The Latest Developments in Food Microbiology - Oxoid

THE LATEST DEVELOPMENTS IN FOOD MICROBIOLOGY
In this issue: Oxoid Dry-Bags TM allow a large contract laboratory to work more efficiently; Earlybird Farm tell us
how the Precis TM Salmonella and Listeria methods save valuable time to results; new ISO compliant media and
Brilliance range of chromogenic media now available; meet the Oxoid Food Award winners; find out about
MicroVal (a European validation & certification organisation); W. H. Pierce Prize awarded; Italian Public Health
Laboratory use Oxoid AGS products.
ISSUE 11
setting standards

Oxoid Dry-Bags TM
as an Effective Alternative to Bulk
Liquid Media Preparation
ALcontrol Laboratories
ALcontrol Laboratories is one of the largest laboratory service
businesses in Europe, with laboratories in the United Kingdom,
Benelux and Sweden offering services covering food, environmental,
potable water, land, oil, asbestos and air testing. ALcontrol
Food is one of the UK market leaders in food analysis, with six
laboratories throughout the UK.
ALcontrol Food laboratories are UKAS group accredited to ISO
17025, which gives them substantial capacity and contingency
capability should unforeseen circumstances arise. The UK
laboratories hold Independent Supplier Audits (ISAs) to facilitate
analysis to M&S requirements and DEFRA approval for the animal
feed industry.
With a fleet of refrigerated vehicles across the network of
laboratories, they provide customer sample collection throughout
the whole of the UK. Their industry-leading electronic reporting
system (AMIS) provides results as rapidly as methods allow, and
enables customers to easily store, manage, report and perform
trend analysis of large amounts of data.
ALcontrol services and capabilities:
1 Comprehensive microbiological analysis utilising rapid
methods where appropriate:
1 Spoilage organisms such as Pseudomonas, yeasts
and moulds
1 Pathogens, for example, E. coli, Salmonella and
Listeria
1 Food poisoning organisms such as Staphylococcus
aureus, Clostridium perfringens and Bacillus cereus
1 Shelf-life testing
1 Testing of products that are the subject of consumer
complaints
1 Bacterial identifications.
2
OPEN
FILL
DISPENSE
Wide range of sample types
ALcontrol analyses a vast range of sample types, from raw meat,
raw fish, mechanically recovered meat and mycoprotein to milk,
snacks, confectionery, ready meals, sandwiches, herbs & spices.
Such a wide range of sample types necessitates a wide range of
microbiological tests and methods. Enumeration tests, such as
Aerobic Colony Count, E. coli, coliforms and S. aureus, are routinely
performed. Some pathogen analyses, such as Salmonella, Listeria
monocytogenes, Campylobacter and E. coli O157 aim to determine the
presence or absence of the organism. These tests require an enrichment
process to ensure detection of even small levels in the product.
With approximately 2.2 million samples analysed each year, it is
imperative that processes are as efficient as possible.

Bulk liquid handling
Buffered Peptone Water (BPW) is the main diluent used by
ALcontrol in the preparation of samples. Each batch takes time to
prepare in terms of weighing out media powder, measuring liquid,
and autoclaving and cooling bottles, which can take four to five
hours. Staff availability, the number of runs per day and the number
of autoclaves available all restrict the production of BPW.
As the number of samples increased at the Shrewsbury laboratory
(now handling 65,000 tests per month), it was evident that the autoclave
capacity for the manufacture of liquid media was becoming
a limiting factor. The autoclaves were not coping well with running
24 hours a day, but the installation of further machines was not
possible within the timescale required.
After considering other ideas, such as ready-filled options, Oxoid
Dry-Bags TM provided an ideal solution. Following a short trial period,
it was clear that they would fit well into current procedures,
enabling an increase in media production and efficiency without
heavy capital investment.
Oxoid provided full training for the laboratory staff and have continued
to support ALcontrol's training requirements.
Easy to use and cost effective
“We are impressed by the Dry-Bags' ease of use," says Paul
Anderson, site manager at ALcontrol, Shrewsbury. "They fit directly
onto Dilumats for dispensing, thereby reducing the risk of contamination.
As a disposable option, they eliminate the time and expense
of recycling glassware associated with in-house production. They
offer cost savings in terms of running the autoclaves (300 litres
saves 3-4 autoclave runs) and through the reduction in QC checks
necessary (the batch numbers of the Dry-Bags change far less
than those of in-house BPW). All these factors lead to more
efficient media production.
“The performance pressures and wear and tear on the autoclaves
is reduced", continues Paul. "The Dry-Bags stack neatly and tidily
into existing laboratory crates and take up far less space than
glass bottles. Media staff time is freed up to concentrate on other
tasks, and the Dry-Bags have been incorporated into our
production without having to make any changes to sample
processes.”
Enhanced flexibility
“Perhaps more importantly, the media can be reconstituted in
around 10 minutes, significantly reducing the lead-time of BPW
production. This allows us to cope seamlessly with large fluctuations
in workload, which are often typical of our business. They enable
the laboratories to be highly flexible and respond efficiently to the
media demands of variable sample through-put.”
For more information about Oxoid Dry-Bags please speak to your
local Oxoid representative, visit www.oxoid.com or tick 1 on the
reply paid card.
REPLY 1
3

