A Guide to

Environmental
Microbiological Testing
for the Food Industry
CONTENTS PAGE

1) Introduction 3

2) Environmental Sampling Programs 4

3) Features and Benefits 9

4) Kit Description 10

5) Procedure 11

6) Performance Characteristics: 15

i. Pre-moistened Swabs 15

ii. Coliform Detection Broth 15

iii. Salmonella Detection Broth 20

iv. Listeria Detection Broth 23

7) Positive Results – What Do They Mean? 29

INTRODUCTION
.
The Food Safety Pyramid (Figure 1.) provides food manufacturers and processors at all levels with
a simple structure to enable the protection of both the products manufactured by the company from
contamination by microorganisms (which have the potential to cause food spoilage and food
poisoning), and the customers who may later consume these products.

SSOP’s

HACCP

Environmental
Monitoring

Good Manufacturing
Practices

Figure 1. Food Safety Pyramid (SSOP’s

– Sanitation Standard Operating Procedures)

Microorganisms are always present in food handling environments. These microorganisms can be
characterised as belonging to 2 distinct groups: transient and resident. Transient microorganisms
are usually introduced into the food environment through raw materials, water and employees.
Normally the routine application of good sanitation practises are able to kill these organisms.
However, if contamination levels are high or sanitation procedures are inadequate, transient
microorganisms may be able to establish themselves, multiply and become resident. Organisms
such as Coliforms and Salmonella spp. and Listeria spp. have a well established history of
becoming residents in food handling environments.

Environmental Monitoring

Food processors should employ environmental sampling programs to monitor for general levels of
hygiene (the efficacy of general cleaning and sanitation for the removal of transient
microorganisms) or indicator testing may be achieved through a variety of methods including visual
inspection, ATP monitoring or the detection of surface protein residues. In addition, pathogen
specific environmental sampling should be undertaken to monitor for the presence of specific
pathogens that may be present as transient or resident microorganisms. The detection of specific
pathogens serves two important roles. Firstly it highlights the presence of important food pathogens
which may have been introduced into a food handling environment but may not have been
eliminated by routine sanitation practises and therefore may be passed onto other food materials
being processed. Secondly, it assists in determining sources of these important pathogens that
may be resident.

3
Environmental Sampling Programs

Introduction
All food handling companies and establishments should employ an environmental
sampling program to monitor for food spoilage microorganisms and food poisoning
pathogens. Such a program, if well designed will enable the detection of unacceptable
microbial contamination in a timely manner. Over the last decade environmental monitoring
has changed from essentially random sampling, employing imaginary grids over a
production area and testing points within each grid, to current methods that are focussed
on risk assessment to determine the most appropriate methods for monitoring. Sampling
programs should include the collection of samples during production on a regular basis
from work surfaces in a randomised manner which will reflect the differing working
conditions. In addition, samples should be taken from these sites after sanitising and from
sites which may serve as harbours of resident organisms.

Sampling should not only be conducted on food contact surfaces, but the evaluation of
non-food contact surfaces such as conveyor belts, rollers, walls, drains and air is equally as
important as there are many ways (aerosols and human intervention) in which
microorganisms can migrate from non-food contact surfaces to food.

The results of these samples should be tabulated as soon as available and in such a way
that they can be compared with previous results in order to highlight trends.

Determining the Frequency of Monitoring

The development of an effective environmental monitoring program should reflect a
balance between employing the available resources efficiently and monitoring at sufficient
intervals so as to ensure that a meaningful picture of the levels and nature of bacterial
contamination can be obtained.

When establishing an environmental monitoring program, the frequency of monitoring

different areas may be determined based on “Criticality Indexes” relevant to each specific
processing area or environment, or by using a Zones of Risk System.

Criticality Indexes

The development of a criticality program on which monitoring frequencies can be

based should be focussed on the targeting of the critical steps in the manufacturing
process. Therefore, the final manufactured product should receive more
monitoring than early manufacturing steps i.e. end product testing of product.

The use of “criticality indexes” provides a means whereby the frequency of

monitoring can be assigned to each designated critical area. The assessment of
risk should be based on the potential impact any risk may have on the final quality
or safety of the products being manufactured e.g. exposure to low temperatures
would constitute a low risk whilst the presence of water or warmer temperatures
would constitute a high risk.

The scheme employed by each food handling or manufacturing environment will

necessarily be unique to the processes and types of food being handled in that
environment. An example of how such a scheme can be constructed is shown in
Table 1.

4
In the development of such a scheme, all manufacturing areas should be
evaluated against a series of guiding questions which may include:

Higher Weighting should be given to:

x Dirtier activities.
x Areas where dirty activities are performed in close relative
proximity to clean areas.
x Areas which are often wet.
x Areas with open drains.
x Areas with high levels of staff activity.

Higher Monitoring Frequencies should be assigned to:

x Warm or ambient handling areas as apposed to cold rooms.

x Areas with sinks, drains or ongoing wetness as opposed to
dry areas.
x Areas where unprocessed raw foods are handled.
x Product filling.
x Packaging
Criticality Index Frequency of Monitoring
1 Daily or Each Batch
2 Weekly
3 Fortnightly
4 Monthly
5 Three Monthly
6 Six Monthly

Table 1. Criticality Indexes and Monitoring Frequency

Once the critical factors have been established a final Monitoring Schedule can be
developed (Table 2.).

