SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL...
Transcript of SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL...
![Page 1: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/1.jpg)
SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN
FOOD ENVIRONMENTS
Christine Faille
International Biofilm SummitREALCO 24-25 march 2015
INRA, UMET UMR 8207Team « Interface Processes and Hygiene of Materials »
369 rue Jules Guesde, 59651 Villeneuve d’Ascq+33 (0)3 20 43 54 24
![Page 2: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/2.jpg)
.2
What is a biofilm?
� Collection of microbial communities enclosed by a matrix of extracellular polymeric substance (EPS) and separated by a network of open water channels
� EPS : proteins, carbohydrates, DNA� Within biofilms, bacteria in ≠ physiological states (dead, cultivable…)
![Page 3: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/3.jpg)
� Different physiological conditions of the microorganism within soils� Viable and cultivable� Viable but non-cultivable� Dead / inactivatedÎ Underestimation of the bacterial load
.3
Physiological state of the microorganisms
![Page 4: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/4.jpg)
� Presence of “injured” bacteria (stationary phase, stress…)=> Increased sensitivity to environmental conditions (components of the isolation medium / enumeration medium…)
� Colour indicator � Antibiotics� NaCl� Incubation T° (5°C < optimal temperature)
=> Length to recover (resuscitation process)�When bacteria are able to repair injuries => increased duration of the lag phase
.400,511,522,533,54
75 80 85 90 95Température de traitement (20 min)
-log(
1-%
endo
mm
agée
s)
Physiological state of the microorganisms
![Page 5: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/5.jpg)
.5
Methods to control surface hygiene(to detect biofilms?)
Soil
Biochemical testsMicrobiological tests(viable and cultivable / VNC)
Contact methods
PlatesSlides
Petrifilms
ProteinsCarbohydrates
LipidsFood
EPS ATPNADPH
![Page 6: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/6.jpg)
.6
Contact methods (microbiological)
![Page 7: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/7.jpg)
.7
Principle: Adherent bacteria transferred by pressing the agaronto the contaminated surface
� Contact plates, RODAC* plates *Replicate Organism Direct Agar Contact
� PétrifilmTM
� Contact Agar Slides
Contact methods (microbiological)
![Page 8: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/8.jpg)
.8
Contact methods (microbiological)
Many available growth media� Total mesophilic counts (PCA)� Staphylococcus (Baird Parker)� Coliforms (VRBL)� Faecal streptococci (Slanetz)� Yeasts & moulds� Listeria…+ neutralizers to deactivate residual disinfectants from the surface to be tested
![Page 9: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/9.jpg)
.9
Contact methods (microbiological)
Contact plates (RODAC)� Surface slightly convex (meniscus of agar extending above the top of the poured plate)
=> contact with the surface to be sampled (ø 56 mm) � Reproducibility of pressure and application time => applicators=> Widely used=> Rather adapted for plane and smooth surfaces
Applicator (500 g, 10 s)
Food Stamp TGSE Agar Staph. aureus
Columbia Agar with Sheep Blood
Staph. / streptococci
![Page 10: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/10.jpg)
.10
Contact methods (microbiological)PetrifilmTM 3M� Flexible film
• Medium in dry form• Medium protected by a polypropylene membrane
�Moisten the medium before use (30 min to 1-2 h)� Reproducibility of pressure and time => applicator to be adapted on flat surfaces=> Possible testing of curved surfaces (tanks, pipes, door handles…)
![Page 11: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/11.jpg)
.11
Contact methods (microbiological)
Incubator
Contact Agar Slides (=dipslides)� Hinged plastic paddle covered with culture medium on both faces (one/two media)� Ready to use�Mini-incubator, no applicator=> Widely used in the food industry=> Rather adapted for plane and smooth surfaces=> Poorly reproducible
![Page 12: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/12.jpg)
.12
Contact methods (microbiological)
RODAC plates : successively applied to stainless steel coupons contaminated with bacterial spores
Partial recovery of adherent bacteria� Up to 60% spores not detached after the first contact � Great differences between strains
![Page 13: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/13.jpg)
.13
MICROBIOLOGICAL METHODS• Very long (18 – 72 h)• Accessibility required
• Unable to detect food residues• Only able to detect cultivable microorganisms• Possible identification of the microorganisms
- - - - - - - - - -
ÎNot suited to check the effectiveness of cleaning procedures
Î Suited to check the effectiveness of disinfection procedures OR to validate routine methods
![Page 14: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/14.jpg)
.14
Biochemical methods
![Page 15: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/15.jpg)
.15
Biochemical methods
Sampling by swabbing followed by a direct dosage(colorimetric tests…)
� of proteins +/- reducing sugars� of carbohydrates� of biofilm EPS� of food soils� of ATP, NAD(H)
![Page 16: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/16.jpg)
.16
Biochemical methodsProtein detection� CleanTrace (3M), Clean Test (LiofilChem), PRO-Clean (Hygiena)…
– Biuret test => detection of protein residues + reducing substances