New Oxoid Precis Culture Methods
Simple – Fast – Approved!
Oxoid Precis TM culture methods allow isolation, detection and confirmed identification of foodborne
pathogens in just 2 days without the need for specialist equipment or training.
Just follow these 3 simple steps:
1. Enrichment – a single broth combining resuscitation and growth in overnight incubation
2. Plating – a single medium incorporating enzyme specific inhibition and differentiation.
Coloured colonies clearly indicate presumptive positive results
3. Confirmation – confirmed test result available in less than
10 minutes, direct from plate.*
Precis methods for Salmonella and Listeria monocytogenes,
validated by AFNOR to ISO 16140:2003, are now available. A
poster1 presented at the 2008 International Association of
Food Protection Meeting concluded:
“According to the ISO 16140 performance assessments, the
Salmonella Precis method represents a valuable alternative
method for the detection of Salmonella spp. in food, animal
feed and environmental samples. The Salmonella Precis
method also offers important economic savings by
minimising the time to obtain results and the number of
‘hands-on’ operations required. Workflow studies demonstrate
a 2 to 3-fold reduction in the manipulation time
using the Salmonella Precis method compared to the
ISO reference method.”
You can download this poster and the Precis Salmonella and
Listeria product datasheets from www.oxoid.com or talk to your
local Oxoid representative, or tick 2 on the reply paid card.
*ISO standard tests for confirmation have also been validated and approved by
AFNOR, offering complete flexibility.
Reference: 1. Poster presentation: Oxoid Salmonella PrecisTM REPLY 2
method for Salmonella spp. detection in food, feed and environmental samples:
EN ISO 16140 performance validation. Danièle Sohier, Maryse Rannou, ADRIA Développement, expert lab at the AFNOR and Microval validation
committees, France; Tamsin Baalham, Alastair Thomas, James Stringer, Oxoid.
Oxoid ONE Broth-Salmonella saves time and increases efficiency
Did you know you can delay the addition of the freeze-dried selective supplement to ONE
Broth-Salmonella Base by up to 45 minutes, allowing you to carry out both viable count and
Salmonella investigations from a single sample preparation? Here's how:
After stomaching the food sample in ONE Broth-Salmonella Base, remove a portion of the
broth and dilute it for viable counts. Add ONE Broth-Salmonella Supplement to the remaining
broth for enrichment according to the AFNOR validated Oxoid Salmonella Precis method.
This eliminates the need to prepare separate broths for each investigation - saving both time
and resources in your laboratory.
To request a datasheet summarising this protocol for both viable counts and presumptive
identification of Salmonella (following the Oxoid Salmonella Precis method), or to obtain the
Oxoid Salmonella Precis method datasheet, please speak to your local Oxoid representative,
visit www.oxoid.com or tick 3 on the reply paid card.
REPLY 3
4

Salmonella and Listeria Precis Methods Save Time
and Money for Earlybird Farm
Earlybird Farm
Earlybird Farm, part of the Astral Foods group, is one of the
largest poultry producers in South Africa, providing frozen,
fresh and value-added chicken products to both the retail and
food services sectors. The two processing plants, in Standerton
and Olifantsfontein, process around 800 tonnes of chicken
every day.
The Earlybird Laboratory at Standerton regularly tests products
from throughout the production process, including finished
product and environmental samples for Salmonella and Listeria,
investigating 400-450 samples every month. Previously, they
were using the full ISO detection methods for these micro-
Day 0: Enrichment
25g or 25ml of sample
+
225ml ONE Broth-Salmonella
Incubate for
16-20 hours at 42°C
Day 0: Enrichment
25g or 25ml of sample +
225ml ONE Broth-Listeria
Incubate for
22-26 hours at 30°C
Protocol for Salmonella Precis Method
Day 1: Plating
Using a 10µl microbiological
loop, inoculate a single Brilliance
Salmonella plate.
Protocol for Listeria Precis Method – Detection
Day
1:
Incubate for
22-26 hours at 37°C
Figure 1. Time Saving Oxoid Precis methods
P lating
Day 2: Results
Using a 10µl microbiological loop,
I f present,
confirm
blue/
green
colonies
with
halos
inoculate a single Brilliance Listeria as L. monocytogenes using the O.B.I.S. mono test.
plate.
Alternatively, confirm using standard ISO methods.*
Incubate for 22-26 hours
at 37°C
(for meat samples re-incubate plates that show no blue colonies with
halos for a further 22-26 hours at 37°C)
organisms, which took five days to obtain a confirmed positive
result for Salmonella and up to seven days for Listeria. In the last
year, however, they have begun to use the Oxoid Salmonella and
Listeria PrecisTM methods, which have reduced the time to result
for both organisms to just two days (Figure 1).
“The faster time to result was very appealing to us and so we
decided to try the new Precis methods,” comments Laboratory
Manager, Louise Zwarts (pictured top left). “After performing our
own in-house verification, we could see that the new method
saved a lot of time, without compromising sensitivity or quality of
results. This, and the ease of performing the Precis methods,
made the decision to change easy.”
AFNOR validated
“The fact that the Precis methods were already AFNOR validated
to ISO 16140:2003 against the ISO reference methods made it
much easier for us to adopt them,” Louise continues. “It gave us
the confidence to try them and reduced the amount of in-house
work we had to do.”
Continues over page
Day 2: Results
If present, select a well isolated purple colony
and test using the Oxoid Salmonella Latex Test.
Alternatively, confirm purple colonies using
standard ISO methods.
Select purple
colonies for confirmation
Select green-blue
colonies with halos for
confirmation
For Listeria monocytogenes enumeration:
Resuscitate any organisms present in the sample by adding 25g or 25ml to 225ml of Buffered Peptone Water and incubate for 1 hour at 20°C. Inoculate a single Brilliance Listeria plate with
100µl and incubate for 45-51 hours at 37°C. Inspect the plate for characteristic blue/green colonies with halos and count. Confirm using O.B.I.S. mono or alternatively, confirm using standard
ISO methods.* Calculate CFU/g or CFU/ml of sample.
*If there is insufficient material to carry out an O.B.I.S. mono test, or if a mixed culture of L. monocytogenes and other Listeria spp. is suspected, first purify suspect colonies by sub-culture onto a second Brilliance Listeria plate.
5

