to the laboratory as soon as soon as possible to ensure it is tested on the same day or at
least within 24 hours of collection.
If indicator organisms are being sought the area swabbed in cm2 or per 100cm2 needs to be known to enable
meaningful examination. If pathogens are being sought the area swabbed is less critical in the examination.
It is essential to clearly indicate which investigations are required.
Figure 1
A B C
Page 19 of 21
Appendix 2: Sponge swabs

instructions for use
1. Check that the swab is 2. Tear open the bag along the
within its shelf-life, the swab perforation. Feed the Sponge handle
appears wet and that the through the opening, by pushing up
bag is intact. from outside the bag. Grasp the
handle above the thumbstop and
Please note: before swabbing ensure remove the Sponge from the bag. To
that the sponge is moist but not over avoid contamination do not touch the
wet by squeezing excess diluents into space between the thumbstop and
the sample bag . sponge, or the sponge itself.
thumbstop
4. Return the sponge part of 5. Discard the

the Sponge to the sterile handle. Roll
sample bag. Do not insert down the top
the handle past the edge of the
thumbstop. Break off the bag and
handle by holding the secure with
sponge in place from wire tabs.
3. Using both sides of the sponge outside the bag and
swab the sides
surface back and Sample
3. Using both of the sponge bending the handle back
forth thefor surface
a period of sponge is
swab back and and forth at the point
approximately ready for
forth for a1 minute.
period If of a where it joins onto the
template is being laboratory
approximately 30 used swabIf in
seconds. a sponge (do not try to
all direction withinused
the template examination
template is being swab in break at thumbstop). The
area. directions and must be
all within the sponge will drop to the transported
template area. bottom of the bag. at 2-8oC
Page 20 of 21
Appendix 3: Detection and enumeration

of bacteria in swabs and other
environmental samples
Enumeration methods Detection methods
-1
For swabs test neat or prepare a For swabs prepare an approximate 10 dilution of
-1
1:2 or 10 dilution in PSD or BPW. the sample in the required enrichment medium.
For cloths add 500mL of ND For cloths add 500mL of ND, homogenise and
aliquot to the required enrichment media
Mix by vortexing or in a stomacher Mix by vortexing or in a stomacher

for 30secs to 3mins for 30secs to 3mins

Incubate and proceed as for the
Prepare further dilutions in PSD if HPA Standard method for food
required
Inoculate appropriate medium for Report as pathogen

organisms of interest and proceed
as for HPA Standard method for DETECTED
food or
Not Detected

Count and confirm colonies as for If required refer isolate to the
the HPA Standard method for food appropriate reference laboratory

2
Calculate Count per swab, per cm ,
per cloth or per item
Page 21 of 21

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Detection and enumeration of bacteria

in swabs and other environmental

About Public Health England

Public Health England

Version number 2 Effective Date 17.12.13

Crown copyright 2014

You can download this publication from

Published June 2014

This document is available in other formats on request.

Version number 2 Effective Date 17.12.13

Version number 2 Effective Date 17.12.13

Status of Microbiology Services Food,

The performance of a standard method depends on the quality of reagents,

Citation for this document:

Version number 2 Effective Date 17.12.13

Controlled document FNES4 (E1)

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

Version number 2 Effective Date 17.12.13

The environment can be contaminated with a variety of micro-organisms derived from

Determination of the number of aerobic viable micro-organisms and the number of

Detection of Listeria species in a food processing premises can be used as an indicator of

Microbiological examination of cleaning cloths can also be useful both in monitoring

Version number 2 Effective Date 17.12.13

3.0 Safety considerations

Normal laboratory precautions apply 5-6.

3.2 Specific safety considerations

Refer to individual food methods for specific safety considerations

3.3 Laboratory containment

Version number 2 Effective Date 17.12.13

5.0 Media,reagents and issue of

10mL neutralising buffer with dacron tipped swab (egTechnical Service

5.2 Commercially prepared sponge swabs with neutralising buffer

SpongeSicle swabs or sponge swabs containing 10mL neutralising buffer (eg

Version number 2 Effective Date 17.12.13

5.3 Commercially prepared viscose tipped swabs with Amies charcoal

5.4 Sampling bags for collection of other environmental samples

Other environmental samples including cloths or utensils can be collected in sampling

5.5 Media and Reagents

Equivalent commercial dehydrated media may be used; follow the manufacturers

Peptone Saline Diluent (PSD)

pH 7.0 0.2 at 250C

Information note: This media is often referred to as Maximum Recovery Diluent

Buffered peptone water (ISO formulation)

Enzymatic digest of casein 10.0 g

pH 7.0 0.2 at 25C

Version number 2 Effective Date 17.12.13

Neutralising Diluent (ISO 18593-3)

Sorbitan monooleate (Polysorbate 80) 30.0 g

pH 7.0 0.2 at 250C

6.0 Sample processing

6.1 Sample preparation and dilutions

6.1.1 Preparation of dacron swabs

The sample comprises a swab in a tube of 10 mL neutralising buffer.

Preparation for enumeration tests

Preparation for detection tests

6.1.2 Preparation of sponge swabs

The sample comprises of a sponge moistened in 10mL of neutralising buffer inside a

Preparation for enumeration tests

Preparation for detection tests