Most food manufacturing processes involve one or more steps that effectively kill
pathogenic bacteria, although manufacturing involving the production of fresh
(salads etc), frozen products (vegetables, meats, poultry and fish) or some dairy
products may not. In manufacturing in which processes designed to kill bacteria
are employed, the challenge is to prevent the processed food from becoming
recontaminated. In these situations, food handling surfaces and possibly
equipment become contaminated by bacteria travelling through the food
processing environment through a series of steps before finally coming into contact
with the food that has been processed. Listeria species for example can multiply
rapidly to high numbers on wet areas such as floors and drains and then be
transferred to conveyor belts and benches through human intervention or the use
of high pressure water hose that both can result in the production aerosols . Any
processed food that subsequently touches these surfaces may the become
recontaminated.

In the development of any monitoring program, post-processing environmental

monitoring should always be considered as likely areas where pathogens may
reappear and contaminate food post-processing. Generally, these post processing
environments should be relatively free of bacteria when production commences.
After periods of production, it should be expected that the level of bacterial

5
contamination of these areas should increase. However, the presence of
microorganisms normally present in the pre-processed foods should not be
expected.

Criticality Likelihood of Impact Definition Frequency of

Index on Finished Product Monitoring
Mixing and Filling
1 Highly Likely Machines work places Daily or Each Batch
are sanitised daily
Packaging areas or
2 Likely areas in which final Weekly
handling is performed
Areas where process
3 Moderately Likely food is exposed to the Fortnightly
environment
Cold areas where little
4 Unlikely or no processing is Monthly
performed
Areas in which indirect
exposure to prepared
5 Very Unlikely Three Monthly
and packaged product
is unlikely
Any are that is
uncontrolled or where
microbial
6 Highly Unlikely Six Monthly
contamination is very
unlikely such as
freezers.

Table 2. Monitoring Schedule Based on the Determination of Criticality

Factors.

Zones of Risk System Approach

A more simplistic approach that can be adopted is to employ a program based

around zones that have different levels of risk.

A typical three zone plan for example would divide a production facility into zones
that cover low, medium and high risk areas within the production facility, with high
risk zones being those in which their is direct contact between food products and
surfaces (International Commission on Microbiological Specifications of Foods.
2002. Microorganisms in Foods. 7. Microbiological Testing in Food Safety
Management. Blackwell Scientific. London.). These high risk areas would be those
most stringently monitored. (See Table 3.)

Zone Food Contact Surfaces

After dryer
Pipes
1
Conveyor belts
Silos
Lids,
Covers
2
External
Surfaces of Silos
Floors
3 Walls
Pipes

Table 3. Zones of Risk Analysis – Milk Powder Manufacturer

6
What to Monitor

Organisations involved in food handling should employ environmental monitoring as a means of :

1. Monitoring the general levels of hygiene within the environment in question. The monitoring
of the general level of hygiene provides an overall impression of the level of cleanliness
within the test environment – it measures the efficiency of the general cleaning and
sanitation procedures in place and their ability to remove food residues and transient
microorganisms. A variety of methods are available to achieve this task, including general
physical inspections, ATP Monitoring Systems and the detection of the presence of food
residues (generally protein).

2. Environmental microbiological monitoring for the presence of specific pathogens within the
processing environment. The detection of specific pathogens serves two important roles:

a. It highlights the presence of important food pathogens which may have been
introduced into the food handling environment generally through human contact or
from raw ingredients, but which may not have been eliminated by routine cleaning
and sanitation procedures.
b. Secondly, it highlights the sources of these pathogens that may be resident in the
environments being tested.

Microbiological environmental monitoring should be used to indicate either unacceptable conditions

or practises which in turn should aid in controlling pathogenic bacteria such as Salmonella and
Listeria. The presence of Coliforms may also be valuable as they will provide an indication of the
general levels of microbiological cleanliness within test environments.

How to Collect Swab Samples for Microbiological Environmental Testing

At this point in time no detailed standard methods (AOAC, USDA FSIS, USFDA etc) exist for the
performance of microbiological environmental monitoring. ISO 18593:2004(E) Microbiology of food
and animal feeding stuffs – Horizontal methods for sampling techniques from surfaces using
contact plates and swabs does however provide a general platform for the critical steps that should
be considered in the development of testing procedures.

The key elements of this standard that should be taken into consideration include:

1. Moistened swabs should be employed for all sampling of surfaces. (Item 4.1.)
2. The solution used to moisten swabs should neutralise any detergents and sanitisers
employed. (Item 6.1.)
3. Swab moisturizer solution must preserves the integrity of the sample i.e. bacterial numbers
should remain constant until the sample collected onto the swab can be evaluated. (Item
6.1. and 7.)
4. Wherever possible the size of the area sampled should be greater than 100cm2. (Item 8.2.)
5. The analysis of the samples for specific pathogens is achieved by transferring the swabs
into an appropriate enrichment broth. (Item 8.2)
6. After enrichment transfer a sample to an appropriate agar plate medium for the target
organism being sought. (Item 8.3.4.)
7. Report the target microorganism as present or absent. (Item 9.2.5.)
8. The contact plate method (including dipslides, RODAC plates and 3M Petrifilm™) shall not
be used for the specific detection of pathogenic microorganisms. (Item 8.1.)

7
Detection of Target Microorganisms
As discussed above, the examination of environmental swabs for specific food pathogens is not
described in any specific standard methods, however the general principle that the analysis of the
samples for specific pathogens can be achieved by transferring swabs into an appropriate
enrichment broth can be applied to any specific pathogen being sought.

In such instances, methods such as AOAC and USDA FSIS methods can be applied to any specific
pathogen. In doing so however, it must be recognized that different methods employ different
culture media and brands (and hence recipes) of different culture media will vary in terms of their
selectivity, sensitivity and specificity. On this basis, although methods such as these may be
applied, results may vary.

Therefore, it should be standard practice that prior to the adoption of any method it should be
thoroughly evaluated to ensure that it is capable of recovering low numbers (<10 cfu) of the target
organism whilst inhibiting high numbers (>103 cfu) of potentially competing organisms. Any system
should also be evaluated to ensure that it is capable of recovering damaged organisms in low
numbers.