Continued from previous page
“For our own verification we performed the Precis methods alongside
the ISO methods on routine samples and on spiked control
samples. The Brilliance TM chromogenic media used in the Precis
methods made the differentiation of Salmonella and Listeria
colonies very easy because the colours were much more distinctive
than with traditional media.
"We found that the Precis methods gave us equivalent results and
were much easier to perform. They also resulted in fewer false
positive colonies, which, as well as giving us greater confidence
in the results, meant that there were less confirmation tests to
carry out. We have actually reduced the number of confirmations
by around 75%, leading to a big saving in time and money.”
Saving time
“We were amazed by how much time we saved. This was a really
important consideration for us as we want to have results back
before products leave the premises. Using the Precis methods, we
get results several days earlier than before, allowing us to get
products out to the market much sooner.
“We also save time in preparation and performing the methods.
We have much less media preparation with the Precis methods,
and there are fewer steps and less subculturing compared to the
ISO methods. There is nothing difficult about the Precis
methods - they are very easy. We love using them.”
O.B.I.S. Salmonella differentiates Salmonella from organisms
with similar colonial appearance, such as Citrobacter, Proteus
and Morganella spp., on standard diagnostic culture media.
It takes just five minutes to perform and confirm that suspect
colonies are NOT Salmonella, 24 hours earlier than by
traditional methods. Simply apply a suspect colony to each
test circle on the reaction card, wait for five minutes and
then add developing solutions.
A colour reaction in either test circle confirms that the
organism is not Salmonella. The lack of PYRase and
deaminase enzyme activity in Salmonella spp. is used to
differentiate them from Citrobacter spp., which possess
PYRase activity, and Proteus, Morganella and Providencia
spp., which have deaminase activity.
PYR
NPA
6
Saving money
“We save money in several ways,” Louise adds. “The need for
fewer manipulations with the Precis methods requires less staff
time. In addition, fewer false positives and fewer confirmations
also means less work and saves on materials; the earlier results
allow us to save on storage costs since products can be released
much sooner.”
New opportunities
“The time we have saved since we have been using the Precis
methods has allowed us to look at expanding our test repertoire,”
comments Louise. “Not only do we have more time for method
development,” she explains, “but our autoclaves and incubators
have also been freed up. This has enabled us to look at performing
Clostridium and Campylobacter tests in-house.
“Our experiences with the Precis methods also encouraged us to
try the Oxoid Brilliance Bacillus cereus chromogenic agar. We
really liked the Brilliance product and started using it almost
straight away.”
Bev Reid, from Oxoid's South African distributor, Quantum
Biotechnologies (Pty) Ltd, says “Louise has to be commended for
her willingness to try new technology and to look positively on the
potential benefits. Setting up the in-house verification was the first
step and after that the products sold themselves.”
“The support we have had from Bev has been fantastic,” Louise
adds. “I don't know what we would do without her.”
To find out how the Oxoid Salmonella and Listeria Precis
methods can save time and money in your laboratory, visit
www.oxoid.com or circle 4 on the reply paid card.
REPLY 4
O.B.I.S. Salmonella: a Rapid Identification Method for Salmonella spp.
L-pyroglutamic acid 7-amino-4-methyl coumarin (7AMC) is a
non-carcinogenic substrate that detects PYRase activity in
the PYR test circle, indicated by a vivid purple reaction.
Nitrophenylalanine (NPA) detects deaminase activity in the
NPA test circle, indicated by an orange/brown reaction.
Since Salmonella spp. do not possess these enzymes, they
will produce no colour reactions.
From 470 plates growing suspect colonies, O.B.I.S.
Salmonella successfully identified 406 as false positives and
detected all true positive Salmonella isolates1 . For more
information, please speak to your local Oxoid representative,
visit www.oxoid.com or tick 5 on the reply paid card.
Reference: 1. Data held on file, Oxoid Limited
REPLY 5