– 1 to 10 min incubation– Detection limit: 50/60 µg on 100 cm² (BSA)– Test reading: visual / luminometer
=> Colour change easy to interpret=> Pro-Clean : very flexibleÎ sometimes, detachmentpoorly efficient (rough materials…)
Luminometer
![Page 17: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/17.jpg)
.17
Biochemical methodsProtein detection� Clean-Trace Surface Protein (instant) [3M]
– Proteins => green colour– Instant reading of the “swab”
=> Very quick reading required=> Difficult to break up the “capsule” (to activate the reaction)
![Page 18: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/18.jpg)
.18
Protein detection� Flash [Biocontrol]
– Press device tip into the hydration pad for 2-3 s (device tip color: from blueto yellow)– After swabbing, observation of the device tip (<10 s) (if proteins, colorfrom yellow to blue [Detection limit: 20 µg])
=> Reading sometimes ambiguous on biofilms=> Excessive pressure while swabbing may damage device membrane
Biochemical methods
- +
![Page 19: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/19.jpg)
.19
Protein detection� Ridacheck (R-Biopharm), Pathcheck (Microgen)…
– Surface sampled = 20 cm²– Detection limit: 20 µg– Result produced in 5 sec to 2 min– Colour change from yellow to blue/green
=> Ease to use=> Colour change easy to interpret
Biochemical methods
![Page 20: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/20.jpg)
.20
Biochemical methodsCarbohydrate detection� SpotCheck Plus [Hygiena]
– Detection of glucose, lactose– Result obtained in 60 s– Reagent turns green in the presence of either sugar residue– Detection limit: 2.5 μM D-glucose, 5.0 μM lactose
=> Useful for dairy plants, confectionary candy and beverage plants=> Unsuitable for biofilm detection
![Page 21: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/21.jpg)
.21
Biochemical methodsEPS detection� Biofilm Detector Kit [REALCO]
– Detection of EPS produced by adherent bacteria– Use on stainless steel surfaces– Result obtained in 10-15 min (5 min reactive 1, rinse, 5 min reactive 2)
– Blue colour visible with naked eye in the presence of EPS
=> Direct visualisation of residual biofilm on surfaces=> Unsuitable on polymers
![Page 22: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/22.jpg)
.22
Biochemical methodsDetection of organic food residues� Vericleen [Charm]
– Detection of organic matter– Surface must be wet(if dry surface => wet with >3 pumps of “wetting Liquid”)– Results obtained in 5-60 s– Development of a purple color in the presence of OM
=> Many false negative results with biofilms=> Unsuitable on rough materials (fragile membrane)
![Page 23: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/23.jpg)
.23
Biochemical methodsDetection of NAD(P)/NAD(P)H (food/microorganisms)� SpeedCheck [Ecolab]
–Development of a blue colour in the presence of NAD– Results obtained in 5 min
=> Colour change easy to interpret=> Unsuitable on rough materials (fragile membrane )
![Page 24: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/24.jpg)
.24
Biochemical methodsDetection of ATP (food / microorganisms)� Principle: enzymatic reaction to detect ATPATP found in all living cells (bacteria, animals, vegetables…) = energy source
ATP + Luciferine + O2
Luciferase + Mg
Oxyluciferine + AMP + CO2 + PPi + photons
– Emitted light proportional to the ATP level– Measurement of the emitted light in a luminometer
![Page 25: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/25.jpg)
.25
Biochemical methodsDetection of ATP (food / microorganisms)� Sampling devices
– Many swabs� Luminometers
– with photodiodes : cost saving, low sensitivity– with photomultiplier tubes : expensive, high sensitivity
=> Detection system can be affected by residual disinfectants– QAC sanitizers known to create false positive readings– Oxidizing sanitizers can create false negatives on freshly sanitized surfaces
=> Great differences in the ATP content in different organisms
=> Few if any detection of dead cells & VNC
=> Quick (≤ 30 min)
![Page 26: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/26.jpg)
.26
Biochemical methods
Some interesting devices able to detect biofilms (results from PIHM)� Positive tests, easy reading (clear change of colour)
� Speed Check (Ecolab)
� Ridacheck, Pathcheck (R-Biopharm / Microgen)
� Clean trace Plus (3M), Clean Test (LiofilChem)
� Biofilm Detector Kit (REALCO)
![Page 27: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/27.jpg)
.27
BIOCHEMICAL METHODS• Rapid
• More or less accessible areas
• Minimal training required• Not affected by the physiological state of the bacteria (except ATP, NAD)
• +/- sensitive, +/- reproducible- - - - - - - - - -
Î Not suited to check the effectiveness of disinfection procedures
Î Suited to check the effectiveness of cleaning procedures
![Page 28: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/28.jpg)
.28
Conclusion
![Page 29: SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING … · SUITABILITY OF BIOCHEMICAL SAMPLING DEVICES FOR MONITORING BIOFILMS IN FOOD ENVIRONMENTS Christine Faille International](https://reader031.fdocuments.us/reader031/viewer/2022041102/5edbc183ad6a402d6666210f/html5/thumbnails/29.jpg)
.29
BIOCHEMICAL METHODS
Could be used routinely in complement to microbiological methods to detect biofilms on
surfaces in food environmentsÎDetection of possible cleaning incidents
ÎImplementation of corrective actions in time