The recovery of Listeria monocytogenese for example may be achieved using one of the following
combinations employed in various standard methods. (Table 4.)

Method 24 Hours 48 Hours

USDA FSIS
UVM Broth Fraser Broth
Health Canada (HFLP-38,
PALCAM Broth UVM2 Broth
2002)
USFDA (BAM)
LEB LEB
AOAC/FDA

Table 4. Culture media suitable for the selective enrichment of Listeria spp.

Evaluation of the Results from Environmental Microbiological Testing

Following sampling and microbiological analysis a series of results will be available that provide an
indication of the overall levels of hygiene in the processing environments evaluated. This body of
information provides a valuable tool for maintaining and improving the quality and safety of
products. In addition, the detection of specific pathogens such as Salmonella and Listeria is critical
in ensuring food safety for the consumer.

It is not uncommon for food manufacturers only to react to unacceptable results when these
pathogens appear when final food products are evaluated however, it is important (particularly in
high risk products) that on-going environmental sampling practices are implemented and
performed. The evaluation of samples and sampling plans and the test data generated over
extended periods should lead to changes in test sample frequency and location, which in turn
should lead to improvements in cleaning and sanitising practises.

8
Path-Chek Hygiene Pathogen Detection
The Path-Chek Hygiene Pathogen Detection product range is a range of microbiological
environmental monitoring products that combine all of the requirements of the ISO Standard, ISO
18593:2004(E) Microbiology of food and animal feeding stuffs – Horizontal methods for sampling
techniques from surfaces using contact plates and swabs and the pathogen isolation, and detection
methods employed in pathogen isolation and detection methods such as those described in AOAC,
USDA FSIS, USFDA methods into a single convenient package.

Features
The following is a list of the key features of the Path-Chek Hygiene Pathogen Systems (Table 5.):

PRODUCT FEATURE BENEFITS TO USERS

x Meets the requirements of ISO 18593:2004(E)
Microbiology of food and animal feeding
stuffs – Horizontal methods for sampling
techniques from surfaces using contact
Pre-Moistened Swabs
plates and swabs.
x This improves the recovery of organisms from
both wet and dry surfaces, increasing the
sensitivity of the test.

The wetting agent neutralises the effects of residual

Swab Moisturiser Neutralises Detergents and detergents and sanitisers remaining on surfaces after
Sanitisers cleaning, this maintains integrity of the sample if such
residues are present.

The wetting agent ensures that the samples

introduced into the various Pathogen Detection
Swab Moisturizer Preserves the Integrity of the
Broths are representative of the sample taken. This is
Sample
especially important if there are delays in transferring
the sample swabs to the testing laboratory.
The shaft of the swabs used have a special “break
Swab sticks have special “break point” point” to simplify the transfer of samples into the
Pathogen Detection Broths.
Three different individual Pathogen Detection Broths
are currently available:
x Coliforms – generally used as an indicator of
overall hygiene conditions.
A Range of Individual Growth Media x Salmonella – an important cause of food
poisoning.
x Listeria – a very important foodborne organism
that causes a range of diseases with potentially
high mortality rates.
x Sensitivity – able to detect <1 organism per
10cm2 of surface tested.
High Sensitivity and Specificity
x Specificity – high levels of specificity minimize
problems with false positive tests.
The Specific Growth Media, the Methods of Use and
the Recommended Confirmation Methods all comply
Detection Media and Confirmation Methods
with recognised Food Standards such as USDA/
Compliant with International Standards
FSIS, US FDA etc. Meets laboratory accreditation
requirements.

Table 5. Key Features of Path-Chek Hygiene Pathogen Systems

9
Kit Description

Path-Chek Hygiene Pathogen Systems are a range of screening tests intended for use in food
handling and manufacturing environments and on food contact surfaces for the detection of
Coliforms, Salmonella ssp. Listeria species. Path-Chek Hygiene Pathogen Systems should be
considered as a fundamental component of Good Manufacturing Practice and an integral
component of any HAACP (Hazard Analysis Critical Control Points) plan, providing a highly
sensitive and specific indication of the presence of the foodborne pathogen being tested for.

Kit Contents
PC-010 Path-Chek Hygiene Coliform Detection Broth (3ml). 100 x 3ml

PC-020 Path-Chek Hygiene Salmonella Detection Broth (3ml). 100 x 3ml

PC-080 Path-Chek Hygiene Listeria Detection Broth (3ml). 100 x 3ml

PCS-100 Pre-moistened Path-Chek Hygiene swabs. 100 swabs

Instructions for use

Additional Materials

Incubator set to appropriate temperature

Rack to hold tubes during incubation
Template 10 x 10cm to assist with sampling

Note: The Path-Chek Hygiene Detection Broths (3ml) and Pre-moistened Path-Chek Hygiene
swabs, (CODE: PCS-100 per 100 swabs) are purchased separately and combined to create the
appropriate Path-Chek system.

Storage and Shelf Life

The Path-Chek Hygiene Detection Broths should be stored at 2 - 8°C when not in use. The
pre-moistened sample swabs should be stored at 4 - 25°C. Both components should not be used
after the expiry date printed on the carton label.

10
Procedure

Step 1

Carefully remove the cap from the pre-moistened Path-Chek Hygiene swab.

Step 2

Thoroughly swab a standard sample area (10 x 10cm), rotating the swab as the sample is
being collected. If sample areas are irregular develop a standard sampling procedure
which is documented and used consistently.