MSRV Salmonella Medium Now ISO Compliant
Salmonellosis constitutes a major public
health burden and represents a significant
cost to society in many countries. Most
national and international food safety
standards state that the presence of
Salmonella is unacceptable in any food
product.
The EU Commission Regulation No.
2073/2005 on Microbiological Criteria for
Foodstuffs 1 states that:
“Food should not contain micro-organisms
or their toxins or metabolites in quantities
that present an unacceptable risk for
human health.“
The acceptable level of Salmonella in food
products placed on the market during their
shelf-life is zero organisms in 25g. This
applies to any food intended to be eaten
raw or cooked and all food raw materials.
The exception to this is for raw materials
or products where the manufacturing
process or the composition of the product
will eliminate the Salmonella risk, for
which zero organisms in 10g will be the
acceptable limit until 2010.
Oxoid Modified Semi-solid Rappaport-
Vassiliadias (MSRV) Agar (ISO) meets the
specifications for formulation and performance
as described in ISO 6579:2002 Annex D
- the Horizontal method for the detection of
Salmonella spp. in animal faeces and in
environmental samples from the primary
production stage.
Oxoid MSRV Agar has been shown to
detect more Salmonella positive samples
than traditional enrichment procedures
for animal faeces and environmental
samples 2-5 .
Motile Salmonella colonies are characterised
by their grey-white colour, with turbid
zones radiating from the point of inoculation.
Zones are surrounded by a white halo
with sharply defined border.
For further information on this product
(order code CM1112), please speak to
your local Oxoid representative, visit
www.oxoid.com or tick 6 on the reply
paid card.
REPLY 6
Summary of ISO 6579:2002 Annex D
Homogenise sample 1:10
in BPW and incubate at 37°C ± 1°C
for 18 hours ± 3 hours
Inoculate MSRV plates with 3
drops totalling 0.1ml. Incubate at
41.5°C ± 1°C for 24 hours ± 3 hours
If plates are negative
re-incubate for a further 24 hours
± 3 hours
Plate typical colonies onto 2 x XLD Agar
and a 2nd medium of choice and
incubate at 37°C ± 1°C
Carry out appropriate biochemical
and serological tests
Confirmed Salmonella spp.
OTHER ADDITIONS TO THE
OXOID RANGE OF ISO COMPLIANT
PRODUCTS
1 Oxoid Baird-Parker Agar (ISO) - a
selective diagnostic medium for
the isolation and enumeration of
coagulase-positive staphylococci
in foods. Conforming to ISO 6888-
1:1999, it is widely recommended by
national and international bodies 6 .
1 Oxoid Violet Red Bile Lactose
(VRBL) Agar (ISO) - for the detection
and enumeration of coliforms in
food, animal feed and environmental
samples, conforms to ISO 4832:2006.
1 Oxoid Violet Red Bile Glucose
(VRBG) Agar (ISO) - for the
detection and enumeration of
Enterobacteriaceae in food, animal
feed and environmental samples,
conforms to ISO 21528-2:2004.
As well as complying with the
recommended formulations, these
media meet the growth and inhibition
specifications given in
ISO/TS 11133:20037 .
REPLY 7
References: 1. Commission Regulation (EC) No 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs.
2. De Smedt J. M., Bolderdijk R., Rappold H. and Lautenschlaeger D. (1986) J. Food. Prot. 49:510-514. 3. De
Smedt J. M., Bolderdijk R. (1987) J. Food. Prot. 50:658-661. 4, De Zutter L. et al. (1991) Int. J. Food Micro. 13:11-20. 5. De
Smedt J. M. et al. (1991) Int. J. Food Micro. 13:301-308. 6. Chopin A., et al. (1985) ICMSF Methods studies XV. J. Food
Protect. 48:21-27. 7. ISO/TS 11133-2:2003 Microbiology of food and animal feeding stuffs - Guidelines on quality assurance
and performance testing of culture media Part 2: Practical guidelines on performance testing of culture media
7

Oxoid Awards: Winners Announced
YOUNG MICROBIOLOGIST OF THE YEAR
We are delighted to announce that Shazia Naz of the Marine
Fisheries Department, part of the Ministry of Food, Agriculture and
Livestock, Karachi, Pakistan, has been awarded the title of Oxoid
Young Microbiologist of the Year 2007/2008. Shazia wins £1,000
and the opportunity to attend the 2009 International Association of
Food Protection Meeting to be held in Grapevine, Texas, USA.
Shazia is in charge of the government laboratory at the fish
harbour at the West Wharf, Karachi. The laboratory monitors fish
processing and other processing sites, as well as process water
and ice at the landing areas.
Her entry demonstrated many achievements, but the work she
undertook to secure ISO 17025 accreditation for the laboratory
(the first accreditation in a laboratory of this kind in Pakistan)
particularly impressed our judges. This involved development of
standard operating procedures; quality and technical manuals;
test validation methods; maintenance of certified reference
materials; evaluation of staff competence and measurement of
non-conformances and corrective actions.
During the accreditation process, Shazia was still responsible for
the day-to-day running of the laboratory, supervision of staff and
also undertook hands-on testing, herself. Her dedication was, highlighted
by the many long days worked to complete the project.
8
Additionally, Shazia is a member of the HACCP monitoring and
audit team at the Marine Fisheries Department and an internal
auditor for both the microbiology and chemical laboratories. She is
also the youngest technical assessor for ISO 17025 (Microbiology)
of the Pakistan National Accreditation Council - which the Awards'
judges cited as a huge achievement.
The judges agreed that Shazia was an outstanding, highly motivated
individual, with one judge commenting, “Shazia has everything you
would want in a young microbiologist”.
Second Prize
Rebecca Moorhouse, (pictured left with Bhavan Mistry, Oxoid)
Laboratory Technician at Young's Seafood Ltd, Grimsby, UK was
awarded the second prize of £500. Our judges recognised that
Rebecca had shown continuous development as a technician and
that she was undertaking the role to a very high standard. Her entry
demonstrated she was a team player, a good communicator and an
ambassador for her laboratory.
Highly Commended
The judges also made additional, Highly Commended Awards to
Beenish Habib, Microbiologist at Dawn Bread, Karachi, Pakistan;
to Mr Arsalan Saeed Zai, microbiologist and GMP coordinator at
Cadbury Pakistan Ltd; and to Mrs Omondi Florence Awino Ouma,
laboratory analyst at Kenya Bureau of Standards, Kenya.
For more information about the Oxoid
Awards, which are organised in conjunction
with International Food Hygiene and the
Institute of Brewing and Distilling, please
visit www.oxoid.com, contact Fiona
Macrae, awards manager, on
oxoid.awards@thermofisher.com, talk to
your local representative of Oxoid products
or tick 8 on the reply paid card.
REPLY 8

