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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
SURFACE MICROBIOLOGYBiofilm Formation & Its Elimination
The Annual Veterinary Congress 2015
Helsinki Fair Center, Helsinki, Finland
December 4, 2015
Gun WirtanenDTU National Food InstituteLyngby, Denmark
National Food Institute, Technical University of Denmark
Example: Poor hygienic design
2 (21)
Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Example: Poor hygienic design
National Food Institute, Technical University of Denmark
Surface Profiles of Various Stainless Surfaces
2 B plus, 400 grit polished surface
2 D plus, cold rolled surface
Cold rolled, annealed and pickled surface
Surface profile
Profile height
The centre of the surface
Size of a bacterial cell
,
# 2D # 2B # 7
Surface profile
-
in m
200
100
0 0
100
200
1
1
2
2
3
3
4
5
4
5
4
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Size of Microbes
Mould Spores – Ø [µm]
Aspergillus niger 2-10
Bacterial strain Length [µm] Thickness - Ø [µm]
Clostridium botulinum 3-8 0.5 – 0.8
Clostridium perfringens 4-8 1.0 – 1.5
Typical representation of a stainless steel surface profile of Ra =0.6 µm roughness achieved by 240 grit mechanical polish.
No surface defects or damage.
Source: Fraunhofer AVV, Leibniz –Institut für Polymerforschung Dresden
National Food Institute, Technical University of Denmark
SIZE OF MICROBIAL CELLS
The average diameter of a microbial cell are:
• bacterial cell 1 m
• mould mycelia 2 – 10 m
• yeast cell 2 – 12 m
• virus 10 – 100 nm
Electron microscope Light microscope
4 (21)
Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Microscopying of 5 d Pseudomonas fragi biofilm stained with acridine orange on various stainless steel surfaces (AISI 304):
(a) glass blasted, b) lapped and c) mechanically polished (according to Wirtanen, Saarela, Mattila-Sandholm. 2000. Biofilms – Impact on hygiene in food industries. In: Bryers (Ed.) Biofilms II: Process analysis
and applications. Wiley-Liss Inc. Pp. 327-372.)
National Food Institute, Technical University of Denmark
Biofilm consists of:
Microbes
Extracellular polymers (EPS; polysaccarides, glycoproteins etc. from microbes)
Water (85 - 98%)
Captured particles and other dissolved materials from the process stream
b) the same biofilm after swabbing
a) 6 d biofilm of Lactobacillus brevis
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Phases in theformation of biofilms
according to Busscher & van der Mei (2000) in "Initial microbial adhesion events: mechanisms and implications".In Proc. 59th SGM Symposium,p. 25-36, Cambridge University Press, ISBN 0 521 79302 5.
National Food Institute, Technical University of Denmark
PALM BEACH
pH
Atmosphere (oxygen)Temperature
Nutrients
Humidity, water
FACTORS AFFECTING MICROBIAL GROWTH
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
National Food Institute, Technical University of Denmark
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Biofilm Problems on Food Contact Surfaces:
on food contact surfaces of stainless-steel
on conveyors
on gaskets
on blancher extractor surfaces
in air handling systems
on floors and in drains
in manufacturing of paper-based packaging material
in packaging machines
in milk transfer lines cont.
National Food Institute, Technical University of Denmark
Biofilm Problems on Food Contact Surfaces:
in heat exchangers, pasteurizers and cooling systems
in ultrafiltration and reverse osmosis membranes
on mixers and slicers
on rubber fingered pluckers
in poultry processing equipment and
on vegetable processing lines etc.