11
Step 3
After swabbing the test area, aseptically remove the cap from the Path-Chek Hygiene
Detection Broth and carefully place the swab into the tube. If the swab cannot be
transferred immediately into the Path-Chek Hygiene Detection Broth, return it to it’s holding
tube and store in a cool place. Label the Path-Chek Hygiene Detection Broth or the holding
tube for the swab.

NOTES:

1. Swabs should be placed into the Path-Chek Hygiene Detection Broth at an angle of
45° with the tip of the swab against the side of the tube. Press down on the shaft of the
swab. The shaft of the swab will break at breakpoint of the swab, 45mm from the swab
tip.

12
 2. If the swab cannot be transferred immediately into the Path-Chek Hygiene Detection
Broth, the swab should be returned it to it’s holding tube and store in a cool place.
Swabs may be held at a maximum temperature of 20°C for up to 24 hours.

Step 4
Place inoculated tubes into a suitable rack and incubate at 35-37°C, for 18-24 hours for
Coliforms and Salmonella, and at 28-30°C, for 24-48 hours, for Listeria.

Note: If Path-Chek Hygiene Listeria is incubated at 35-37°C there will, in certain

circumstances, be an increased risk of false positives.

Step 5
Observe for colour changes and record the results. A positive result may be interpreted as
early as 18 hours however, results must not be considered as negative until the Path-Chek
Hygiene Detection Broth has been incubated for up to 24 hours for the Coliform and
Salmonella spp. systems and 48 hours for the Listeria spp. system.

Listeria Coliforms Salmonella

(+) (-) (+) (-) (+) (-)

13
Step 6

Interpretation:

System Positive Negative

Coliform Yellow Purple
Salmonella Black Purple/ Yellow
Listeria Black Straw Colour

Table 6. Interpretation Criteria for Path-Chek Hygiene Detection Broths.

Step 7

OPTIONAL CONFIRMATION PROCEDURES

Presumptive positive tests may be confirmed by sub-culturing a drop of the growth medium
onto an appropriate selective agar plate medium for the organism being tested.

The use of the following media with provide compliance with standard testing methods
such as BAM and USDA/ FSIS etc.

System Media
Coliform/ E.coli mENDO
VRBA
Salmonella XLD
Bismuth Sulphite
Listeria Oxford
Palcam
ALOA

Table 7. Culture media for Confirmation of Positive Detection Broths

After incubation at 35 - 37°C for 24 – 48 hours, plates should be examined for colonies
resembling the targets being sought.

Any suspect colonies should be further identified using more definitive tests such as
microscopy and biochemical tests such as the Microgen® GNA ID (MID-64) and GNB ID
(MID-65) and Listeria ID (MID-67).

14
Performance Characteristics

Pre-Moistened Swabs

Preservative Efficiency of Neutralizing Buffer

Eight species of commonly encountered environmental bacteria and food pathogens

bacteria were grown overnight on Tryptone Soya Agar plates and suspended in 10ml
Ringers solution to an approximate turbidity of Browns Opacity Standard No 1. 0. 1ml of
these dilutions were transferred into the 100ml neutraliser. Six sponges were inoculated
with 5ml neutraliser for each type of bacteria. The sponges were incubated at 22oC for the
duration of the test.

A semi-quantitative total viable counting method was used to test the number of bacteria
surviving in the neutraliser. Bacterial levels were tested at 0, 24, 48, 72 and 168 hours.

RESULTS

Organism 0 hrs 24 hrs 48 hrs 72 hrs 168 hrs

S. enteritidis (NCIMB 50073) 100% 100% 70% 70% 70%

B. cereus (ATCC 11778) 100% 70% 70% 50% 30%

S. typhimurium (ATCC 14028) 100% 100% 100% 70% 50%

S. aureus (NCTC 6571) 100% 100% 50% 25% 0%

E. coli (NCIMB 11943) 100% 100% 100% 70% 70%

L. innocua (NCTC 11288) 100% 100% 100% 100% 100%

L. monocytogenes (NCTC 11994) 100% 100% 100% 70% 70%

L. monocytogenes (ATCC 7645) 100% 100% 70% 70% 70%

DISCUSSION
All of the organisms in this test maintained constant numbers up to 24 hours when stored
at 22ºC. Beyond 24 hours a gradual decrease in the numbers of organisms recovered
occurred with all species tested, however S. aureus was the only organism to suffer
significant reductions in numbers.

The results show that the swab neutralising buffer is able to preserve the viability of the
bacteria used in the test whilst also preventing over growth.

15
Path-Chek Hygiene Coliform Detection Broth
Sensitivity

Organism Dilution cfu/swab Results

E.coli MBCC 69 104 TNC Positive

103 230 Positive
102 30 Positive
101 2 Positive
100 0 Negative
E.coli MBCC 70 104 TNC Positive
103 290 Positive
102 50 Positive
101 3 Positive
100 0 Negative
E.coli MBCC 71 104 TNC Positive
103 356 Positive
102 35 Positive
101 5 Positive
100 0 Negative
E.coli MBCC 72 104 TNC Positive
103 430 Positive
102 20 Positive
101 1 Positive
100 0 Negative
E.coli MBCC 73 104 TNC Positive
103 190 Positive
102 10 Positive
101 0 Positive
100 0 Positive
K. oxytoca MBCC 46 104 500 Positive
103 60 Positive
102 6 Positive
101 <1 Positive
K. pnuemoniae MBCC 47 104 TNC Positive
103 150 Positive
102 15 Positive
101 1 Positive
K. pnuemoniae MBCC 161 104 TNC Positive
103 100 Positive
102 12 Positive (weak)
101 1 Purple

Pre-moistened Path-Chek swabs were inoculated with a range of coliform species at

dilutions down to zero cfu. The swabs were transferred into Path-Chek Hygiene Coliform
Detection Broth and the broths incubated at 35 - 37ºC for 24 hours and then observed for
any colour changes.