Oxoid Food Safety Teams of the Year
First Prize
The food safety team based at Cadbury Trebor Bassett's Chirk site
in Wales are the 2007/2008 Oxoid Food Safety Team of the Year,
winning £1,000.
The Cadbury's entry demonstrated the team's determination to
drive forward a continuous quality improvement process which
involved improvements in methodologies and changes in factory
processes. The Chirk site is a feedstock site for the larger Cadbury
organisation, with the laboratory handling samples of butter and
chocolate liquor.
The team's work brought major benefits to the company in terms of
food safety:
1 A positive clearance system was introduced, which improved
laboratory inspection processes and provided enhanced
traceability
1 With no increase in staffing levels, the frequency of sample
testing went up - creating a 200% increase in workload
1 A laboratory extension was built and commissioned with no
disruption to workflow
1 New and improved environmental monitoring practices were put
in place, including training of maintenance, supply chain partners
and contractors, who all now work to Cadbury GMP standards.
Second Prize
Fonterra Clandeboye, Timaru, New Zealand. The second prize of
£500 was awarded to the laboratory team at Fonterra Clandeboye,
Timaru, New Zealand. The Fonterra Co-operative Group is a major
producer of dairy products with 97% of products being exported,
globally. The laboratory at Clandeboye is responsible for testing all
products produced in the South Island's manufacturing plants and
also from external companies. During 2007/2008, the laboratory
workload increased to receive samples from the North Island, as
well. The judges were impressed by the way in which this laboratory
treated the increasing workload as a challenge and restructured
working practices and incorporated quality management principles
to maximise effectiveness and increase productivity.
Judges' Special Award
Westward Laboratories, Callington, UK. A Judges' Special Award
was made to Westward Laboratories, Callington, Cornwall, UK,
previous winners of the Oxoid Food Safety Team of the Year Award,
for their continued contribution to food safety. Amongst the recent
initiatives undertaken by Westward, the judges cited the team's
willingness to promote food safety issues to the local community,
by involvement with young farmers groups and local colleges, as
being worthy of special recognition.
9
All of these factors contributed to reductions in out of specification
microbiological results compared to previous years.
“The commitment and dedication of the Cadbury Chirk team have
had a significant impact on food quality and safety that was
demonstrated by the facts and figures contained in their entry”,
commented Cheryl Mooney, chairman of the judging panel. “The
team has also raised its own morale and through the enthusiasm
and initiatives of its members, has raised morale and levels of food
hygiene and safety within the production environment. We are
delighted, therefore, to name them as the Oxoid Food Safety Team
of the Year for 2008/2009.”

The Brilliance of Oxoid Chromogenic Media
To reflect the innovative and ingenious nature of the
products as well as the brightly coloured colonies
produced, we are pleased to announce that our
chromogenic agar media for food microbiology
laboratories are becoming part of our Brilliance range.
The vivid colour reactions produced by all media in the Brilliance range allow rapid, easy differentiation and presumptive
identification of the organism in question.
Brilliance Listeria Agar
order codes: CM1080, SR0227 & SR0228
Brilliance Listeria Agar is a chromogenic
medium incorporating a chromogen that is
cleaved by the enzyme ß-glucosidase, common
to all Listeria spp., giving rise to blue-green
colonies. Other organisms that are positive for
this enzyme are inhibited by the selective agents in the medium
(lithium chloride, polymyxin B and nalidixic acid), whilst inclusion of
amphotericin inhibits the growth of any yeasts and moulds present.
Listeria monocytogenes and pathogenic L. ivanovii are then further
differentiated by their ability to produce the phospholipase enzyme,
lecithinase. This enzyme hydrolyses lecithin in the medium,
producing an opaque white halo around the colony.
Brilliance Listeria Agar also forms part of the Listeria Precis
method; a protocol using just one broth, one plate and a simple 10
minute confirmation step to deliver results in only two days.
Listeria Precis is validated by AFNOR to ISO 16140:2003 for all food
and environmental samples.
Brilliance Salmonella Agar
order code: CM1092 & SR0194
Brilliance Salmonella Agar is the first in a new
class of chromogenic media to incorporate
novel Inhibigen technology. This new technology
improves recovery of Salmonella by reducing
background flora. The medium provides easy
identification and differentiation by producing brightly coloured
purple colonies. Brilliance Salmonella Agar also forms part of the
Salmonella Precis TM method which can give a confirmed result for
Salmonella from food and environmental samples in just two days.
This ISO 16140:2003 AFNOR validated method uses a single broth
and plate followed by a simple latex test.
Brilliance E. coli/coliform Agar
order code: CM0956
Brilliance E. coli/coliform Agar is a non-selective agar that can be
used when performing TVC (total viable count) on a sample. The
combination of chromogens used means it will give an indication of
any Escherichia coli and other coliforms in the sample, as they will
produce brightly coloured purple or pink colonies respectively.
10
Brilliance E. coli/coliform Selective Agar
order code: CM1046
Brilliance E. coli/coliform Selective Agar is used
for the detection and enumeration of E. coli
and other coliforms from food and water, while
inhibiting the growth of Gram-positive organisms.
Optimisation of the peptone composition of the
medium now allows an indole test to be preformed directly on the
plate. This test is commonly used as a confirmatory test for E. coli.
Kovac's solution is added directly to purple, presumptive E. coli
colonies, which then produce a characteristic cherry red
colouration, allowing rapid and easy confirmed enumeration
from the plate.
Brilliance Bacillus cereus Agar
order codes: CM1036 & SR0230
Brilliance Bacillus cereus Agar incorporates a
chromogenic substrate that is cleaved by the
enzyme ß-glucosidase expressed by Bacillus
cereus, resulting in the formation of an intense
blue colour. The medium also incorporates the
selective agents polymyxin B and trimethoprim, which inhibit
the growth of non-Bacillus spp. and other organisms capable of
utilising the chromogenic substrate.
Brilliance Enterobacter sakazakii Agar (DFI)
order code: CM1055
Brilliance Enterobacter sakazakii Agar
allows recovery and detection of Cronobacter
sakazakii (previously known as Enterobacter
sakazakii) in just three days. This innovative
medium contains a chromogen that is cleaved
by the enzyme, α-glucosidase, expressed by C. sakazakii, to form
easily distinguishable blue-green colonies.
So when you are next talking to your Oxoid representative or
ready to place an order remember Brilliance - chromogenic media
that deliver clearly visible answers on a single culture plate. For
more details visit www.oxoid.com or tick 9 on the reply paid card.
REPLY 9

