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BacTracBacTrac CalibrationCalibration, , StandardisationStandardisation and Validationand Validation
Dr. Manfred SchinkingerDr. Manfred SchinkingerSYSY--LAB GerLAB Gerääte GmbH., te GmbH., NeupurkersdorfNeupurkersdorf, , AustriaAustria
MICROBIAL ENUMERATION MICROBIAL ENUMERATION
PLATEPLATE
•• GivesGives a a numbernumber of so of so calledcalled „„colonycolony formingformingunitsunits = CFU= CFU““
BacTracBacTrac
•• GivesGives a a detectiondetection time time dependingdepending of of thethe activityactivityand and nnúúmbermber of of microbialmicrobialcellscells presentpresent in a in a samplesample
•• NOTE: Short NOTE: Short detectiondetection = = high high numbernumber ((activityactivity))
TheThe MysteryMystery of CFU Determinationof CFU Determination
5 15
2 38
17
3
TVC = 71 CFUTVC = 7 CFU
BacTracBacTrac CalibrationCalibration –– MethodMethod. . differencesdifferences
PLATEPLATE
•• Definition of Definition of „„CFUCFU““ –– notnotalwaysalways originatingoriginating fromfrom a a singlesingle cellcell
•• ResultResult calculatedcalculated usingusing a a statisticalstatistical approachapproach in a in a countablecountable rangerange
•• SemiSemi solid solid substratesubstrate
BacTracBacTrac
•• Different Different metabolicmetabolicactivitiesactivities of different of different microbesmicrobes
•• DirectDirect resultresult withoutwithoutdilutiondilution errorerror
•• Liquid Liquid substratessubstrates
RelationshipRelationship BacTracBacTrac -- PlatePlate
log CFUlog CFUy =y =
--0,96340,9634r =r =
0,350,35SyxSyx ==
BacTracBacTrac CalibrationCalibration --FundamentalsFundamentals
•• identicalidentical startingstarting material (material (samesame homogenatehomogenate))•• standardisedstandardised referencereference methodmethod•• suitablesuitable rangerange of of contaminationcontamination ((maxmax 101099 -- 10)10)•• adequateadequate distributiondistribution overover a 4 a 4 -- 5 log 5 log rangerange•• preciseprecise voluminavolumina•• contaminationcontamination floraflora and and metabolicmetabolic situationsituation
of of thethe contaminationcontamination floraflora•• geograficalgeografical variationsvariations of of thethe floraflora
Flora and Flora and metabolicmetabolic situationsituationExampleExample 11
RawRaw milkmilk
•• gram. gram. negneg germsgerms arearedominatingdominating
•• liquidliquid
PastPast. milk. milk
•• SelectionSelection forfor gram gram pos. pos. ThermoduricThermoduricgermsgerms
•• liquidliquid
Different flora = no combined calibration !
Flora and Flora and metabolicmetabolic situationsituationExampleExample 22
Milk Milk powderpowder
•• selectionselection forfor gram gram pos. pos. ThermoduricThermoduricgermsgerms
•• drydry lowlow aaww--valuevalue
PastPast. milk. milk
•• selectionselection forfor gram gram pos. pos. ThermoduricThermoduricgermsgerms
•• LiquidLiquid
Different metabolic situation = no combined calibration !
CalibrationsCalibrations –– Evaluation Evaluation criteriacriteria
•• CoefficientCoefficient of of correlationcorrelation rr•• Dispersion Dispersion ssyxyx
•• ExpectedExpected resultsresults::
r = r = --0,85 to 0,85 to --1.01.0ssyxyx = = < 0.5 (< 0.5 (unitunit = log)= log)
CalibrationCalibration ColiformsColiforms Soft Soft CheeseCheese
Linear regression
y = -0,2566x + 6,6508R2 = 0,8036
0
1
2
3
4
5
6
0 5 10 15 20 25
detection time (µ-Trac oder BacTrac)
log
TVC
-0,8964r =
0,479Syx =
IMPEDANCE and IMPEDANCE and STANDARDISATION (Europe)STANDARDISATION (Europe)
DIN and DIN and §§64 LMBG, 64 LMBG, ÖÖNORMNORMDIN 10115:DIN 10115: basicbasic standardstandard ((principlesprinciples))
DIN 10120: DIN 10120: SalmonellaSalmonella detectiondetection (horizontal)(horizontal)
DIN 10122: TVC DIN 10122: TVC enumerationenumeration
AFNORAFNORNF V08NF V08--105: 105: basicbasic standardstandard ((principlesprinciples))
NF V08NF V08--106: 106: E.coliE.coli in sea foodin sea food
Validation Validation –– legal legal requirementsrequirements
•• ISO 16140ISO 16140 -- Validation Validation guidelinesguidelines foodfood
•• EG 2073/2005EG 2073/2005 –– Validation Validation requirementsrequirements
•• Pharm. Pharm. EurEur. 6. 6 –– PharmaceuticalPharmaceutical applicationsapplications
OfficialOfficial thirdthird party party validationsvalidations(ISO 16140)(ISO 16140)
EnumerationEnumeration of of E.coliE.coli in in shellfishshellfish (IFREMER, (IFREMER, finishedfinished))
DetectionDetection of of EnterobacteriaceaeEnterobacteriaceae((AFNOR,finishedAFNOR,finished))
Determination of Determination of aerobicaerobic mesophilicmesophilic countscounts(AFNOR in (AFNOR in progressprogress))
DetectionDetection of of SalmonellaSalmonella(AOAC (AOAC plannedplanned))