RESULTS

Based on these studies, the Path-Chek Hygiene Coliform Detection Broth is capable of
detecting levels as low as 1 coliform organism per swab.

16
Specificity

A total of 31 isolates of E.coli were inoculated onto pre-moistened swabs at a level of 10 <
cfu. Inoculated swabs were then transferred into Path-Chek Hygiene Coliform Detection
Broth and the broths incubated at 35 - 37ºC for 24 hours and then observed for any colour
changes.

Target Bacteria Number Positive Negative Comment

E.coli 31 30 1
Klebsiella sp. 9 9 0
Enterobacter sp. 9 7 2 2 x non lactose fermenting strains
Citrobacter sp. 13 8 5 5 x non lactose fermenting strains
Total Coliform 62 54 1

The Path-Chek Hygiene Coliform Detection Broth detected 30/31 (97%) of the E.coli
examined after 24 hours incubation at initial levels of < 10 cfu per swab. The one isolate
that was not detected was or clinical origins and may have developed unique resistance
patterns. In addition, a range of other “Coliform” organisms were examined, some of which
were non lactose fermenting and failed to be detected by the indicator system employed in
the Path-Chek Hygiene Coliform Detection Broth

In a second study, 49 non Coliform species comprising both Gram negative and Gram
positive organisms were inoculated onto pre-moistened swabs at a level of 102 cfu.
Inoculated swabs were then transferred into Path-Chek Hygiene Coliform Detection Broth
and the broths incubated at 35 - 37ºC for 24 hours and then observed for any colour
changes.

Non-Target Bacteria Number Positive Negative Comment

Salmonella spp. 5 0 5
Listeria spp. 4 0 4
Enterococcus spp. 4 0 4
Staphylococcus spp. 5 0 5
Streptococcus spp. 3 0 3
Bacillus spp. 6 0 6
Pseudomonas spp. 3 0 3
Burkholderia spp. 8 0 8
Morganella spp. 3 0 3
Serratia spp. 2 0 2
Providencia spp. 2 0 2
Acinetobacter spp. 1 0 1
Aeromonas spp. 1 0 1
Proteus spp. 1 0 1
Micrococcus spp. 1 0 1
Total Non-Coliform 49 0 49

17
Competitor Analysis
The Path-Chek Coliform and the Medical Wire® Coliform Environmental swab system were
compared to determine both their efficiency in the recovery of Coliform organisms and their
specificity.

Individual swabs of each system were challenged with <10 cfu of a total 28 different
species of Coliform, or 100 cfu of a range of non-Coliform species. Both tests were read
after 24 hours incubation.

Sensitivity Comparison

Path- Chek Medical Wire®

Organism Total
Hygiene Coliform Coliform
E. coli 14 14 14
K. pneumoniae 6 6 6
K. oxytoca 1 1 1
C. youngii 1 1 1
C. freundii 2 2 2
E. cloacae 4 4 4
Total 28 28 28

Both the the Path-Chek Hygiene Coliform and the Medical Wire® Coliform were able to
detect 28/28 (100%) of the isolates tested after 24 hours incubaction.

Specificity Comparison

Path-Chek Medical
Organism Comments
Coliform Wire®
L. monocytogenes Negative Positive
L. innocua Negative Negative
L. grayi Negative Weak Positive
Micrococcus sp. Negative Negative
B. cereus Negative Negative
B. licheniformis Negative Negative
E. avium Negative Negative
E. faecalis Negative Positive
E. faecium Negative Positive
E. gallenarum Negative Negative
Bacillus sp. Negative Negative
Staphylococcus sp. Negative Positive
M. morganii Negative Positive
S. maltophilia Negative Negative
A. baumannii Negative Negative
S. seftenberg Negative Positive
P. mirabilis Negative Positive
P. stuartii Negative Positive
P. aeruginosa Negative Negative
S. typhimurium Negative Positive
S. marcesens Negative Positive
Total 21 10

18
A total of 21 species of both Gram negative and Gram positive organisms were examined.
The Path-Chek Coliform inhibited the growth of all species tested i.e. 100% specificity.

The Medical Wire® Coliform failed to inhibit the growth of 11 species which were also able
to produce positive reactions similar to Coliforms. “False positive” results were caused by a
range of both Gram negative and Gram positive organisms. On the basis of this trial, the
specificity of the Pat-Chek Coliform was 94% and the Medical Wire® Coliform was 48%.

19
Path-Chek Hygiene Salmonella Detection Broth

Sensitivity

Pre-moistened Path-Chek swabs were inoculated with a range of Salmonella species at

dilutions down to zero cfu. The swabs were transferred into Path-Chek Hygiene
Salmonella Detection Broth and the broths incubated at 35 - 37ºC for 24 hours and then
observed for any colour changes.

RESULTS

Organisms Dilution cfu/swab Results

Salmonella typhimuruim MBCC 215 104 TNC Black

103 TNC Black
102 120 Black
101 10 Black
100 1 Black
Salmonella bispeberg MBCC 274 104 TNC Black
103 250 Black
102 20 Black
101 2 Black
100 0 Purple
Salmonella java MBCC 275 104 TNC Black
103 180 Black
102 20 Black
101 1 Black
100 0 Purple
Salmonella virginia MBCC 277 104 TNC Black
103 420 Black
102 30 Black
101 2 Black
100 0 Purple
Salmonella saint-paul MBCC 278 104 500 Black
103 40 Black
102 10 Black
101 1 Black
100 0 Purple
Salmonella derby MBCC 281 104 TNC Black
103 270 Black
102 30 Black
101 1 Black
100 0 Purple
Salmonella senftenberg MBCC 282 104 TNC Black
103 290 Black
102 50 Black
101 3 Black
100 0 Purple

20
 Organisms Dilution cfu/swab Results

Salmonella derby MBCC 281 104 TNC Black

103 270 Black
102 30 Black
101 1 Black
100 0 Purple
Salmonella senftenberg MBCC 282 104 TNC Black
103 290 Black
102 50 Black
101 3 Black
100 0 Purple
Salmonella senftenberg MBCC 282 104 TNC Black
103 250 Black
102 30 Black
101 2 Black
100 0 Purple
Salmonella rostock MBCC 283 104 TNC Black
103 500 Black
102 70 Black
101 10 Black
100 1 Black

This study demonstrated that the Path-Chek Hygiene Salmonella Detection Broth is
capable of detecting levels as low as 1 Salmonella spp. organism per swab.