MicroVal, a European Validation and
Certification Organisation
MicroVal secretariat, Pauline
Kalkman, explains the validation
process for alternative
microbiological methods.
The history of MicroVal
MicroVal started as a Eureka project in the nineties. In its final
stages the project consisted of 21 full partners from seven
European countries (Denmark, Germany, UK, Netherlands,
France, Spain and Portugal) and delivered:
1 Technical rules for validation – at a later stage brought into
European standardisation channels and finally published in
2003 as: EN/ISO 16140 – the protocol for the validation of
alternative microbiological methods
1 The MicroVal Rules and Certification Scheme for the quality
control of validation.
In 2006 the organisation became operational. The first certificate
was handed out in 2007 and in 2008 another four certificates
were approved.
MicroVal organisation
MicroVal is a European validation and certification organisation
for the validation of alternative microbiological methods. The aim
is to provide a single validation system within Europe. Stakeholders
in the validation process are represented in the organisation.
The MicroVal organisation is based on an impartial European
structure (Figure 1) which consists of:
1 An Impartial European Board of Experts, the MicroVal General
Committee (MGC), in which the interests of stakeholders are
evenly covered. The most important focus of the Board is to
develop and maintain the MicroVal validation and certification
system
Figure 1: Organogram of the MicroVal organisation
Public Authorities Users Manufacturers Third Parties
EU Expert Labs
MGC
Expert Committees MV Secretariat
EU Collaborative Labs
MCB Group
EU Method Reviewers EU Auditors
11
1 The MicroVal Certification Body (MCB) Group, which is open for
participation by all European Certification Bodies e.g. Lloyd's
Register QA or DNV Certification
1 A Neutral MicroVal secretariat, for which NEN (Dutch standardisation
body) is responsible
1 The European network consisting of Expert Laboratories,
Collaborative Laboratories, reviewers and auditors. The MicroVal
Expert Laboratories that are currently active are UGent (Belgium),
RIKILT (NL), and Campden-BRI (UK)
1 The MicroVal Technical Committee, in which all stakeholders
are represented, for the evaluation of validation studies.
Why use a product with MicroVal validation?
The EU Legislation: Commission Regulation (EC) 2073/2005 on microbiological
criteria for foodstuffs of 15th November 2005, published
in the Official Journal of the European Union on 22nd December
2005, states in Article 5 ‘Specific rules for testing and sampling’:
1 The use of alternative analytical methods is acceptable when
the methods are validated against the reference method in
Annex I and if a proprietary method, certified by a third party in
accordance with the protocol set out in EN/ISO standard 16140
or other internationally accepted similar protocols, is used.
MicroVal Certification conforms to these requirements
as follows:
1 Validation is performed against a CEN method or ISO
method as stated in Annex I, or, if no CEN/ISO method is
available, against a European official method
1 Validation is granted by third party MicroVal certification
bodies
1 Validation is in accordance with the protocol set out in
EN/ISO standard 16140 – Protocol for the validation of
alternative methods, which also forms the basis of
MicroVal.
Therefore, alternative methods certified with the MicroVal
organisation fulfill the requirements as stated in the
European Commission regulation.

MicroVal certification principles
The MicroVal certification procedure is based on three principles:
1. To perform a method comparison study and collaborative study
according to EN/ISO 16140
2. The organisational quality of the manufacturer where the
materials are produced must be in conformance with
ISO 9001/ISO 13485
3. A regular verification of the quality of the certified methods,
which is made after the certification, is granted. Validity of the
certificate is four years. Thereafter a renewal has to be carried out.
Validation procedure
The validation procedure consists of three important decisions
stages. First of all, the protocol for both studies is developed by
the MicroVal Expert Laboratory. Thereafter, the first study is performed,
in which the alternative method is compared to the
reference method. The final part is the interlaboratory study,
where the submitted test method is validated by eight or ten
Collaborative Laboratories (for a quantitative or a qualitative
method respectively) from three different European countries.
Each of the stages requires an approval before the next one can
be performed. Two MicroVal method reviewers and the MicroVal
Technical Committee evaluate the protocol and report on the
studies. On completion, the legal decision to grant the certificate
is taken by the MicroVal Certification Body (Figure 2).
Validation for qualitative and quantitative methods
Qualitative alternative methods
The method comparison study is performed by the MicroVal
Expert Laboratory. If the alternative method is to be validated for
all foods, five categories of food must be involved. For each food
category, 60 test samples should be analysed. Inclusivity and
exclusivity studies, with target and non-target organisms, and
determination of the minimum level of detection are also necessary.
The samples used should be naturally contaminated but, if
that is not possible, a limited number of artificially contaminated
samples may be used with cells in a similar state of stress and a
similar type of background microflora. The availability of naturally
contaminated samples depends strongly on the food categories
selected as relevant for the target organism and for the scope of
the method.
The interlaboratory performance study is organised by the Expert
Laboratory. At least 24 samples are analysed by ten Collaborative
Laboratories, with a minimum of three contamination levels and
at least eight samples per level. Samples are analysed by the
alternative and the reference method, with results returned to
the Expert Laboratory for analysis. On completion, the Expert
Laboratory submits a final report to the method reviewers and
the MicroVal Technical Committee for assessment.
12
Figure 2: Flow Chart Microval Validation and Certification Scheme
Selection of
Expert Lab
and method
reviewers
ISO 9001/ISO 13485
approved?
NO
Audit
manufacturing
site
YES
Application manufacturer
Check administrative conformity by MCB
Draft protocols preliminary and interlaboratory study
by Expert Lab & evaluation by method reviewers &
decision by MV Technical Committee
Protocol
approved?
YES
Expert laboratory performs preliminary study
Results preliminary study evaluated by method
reviewers and decision of Technical Committee
Report
approved?
YES
Interlaboratory study performed by Expert Lab
Results Interlaboratory study evaluated by
method reviewers
Advice of Technical Committee to MCB
Report
approved?
YES
NO
NO
NO
Certification decision by MCB
Certificate published by MV secretariat
Validity 4 years