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Contamination Routes - Surfaces
Source: Roland Cocker, Cocker Consulting
Source: Roland Cocker, Cocker Consulting
National Food Institute, Technical University of Denmark
– condensate / wet films
– stagnant water or liquid, drops
• poor installations allowing backflow
• non potable water
Source: Soro, AiniaSource: Soro, Ainia
Drain opened
Contamination Routes - Waterborne
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
1. Packaging machines (20/44)2. Conveyors (17/44)3. Slicing machines (15/44)4. Dispensers (15/44)5. Cooling equipment (9/44)6. Proofing drawers (6/44)7. Brining equipment (5/44)
Problematic equipment in various food industries based on a Finnish survey
Aarnisalo et al., 2006. The hygienic working practices of maintenance personnel and equipment hygiene in the Finnish food industry. Food Control 17, 1001-1011
National Food Institute, Technical University of Denmark
Biofilm Effects in Food Processing
energy losses in processing e.g. prolongedpasteurisation
deterioration on spesific chemical reactionse.g. contamination of immobilised cell systems
deterioration of products e.g. the product can beof lowered quality already after production
limited shelf-life of the products
occurrence of pathogenic microbes whichincrease the risks of foodborne poisoning
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
cleaning and disinfection processes are affected and the choice of chemicals, flow, time and temperature must be altered
increased need of maintenance in process lines and equipment e.g. prolonged down-time or laborious demounting of equipment e.g. heat exchangers in the process lines, in water supply systems, in granular activated carbon columns, in reverse osmosis membranes, in ion exchange systems, in degasifiers, in water storage tanks and in microporous membrane filters due to cleaning and hygiene, energy losses & blockages.
Biofilm Effects in Food Processing
National Food Institute, Technical University of Denmark
Biofilm Problems in Food Products:
RTE-products of meat and poultry
fermented meat sausages
fish cakes
unpasteurized milk
dairy products e.g. ice-cream & cheese
spices & herbs
iceberg lettuce, minimally processed vegetable salads & various deli
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Biofilm Problems in Food Products:
vegetables
caramel apples
canned products
pastries, biscuits & pizza
fruit juices, apple cider & beer etc.
National Food Institute, Technical University of Denmark
Airborne - Problem high pressure cleaning
Contamination routes
Sou
rce:
Tra
iner
Too
lbox
DVD
10
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Phases in an open, wet cleaning & disinfection procedure
in a food factory
Dry cleaning e.g. brushing or vacuum cleaning
Removal of gross soil from process equipment and facilities through rinsing
Removal of visible soil in process facilities through cleaning and rinsing
National Food Institute, Technical University of Denmark
Removal of visible soil from production equipment (i.e. cleaning of equipment ) foam/gel in open processes
Rinsing of equipment - Removal of remaining soil and cleaning agents from process surfaces through multipressure in open system
Phases in an open, wet cleaning & disinfection procedure
in a food factory
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Disinfection to kill living microbial residues left on surfaces and inhibition of microbial growth in periods when no processing is on
Fogging i.e. disinfection of process facilities
Drying of equipment and processing facilities using efficient air conditioning
Phases in an open, wet cleaning & disinfection procedure
in a food factory
National Food Institute, Technical University of Denmark
Hygiene Assessment -Physical Observation
•Use mirrors (also endoscopic e.g. inside pipelines) and lamps to get a view of hard-to-see places
•Use special light e.g. UV-light to see residues of organic soil
•Feel if the surfaces are greasy or encrusted
•Smell inside containers and tanks
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Hygiene Assessment –Visual Observation
•Look for product residues or raw material
•Look for scale
•Feel if the surfaces are greasy or encrusted
•Use camera to take pictures for documentation
•Smell in premises
National Food Institute, Technical University of Denmark
Problems in Hygiene Monitoring
• Attached microorganisms and biofilms are difficult to detach from surfaces
Improved sampling methods are needed
• Conventional hygiene control often underestimates the number of microorganisms or amount of soil
• Methods based on culturing are slow
Improved detection methods are needed
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Microbiological Assessment