Specificity

A total of 97 different serotypes of Salmonella spp. were inoculated onto pre-moistened

swabs at a level of 10 < cfu. Inoculated swabs were then transferred into Path-Chek
Hygiene Salmonella Detection Broth and the broths incubated at 35 - 37ºC for 24 hours
and then observed for any colour changes.

Target Bacteria Number Positive Negative Comment

S. enteritidis 5 5 0
S. typhimurium 8 8 0
S. dublin 7 7 0
Salmonella spp. 77 76 1 1 Salmonella spp. H2S Negative
Total Salmonella 97 96 1

The Path-Chek Hygiene Salmonella Detection Broth detected 99% of the Salmonella spp.
examined after 24 hours incubation at initial levels of < 10 cfu per swab. The one isolate
that was not detected was found to be H2S negative.

In a second study, 88 non Salmonella spp., comprising both Gram negative and Gram
positive organisms were inoculated onto pre-moistened swabs at a level of 102 cfu.
Inoculated swabs were then transferred into Path-Chek Hygiene Salmonella Detection
Broth and the broths incubated at 35 - 37ºC for 24 hours and then observed for any colour
changes.

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 Non-Target Bacteria Number Positive Negative Comment
E. coli 15 0 15
Klebsiella spp. 5 0 5
Enterobacter spp. 6 0 6
Citrobacter spp. 14 11 3
Listeria spp. 9 0 9
Enterococcus spp. 4 0 4
Staphylococcus spp. 5 0 5
Streptococcus spp. 3 0 3
Bacillus spp. 6 0 6
Pseudomonas spp. 3 0 3
Burkholderia spp. 6 0 6
Morganii spp. 3 0 3
Maltophilia spp. 2 0 2
Serratia spp. 2 0 2
Providencia spp. 2 0 2
Acinetobacter spp. 1 0 1
Aeromonas spp. 1 0 1
Proteus spp. 1 0 1
Micrococcus spp. 1 0 1
Total Non-Salmonella 88 11 77

The Path-Chek Hygiene Salmonella Detection Broth demonstrated a high degree of

selectivity for the Gram positive isolates tested and most of the Gram negative isolates.
Some isolates of Citrobacter spp. were not inhibited by the selective agents incorporated
into the Path-Chek Salmonella Detection Broth, resulting in false positive results.

The occurrence of false positive results due to detection of Citrobacter spp. should
still be considered as a significant result. The detection of significant levels of
Citrobacter spp. from surfaces is an important indication of faecal contamination
and/ or poor cleaning and sanitising and should therefore be investigated.

Recovery from Surfaces

A study was performed using the method of G. Moore and C. Griffith (A Comparison of
Surface Sampling Methods for Detecting Coliforms on Food Contact Surfaces. Food
Microbiology. 2002: 19, - 73), to determine the efficiency of the swabbing process
combined with the Path-Chek Salmonella Detection Broth when used to sample both wet
and dry surfaces.

Wet Surface Dry Surface

Organism
Results cfu Results cfu
Positive 306 Positive 520
Positive 40 Positive 52
S. tranora MBCC 171
Positive 10 Positive 4
Negative 2 Negative 0

The combination of the pre-moistened swabs and the Path-Chek Salmonella Detection
Broth was successful in detecting < 10 cfu recovered from both wet and dry sample
surface areas of 100 cm2.

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Competitor Analysis
No competitor products are available for comparison purposes.

Path-Chek Hygiene Listeria Detection Broth

Sensitivity

Pre-moistened Path-Chek swabs were inoculated with a range of Listeria species at

dilutions down to zero cfu. The swabs were transferred into Path-Chek Hygiene Listeria
Detection Broth and the broths incubated at 35 - 37ºC for up to 48 hours and then
observed for any colour changes.
Note: If Path-Chek Hygiene Listeria is incubated at 35-37 ºC, there will, in certain
circumstances, be an increased risk of false positives, compared to incubation at 28-30°C.

RESULTS

Organism Dilution cfu/ Swab 24 Hours 48 Hours

L. monocytogenes MBCC 97 104 TNC Positive Positive

103 800 Positive Positive
102 110 Positive Positive
101 10 Negative Positive
100 0 Negative Negative
L. monocytogenes MBCC 98 104 TNC Positive Positive
103 250 Positive Positive
102 20 Positive Positive
101 2 Negative Positive
100 0 Negative Positive
L. monocytogenes MBCC 99 104 TNC Positive Positive
103 560 Positive Positive
102 60 Positive Positive
101 2 Negative Positive
100 0 Negative Positive
L. innocua MBCC 93 104 TNC Positive Positive
103 700 Positive Positive
102 45 Positive Positive
101 5 Positive Positive
100 0 Positive Negative
L. innocua MBCC 94 104 TNC Positive Positive
103 440 Positive Positive
102 20 Positive Positive
101 3 Positive Positive
100 0 Negative Negative
L. innocua MBCC 95 104 TNC Positive Positive
103 450 Positive Positive
102 30 Positive Positive
101 2 Positive Positive
100 0 Negative Negative
L.. seeligeri MBCC 110 104 TNC Negative Positive
103 400 Negative Positive
102 60 Negative Positive
101 0 Negative Negative
100 0 Negative Negative