Quantitative alternative methods
Again, comparison and interlaboratory studies are organised by
the Expert Laboratory. The comparison study determines linearity,
relative accuracy, detection and quantification limit. The rest of the
validation procedure is the same as for qualitative methods.
If methods have already been validated and/or certified by another
organisation, specific rules apply in order to consider such results.
Expert laboratories
The independent Expert Laboratory must be accredited to EN/ISO
17025 for the reference method involved in the validation study. It
is also important that the Expert Laboratory has experience in the
development of new techniques; is involved in organising interlaboratory
studies; and has statistical knowledge. It is selected by the
manufacturer (or the MCB) from the database of laboratories
established by the MGC. The qualification ‘Expert Laboratory’ is
only valid for the MicroVal certification of the alternative method
it was selected for.
Collaborative Laboratories
Collaborative Laboratories participating in the interlaboratory study
are selected by the MicroVal Expert Laboratory on the basis of
their capability and geographic distribution (at least three different
European countres). The Expert Laboratory coordinates the interlaboratory
study in which the Collaborative Laboratories are
involved.
DEDICATED TO MICROBIOLOGY
To find out more contact:
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Hants RG24 8PW
Summary
Validation systems are developed to provide fully validated methods
that can be used with confidence by public authorities, food industry,
laboratories, and end users. A certified method provides a level of
confidence to all parties because of its reliability.
MicroVal offers the opportunity for test kit manufacturers to undertake
a third party evaluation programme according to EU legislation
to gain acceptance for the product across Europe. A MicroVal
certificate will show, to the method user, that an alternative method
has been thoroughly tested using an approved and standardised
procedure. It will mean that a method can be used confidently and
with the knowledge that the results of that method will be accepted
without question throughout Europe.
At the international level MicroVal is actively working with AOAC
trying to harmonise validation requirements and allow results of
certifications to pass between these international markets. With
NordVal we are working towards validations that are being performed
at the same time for both MicroVal and NordVal. This benefits
the manufacturers at least with respect to the validation costs.
Furthermore MicroVal validations are being performed by Expert
Laboratories in Europe.
For more information please contact Pauline Kalkman at MicroVal:
e-mail: pauline.kalkman@nen.nl, tel. +31 5 2690 112,
www.microval.org
Oxoid Prepared
Media Service
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Food microbiologists worldwide rely on Oxoid Prepared Media to
save time and resources in their laboratory . With 7 prepared media
manufacturing sites on 4 continents, we make microbiology
easier every day.
13
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Oxoid AGS Products Improve Performance
in Italian Public Health Laboratory
The Oxoid AGS products provided superior results and greater convenience in the
IZSM laboratory in Italy.
The Zooprophylaxis Institute of Southern Italy (IZSM) is a public
health laboratory that operates in the areas of public hygiene and
veterinary health as a technical-scientific centre, in the regions of
Campania and Calabria, for the Italian State.
Following an evaluation four years ago, the IZSM began to use
the Oxoid Atmosphere Generation System (AGS) products in two
different departments within the organisation:
1 The Department of Animal Health - Diagnostic Unit
1 The Department of Food Control - Microbiological Controls of
Foodstuffs of Animal Origin Unit.
The Department of Animal Health is directed by veterinarian,
Dr. Giorgio Galiero. The Diagnostic Unit has six members of staff
and carries out about 350 analyses each week on animal samples,
such as faeces, urine, swabs, organs excised during autopsies,
various other biological samples and housing fluids (e.g. water
from tanks containing tropical fish, turtles, etc.).
The Department of Food Control - Microbiological Controls of
Foodstuffs of Animal Origin Unit, coordinated by Dr. Daniela Bove,
is a specialist facility that performs about 400 microbiological
analyses every week on food and animal feed samples. The aim
of these investigations is to evaluate the level of hygienic quality
and the risk of microbial contamination in these samples, in order
to prevent food-related diseases in humans and animals. This
department is divided into four units:
1 Microbiological Controls of Foodstuffs of Animal Origin
1 Quality Control of Milk and Laboratory for the Control of Honey
and Apian Diseases
1 Microbiological Controls of Foodstuffs for Animal Use
1 Laboratory for the Control of BSE in Animal Ground Meal.
Evaluation
For several years, these laboratories
used jar systems that required the
addition of water and the use of a
catalyst (Oxoid Gas Generating Kits)
for the incubation of media in controlled
atmospheric conditions.
Then, four years ago, they performed
a series of evaluations on the newer
Oxoid AGS products (Table 1) that do
not require a catalyst or water.
The evaluations consisted of recovery tests performed on culture
media that were prepared in the different laboratories and inoculated
with various micro-organisms, including microaerophilic
(Campylobacter jejuni ATCC ® 33291TM *) and anaerobic
(Clostridium sordelli ATCC ® 9714TM *) strains. The plates were
incubated in the appropriate jars using Oxoid AnaeroGenTM ,
CampyGenTM and CO2GenTM products and in pouches using the
corresponding Compact products. Control strains were obtained
from the ATCC ® * collection and appropriate external quality
assessment criteria were applied.
*
The ATCC Licensed Derivative Emblem ® , the ATCC Licensed Derivative word mark ® , and the
ATCC catalog marks are trademarks of ATCC. Oxoid Ltd is licensed to use these trademarks
and sell products derived from ATCC ® cultures.
14
Oxoid AGS products
Oxoid AGS products are activated on
contact with air (no water or catalyst
is required) and the required gaseous
conditions are created quickly and
safely, with no pressure build-up or
generation of hydrogen. Only nonhazardous
materials are used, which
ensures safe and easy transportation,
storage and disposal.
Table 1. Oxoid Atmosphere
Generation System range
Gaseous conditions
required
AnaeroGen
(2.5 & 3.5 L)
CampyGen
(2.5 & 3.5 L)
CO2Gen (2.5 L)
AnaeroGen
Compact
CampyGen
Compact
CO2Gen Compact
AnaeroJar
(2.5 L)
Anaerobic
Jar (3.5 L)
Anaerobic 1 1 1 1
Microaerobic 1 1 1 1
CO2-enriched 1 1 1
Improved performance
“We found that the growth of the ATCC ® strains was more
luxuriant and faster with the Oxoid AGS products, compared to our
previous method, particularly with anaerobic micro-organisms,”
comments Dr Ascione, who coordinated the AGS evaluations in
the Diagnostic Unit. “The excellent qualitative results that we
obtained, combined with the reliability and greater practicality
of the products and speed with which they could generate the
required gaseous atmosphere, led us to adopt the Oxoid AGS
products (both jar and Compact formats) in our daily, routine
work.
“In particular,” Dr. Bove adds, “the AGS Compact line has
improved our laboratory performance, reducing the space
required for both incubating plates and storing the kits. We have
also found the Compact formats to be particularly convenient in
situations where we have a small numbers of samples.”
Convenient
Whereas anaerobic jars are designed to hold 12-15 plates at a
time, the Oxoid AGS Compact products are more convenient for
individual or small numbers of plates: Oxoid AnaeroGen Compact
can hold up to four plates; Oxoid CampyGen Compact and Oxoid
CO2Gen Compact will hold one or two plates.
“We have also recently adopted the new W-zip pouches, because
they are simpler to use,” adds Dr. Bove.
“Given all of these factors, combined with ease of disposal,” Dr
Bove concludes, “we are happy to recommend the Oxoid AGS
products to other laboratories.”
For further information about the
Oxoid AGS product range, visit
www.oxoid.com or tick 10 on the
reply paid card. REPLY 10

