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))