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 Organism Dilution cfu/ Swab 24 Hours 48 Hours

L.. ivanovii MBCC 111 104 TNC Positive Positive

103 560 Negative Positive
102 40 Negative Positive
101 3 Negative Positive
100 0 Negative Negative
L. welshmeri MBCC 114 104 TNC Positive Positive
103 880 Positive Positive
102 100 Positive Positive
101 10 Positive Positive
100 1 Positive Positive

This study demonstrated that the Path-Chek Hygiene Listeria Detection Broth is capable
of detecting levels as low as 1 Listeria spp. organism per swab.

Specificity

A total of 97 different species of Listeria were inoculated onto pre-moistened swabs at a

level of 10 < cfu. Inoculated swabs were then transferred into Path-Chek Hygiene Listeria
Detection Broth and the broths incubated at 35 - 37ºC for up to 48 hours and then
observed for any colour changes.

Target Bacteria Number Positive Negative Comment

L. monocytogenes 42 42 0
L. innocua 24 24 0
L. seeligeri 12 12 0
L. ivanovii 8 8 0
L. welshimeri 11 11 0
Total Listeria 97 97 0

This investigation demonstrated that 100% of the Listeria spp. examined could be
detected after 48 hours incubation at initial levels < 10cfu per swab.

In a second study, 76 non Listeria spp. comprising both Gram negative and Gram positive
organisms were inoculated onto pre-moistened swabs at a level of > 5 x 102 cfu.
Inoculated swabs were then transferred into Path-Chek Hygiene Listeria Detection Broth
and the broths incubated at 35 - 37ºC for 48 hours and then observed for any colour
changes.

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 Non-Target Bacteria Number Positive Negative Comment
E. coli 4 0 4
Klebsiella spp. 10 4 6
Enterobacter spp. 5 0 5
Citrobacter spp. 3 0 3
Salmonella spp. 9 0 9
6/6 Negative when
Enterococcus spp. 6 4 2 challenged with < 5 x 102
Staphylococcus spp. 5 0 5
Streptococcus spp. 3 0 3
5/5 Negative when
Bacillus spp. 5 1 4 challenged with < 1 x 102
Pseudomonas spp. 4 0 4
Burkholderia spp. 7 0 7
Lactobacillus spp. 3 0 3
Carnobacterium spp. 2 0 2
Cornebacterium spp. 1 0 1
Kurthia spp. 1 0 1
Acinetobacter spp. 1 0 1
Achromobacter spp. 1 0 1
Proteus spp. 2 0 2
Rhodococcus equi 1 0 1
TMicrococcus spp. 1 0 1
hMorganella spp. 1 0 1
eStenotrophomonas spp. 1 0 1
Total Non-Listerias 76 9 67
Path-Chek Hygiene Listeria Detection Broth demonstrated a high degree of selectivity for
the all of the Gram positive isolates tested with the exception of 2/6 Enterococcus spp. and
1/4 Bacillus spp. which produced positive reactions at levels > 5 x 102 but failed to grow at
lower levels (< 1 x 102 ) after 48 hours incubation.

With the exception of Klebsiella spp, all other Gram negative isolates either failed to grow
or produced negative results after 48 hours incubation. The Path-Chek Listeria Detection
Broth, resulting in false positive results.

The occurrence of false positive results due to detection of Enterococcus spp.

should still be considered as a significant result. The detection of significant levels
of Enterococcus spp. from surfaces is an important indication of faecal
contamination and/ or poor cleaning and sanitising and should therefore be
investigated.

Recovery from Surfaces

A study was performed using the method of G. Moore and C. Griffith (A Comparison of
Surface Sampling Methods for Detecting Coliforms on Food Contact Surfaces. Food
Microbiology. 2002: 19, - 73), to determine the efficiency of the swabbing process
combined with the Path -Chek Salmonella Detection Broth when used to sample both wet
and dry surfaces.

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 Wet Surface Dry Surface
Organism
Results cfu Results cfu
Positive 300 Positive 100
L. monocytogenes Positive 30 Positive 10
MBCC 305 Positive 6 Negative 1
Positive (weak) 2 Negative 0

The combination of the pre-moistened swabs and the Path-Chek Listeria Detection Broth
was successful in detecting < 10 cfu recovered from both wet surfaces and 10 cfu from dry
sample surface areas of 100 cm2.

Competitor Analysis
The Path-Chek Listeria and the Medical Wire® Listeria Environmental swab system were
compared to determine both their efficiency in the recovery of Listeria spp. and their specificity.

Individual swabs of each system were challenged with <10 cfu of a total 62 different
species of Listeria species, or 100 cfu of a range of non-Listeria species. Both tests were
read after 24 hours and 48 hours incubation.

Sensitivity Comparison

Path- Chek Hygiene Listeria Medical Wire® Listeria

Organism Total
24 Hours 48 hours 24 Hours 48 hours
L. monocytogenes 30 29 30 20 30
L. innocua 18 18 18 16 18
L. ivanovii 4 3 4 0 4
L. seeligeri 4 4 4 0 4
L. welshimeri 6 6 6 0 6
Total 62 60 62 36 62

The Path-Chek Hygiene Listeria detected 60 (97%) of the Listeria isolates tested after 24
hours incubation and 62/62 (100%) of the isolates after 48 hours incubation. The Medical
Wire® Listeria was able to detect 36/62 (58%) of the isolates tested after 24 hours
incubaction and 62/62 (100%) after 48 hours incubation. The Medical Wire® was only able
to detect L. monocytogenes and L. innocua species after 24 hours incubaction.