Oxoid W. H. Pierce Memorial Prize Acknowledges
Research into Modified Peptides
University College Cork microbiologist, Dr Paul Cotter, has won
this year's prestigious W. H. Pierce Memorial Prize following his
research in the area of modified peptides, both those which function
as antimicrobials and a newly identified group of such peptides
which contribute to the virulence factors of food-poisoning
bacteria such as Listeria monocytogenes.
Sponsored by Oxoid, under the auspices of the Society for Applied
Microbiology, the £2,000 prize is given every year to a young
Society member, under 40 years old, who has made a substantial
contribution to this area of science.
Dr Cotter lectures at the University's department of microbiology
and is a principal investigator receiving funding from Science
Foundation Ireland, the Health Research Board and Enterprise
Ireland. He has been undertaking research on food microbiology in
general, and on peptides and L. monocytogenes in particular, for
over 12 years now.
“It is exciting news and a great privilege to receive the prize.
It is the culmination of many years hard work,” said Dr Cotter on
winning the award.
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He added: “The congratulations and acknowledgement I have
received from my colleagues and fellow microbiologists have been
really encouraging and demonstrate the esteem in which the
award is held.”
Dr Cotter's most recent work involved the identification of a toxic
factor that is produced exclusively in the subgroup of L. monocytogenes
strains that are most frequently responsible for epidemic
outbreaks of listeriosis.
The discovery of these factors brings the University College
Cork-based team a step closer to understanding the pathogenesis
of the organism. Furthermore, since the toxic factor is only
produced in the subgroup responsible for outbreaks, Dr Cotter's
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FSS11

work will also be of value in the development of diagnostic
techniques to detect this food-borne pathogen.
“The identification of this toxic factor represents a significant
breakthrough in Listeria research,” explains Dr Cotter. “Scientists
have tried for over twenty years to determine why some
L. monocytogenes strains are more dangerous than others. Now,
for the first time, a factor that may explain this phenomenon has
been identified.
“These findings have even broader implications for the research
of disease-causing bacteria in general, as initial investigations
indicate that these toxic factors may be much more widely
distributed than previously thought.” he added.
Dr Cotter's work on antimicrobial peptides, or bacteriocins, against
Listeria and organisms such as meticillin-resistant Staphylococcus
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aureus (MRSA) and Streptococcus was also recognised by the
award judges.
Unlike the virulence factors that attack healthy eukaryotic cells,
bacteriocins attack bacterial cells. Some bacteriocins, known as
lantibiotics or lanthionine-containing antibiotics, show powerful
antimicrobial activity. Dr Cotter's team is working to identify novel
lantibiotics, to generate derivatives with enhanced activity, in an
attempt to combat antibiotic-resistance in pathogenic bacteria.
The W. H. Pierce Memorial Prize was instituted in 1984 by the
directors of Oxoid to commemorate the life and works of the late
W. H. (Bill) Pierce, former chief bacteriologist of OXO Limited and
long-time member of the Society. Bill Pierce was a pioneer in the
development of dehydrated culture media and was a great
contributor to the foundation of the Oxoid range.
Do you use Oxoid products or have an interesting insight to share with
other food microbiologists in Setting Standards? If so, we'd love to hear
from you - just fill in the reply paid card on the previous page and we'll
contact you to discuss your ideas.
Tel: +44 (0) 1256 841144
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© 2008, Oxoid Ltd.; copyrights to photographs held separately; contact Oxoid Ltd for details. Photographs may not be extracted or reproduced in any way. Folio No 1271/AM/11/08
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