• What to sample in the process? –Surfaces (also air, water, raw material & semi-ready products)
• Where to sample? – Avoid easy-to-sample areas, “worst case” areas are important
• When to sample ? – Before, during and after production
• How to sample ? – Consistent sampling, use contact agar applications, final rinse, swabs, sponges or gauzes
• How to interpret the results? – Internal standards
National Food Institute, Technical University of Denmark
Basic Idea of On-site Microbial Testing
n hours
Sample transported in liquid Sample transported on contact agar e.g. dipslide
x bacteria x * 23n bacteria
x colonies x colonies
Colony number unchanged
n hours01234
Factor 23n
20=123=826=6429=512212=4096
n hoursExample: x=55*1=55*8=405*64=3205*512=25605*4096=20480
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Source: Diversey Inc
Aerosols are efficient transport vehicle for airborne contaminants to surfaces, where the biofilm growth starts
National Food Institute, Technical University of Denmark
Sizes of Airborne Particles (Ø in μm)
Viruses
Brown, 1996
Bacterial spores Fungal spores
Bacteria
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Sedimentation of particles in still air / 1 meter
Diameter of particle Sedimentation timem min
2 1304 338 8
16 2
Movements of Particles in Air
National Food Institute, Technical University of Denmark
Removal of Microorganisms from Surfaces
Technique Suitability Limitations
Swab, sponge & gauze
Open surfaces, large areas or
irregular surfaces
Need suspension which will lower the sensitivity
RinseEnclosed areas or
small items
Dilution of the contamination is inevitable decreasing
the sensitivity
Direct contactCritical spot areas
with low contamination
Only small areascan be examined
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
Enumeration of Relevant Microbes
SWAB
RINSE
CONTACT
Suspension Spread on agar Incubation Counting
Spread on agar Incubation Counting
Incubation Counting(Spread on agar)
• Usually sufficient to know the total amount of microbes
• Sometimes important to know what kind of microbes
• Choice of agar
• Choice of incubation conditions (time, temperature & atmosphere)
National Food Institute, Technical University of Denmark
• Quantification of the actual number of microbes from surfaces is difficult due to strong microbial adherence of biofilms - traditional swabbing method is often improper
• The process hygiene may not be spoiled during sampling of surface
• Rapid results are needed to be able to act upon the results and prevent hazards
How to Sample? –Microbial Sampling of Surfaces
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
• Visual observation of cleaning results is an important practical method for: 1) open food contact surfaces, 2) difficult-to-see areas & 3) surfaces in closed equipment
• Soil can also be visualised with UV-light. This type of assessment is a practical choice especially in large equipment such as fermentation tanks.
• Surface hygiene can be carried out using ATP and protein based kits
National Food Institute, Technical University of Denmark
Establish internal standards
– Conduct a series of min 10 correct cleaning programmes
– Determine the level of microbes after each programme
– Mean result provides a standard
Trend analysis
– Repeated sampling and testing over a period of time
– Trend analysis provides a baseline
These concepts apply to both equipment and environment
Microbiological Assessments in Cleaning Validation –
How to Interpret the Results?
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
National Food Institute, Technical University of Denmark
We can see that biofilm can generate problems anywhere in the food
process if the design and maintenance is improper!
European Hygienic Engineering & Design Group
Advanced Course in Hygienic Designheld at DTU on 24th of November 2015Hazards in Hygienic Food Processing
© 2010 EHEDG
Microbial growth after pasteurisation
Hygienic filling line
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Surface Microbiology – Biofilm Formation & Its EliminationThe Annual Veterinary Congress in Helsinki on 4th of December 2015
Gun Wirtanen , DTU National Food Institute
European Hygienic Engineering & Design Group
Advanced Course in Hygienic Designheld at DTU on 24th of November 2015Hazards in Hygienic Food Processing
© 2010 EHEDG
Microbial growth after sterilisation without recontamination after heat treatment
Aseptic filling line
European Hygienic Engineering & Design Group
Advanced Course in Hygienic Designheld at DTU on 24th of November 2015Hazards in Hygienic Food Processing
© 2010 EHEDG
Microbial growth after sterilisation, when recontamination has occurred
after heat treatment
Filling line
Recontamination due to process air, process surfaces or packaging material
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