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 Specificity Comparison

Path-Chek Listeria Medical Wire

Organism
24 Hour 48 Hour 24 Hour 48 Hour
C. diversus Negative Negative Negative Negative
S. enteritidis Negative Negative Negative Negative
S. enteritidis Negative Negative Negative Negative
S. hadar Negative Negative Negative Negative
S. indiana Negative Negative Negative Negative
S. infantis Negative Negative Negative Negative
S. typhimurium Negative Negative Negative Negative
P. mirabilis Negative Negative Negative Negative
P. stuartii Negative Negative Negative Negative
P. aeruginosa Negative Negative Negative Negative
E. aerogenes Negative Negative Negative Negative
E. coli Negative Negative Negative Negative
E. coli Negative Negative Negative Negative
K. oxytoca Negative Negative Negative Negative
K. oxytoca Negative Negative Negative Negative
K. pneumoniae Negative Negative Negative Negative
K. pneumoniae Negative Negative Negative Negative
K. pneumoniae Negative Negative Negative Negative
K. pneumoniae Positive Positive Positive Positive
K. pneumoniae Negative Positive Negative Positive
K. pneumoniae Positive Positive Negative Negative
K. pneumoniae Negative Negative Negative Negative
K. pneumoniae Positive Positive Negative Negative
E. cloacae Negative Negative Negative Negative
E. cloacae Negative Negative Negative Negative
E. cloacae Negative Negative Negative Negative
E. cloacae Negative Negative Negative Negative
Carnobacterium divergens Negative Negative Positive Positive
Carnobacterium piscicola Negative Negative Negative Negative
Lactobacillus casei Negative Negative Negative Negative
Lactobacillus lactis Negative Negative Negative Negative
Lactobacillus plantarum Negative Negative Negative Negative
Bacillus mycoides Negative Negative Negative Negative
Kurthia zopfii Negative Negative Negative Negative
Micrococcus spp. Negative Negative Negative Negative
Rhodococcus equi Negative Negative Negative Negative
Enterococcus durans Negative Negative Negative Positive
Pseudomonas fluorescens Negative Negative Negative Negative
Corynebacterium renale Negative Negative Negative Negative
Bacillus cereus Negative Negative Negative Negative
Bacillus licheniformis Negative Negative Negative Negative
Enterococcus avium Negative Negative Positive Positive
Enterococcus faecalis Negative Negative Positive Positive
Enterococcus faecium Negative Negative Positive Positive
Enterococcus gallinarum Negative Negative Negative Positive

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 Organism Path-Chek Listeria Medical Wire
24 Hour 48 Hour 24 Hour 48 Hour
Streptococcus spp. Negative Negative Negative Negative
Streptococcus spp. Negative Negative Negative Negative
Streptococcus spp. Negative Negative Negative Negative
Bacillus spp Negative Negative Negative Negative
Bacillus spp Negative Negative Negative Negative
Bacillus spp Negative Negative Negative Negative
Staphylococcus spp. Negative Negative Negative Negative
S. saprophyticus. Negative Negative Negative Negative
S. aureus Negative Negative Negative Negative
S. aureus Negative Negative Negative Negative
S. hyicus ss hyicus Negative Negative Negative Negative
B. stabilis Negative Negative Negative Negative
B. cenocepacia Negative Negative Negative Negative
B. multivorans Negative Negative Negative Negative
B. cenocepacia Negative Negative Negative Negative
A. xylosoxidans Negative Negative Negative Negative
B. cepacia Negative Negative Negative Negative
R. mannitolilytica Negative Negative Negative Negative
B. cenocepacia Negative Negative Negative Negative
P. aeruginosa Negative Negative Negative Negative
P. putida Negative Negative Negative Negative
B. vietnamiensis Negative Negative Negative Negative
Total 58 58 54 54

A total of 62 species of both Gram negative and Gram positive organisms were examined.
The Path-Chek Listeria failed to inhibit the growth of 4 species which were also able to
produce positive reactions similar to Listeria spp.. These “false positive” results were
caused By K. pneuomoniae (3) and Enterococcus spp.(1).

The Medical Wire® Listeria failed to inhibit the growth of 8 species which were also able to
produce positive reactions similar to Listeria spp.. “Falso positive” results were caused by
K. pneuomoniae (1), Enterococcus spp.(6) and C. divergens (1).

On the basis of this trial, the specificity of the Pat-Chek Listeria was 94% and the Medical
Wire® Listeria was 87%.

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POSITIVE RESULTS – WHAT DO THEY MEAN?
With any of the Path-Chek Hygiene Pathogen systems, a positive result for a specific pathogen can
mean one of two things:

1. The presence of a specific pathogen such as Salmonella or Listeria at levels as low as

1 – 2 cfu per sampled area which should be at least 100cm2, OR
2. The presence in high numbers, usually > 102 of potentially cross reacting organisms

Path-Chek Detection System Most Probable Cause of “False Positive Tests

Coliform Nil
Salmonella Citrobacter spp.
Listeria K. pneumoniae
Enterococcus spp.

Table 8. Most Probable Cause of “False Positive Tests

In each of these cases, the potential causes of “False Positive” tests are organisms of
faecal origin.

As such, ALL Positive Tests should be confirmed by subculture and identification of

suspicious colonies. If a specific target pathogen cannot be isolated, the results
should still be considered as significant as they indicate the presence of significant
levels of organisms of faecal origin on the test surfaces. These organisms should not
be present if adequate cleaning and sanitising procedures are in place and being
performed correctly.

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