This paper considers the main factors in the assessment of microbiological examination of food and discusses a few points related to validation of quantitative and qualitative microbiological methods. Within the scope of accredited methods, the author defines the terms such as conform reference, equivalence of reference method, and in-house method. The paper describes evaluation of a routine method with respect to the official method based on results obtained by automatic epifluorescent microscopy using the BactoScan 8000 instrument for determination of bacteriological quality of milk and provides general guidance for the establishment of a conversion relationship between the two methods. The paper gives an overview of the quality assurance aspects involved in the application of the routine method and concludes with an example of interlaboratory proficiency study for the epifluorescent microscopic method which is regularly applied in dairy laboratories.U ovome su članku prikazani glavni čimbenici u procjeni mikrobiološkog ispitivanja hrane. Prikazano je nekoliko točaka validacije kvantitativnih i kvalitativnih mikrobioloških metoda. Osim toga, definiraju se izrazi "sukladnost" i "istovjetnost" referentne metode s "internom" metodom u smislu akreditacija metoda. Članak opisuje kako evaluirati rutinsku metodu u odnosu na referentne/službene metode s obzirom na rezultate dobivene brojenjem automatskim epifluorescentnim mikroskopom s pomoću uređaja BactoScan 8000 namijenjenog za utvrđivanje bakteriološke kakvoće mlijeka te daje opće upute za usporedbu navedenih metoda. Tu je i pregled aspekata osiguranja kakvoće koji se uzimaju u obzir u primjeni rutinske metode te primjer međulaboratorijske prosudbe vrsnosti uz pomoć epifluorescentne mikroskopske metode koja je uobičajena u laboratorijima za mliječne proizvode

Slavica Golc-Teger

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61Arh Hig Rada Toksikol 2001;52:61-67MICROBIOLOGICALEXAMINATION ANDPROFICIENCY TESTING INDAIRY LABORATORIES Slavica GOLC TEGER,University of Ljubljana, BiotechnicalFaculty, Dom`ale, SloveniaReceived December 2000CONFERENCE PAPERThis paper considers the main factors in theassessment of microbiological examination offood and discusses a few points related tovalidation of quantitative and qualitativemicrobiological methods. Within the scope ofaccredited methods, the author defines the termssuch as conform reference, equivalence ofreference method, and in-house method. Thepaper describes evaluation of a routine methodwith respect to the official method based onresults obtained by automatic epifluorescentmicroscopy using the BactoScan 8000 instrumentfor determination of bacteriological quality of milkand provides general guidance for theestablishment of a conversion relationshipbetween the two methods. The paper gives anoverview of the quality assurance aspectsinvolved in the application of the routine methodand concludes with an example of interlaboratoryproficiency study for the epifluorescentmicroscopic method which is regularly applied indairy laboratories.Key words:conform reference, dairy laboratories,epifluorescent miscroscopy, microbiological methods,proficiency testing, quality assuranceThe most important aspects in the assessment of microbiological examination arethe validation of microbiological methods, implementation of quality controls, use ofthe terms conform, equivalent of, and in-house method and arrangements regardingthe scope of accredited methods. The paper will present the practical use of qualityassurance aspects in the application of routine method using the BactoScan 8000Presented at the ICS-UNIDO-Workshop on Contamination of Food and Agroproducts, 28 September – 1 October 2000, Vara`din, Croatia62 Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67instrument for quantitative determination of the bacteriological quality of milk. Specialemphasis will be placed on the importance of interlaboratory proficiency testing.VALIDATION OF MICROBIOLOGICAL METHODSThe term validation is used for a process demonstrating that a particular method issuitable for the intended purpose. There are few sources in the field of microbiologythat cover method evaluation. Nevertheless, methods submitted for accreditation mustbe validated. The validation of microbiological methods should not rely on the sameprinciple as of chemical methods. This is why the Dutch Accreditation Council orga-nises the preparation of the international Explanatory Document on Microbiology (RvA-T2) that would include a selection of performance characteristics dependent on thetesting method.In reality, only in a very limited number of cases will a laboratory fully develop andvalidate a new microbiological method. Often the laboratory itself applies limited valida-tion. When a laboratory adopts a reference method or a method that has been developedand validated elsewhere, it has to demonstrate that it can apply the method properly in itsown environment and obtain correct results. The verification of limited validation must beaccomplished by the use of proper internal and external quality controls.Microbiological methods are divided into either qualitative methods that demon-strate the presence or absence of the target microorganism, either directly or indirect-ly, in a defined quantity of test material or quantitative methods that determine thenumber of microorganisms present by direct enumeration (colony forming units) orindirectly (most probable number counts, colour absorbance, impedance) in a definedquantity of material. When a reference method is used, a laboratory should demon-strate its competence to meet the performance characteristics described in the na-tional or international standard. For qualitative microbiological standard methods theperformance characteristic is the limit of detection, while for quantitative microbiolog-ical standard methods these are trueness, repeatability, and reproducibility.Microbiological investigations may be undertaken using alternative (rapid) meth-ods such as immunological, molecular biological, or instrumental. The validation ofthese methods includes the assessment of their equivalence to the correspondingreference method (1).Equivalence can be applied to the use of alternative (rapid) confirmation tech-niques; use of a different range of colony count than stated in the relevant standard;use of a different method for counting colonies or for calculation and expression ofresults than those stated in the relevant standard; and use of alternative (rapid) meth-ods validated according to the relevant standard.The in-house method is applicable to the use of a non-standard methods or theuse of a standard method on a matrix that differs from the one specified by thestandard method. When an in-house method is adopted it is recommended to selecta non-standard method that has already been validated by a national or internationalorganisation, or an accepted method that has already been in use in particular pro-fessional branches (e.g. the International Dairy Federation, IDF).63Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67APLICATION OF QUALITY CONTROLSOne requirement for competence of laboratories in the International standard (2) isthat a laboratory should ensure the quality of its results by using and assessing differ-ent quality controls. In the field of microbiology the use of correct quality controls isof particular importance, because the translation of the performance characteristic tomicrobiological examination is not always possible and it depends on the test matrix.The terms »trueness« and »precision«, for example, are more difficult to definefor microbiological examination than for analytical chemistry investigations. However,it is possible to demonstrate the technical control of the microbiological methodsthrough control samples. Good microbiological reference samples are not yet avail-able for all types of microorganisms.Quality control can be implemented in a number of ways: internal serial (alsocalled first line control) i.e. internal control of serial analyses assessed by technicians(using blanks, positive controls, negative controls, multiple observations); internal pro-cess (also called second line control) i.e. internal process control conducted by tech-nicians, but assessed by laboratory management staff, e.g. the quality officer (usingreference materials and spiked samples); and external (also called third line control)i.e. participation in an externally organised proficiency testing scheme. The qualitycontrols will be described in the international Explanatory Document on Microbiology(RvA-T2 organised by Dutch Accreditation Council).ROUTINE METHODS FOR QUANTITATIVE DETERMINATION OF THEBACTERIOLOGICAL QUALITY OF MILKParticular routine methods are used for quantitative determination of the bacteriolog-ical quality of milk for a number different reasons such as: speed of analysis and/orresponse, ease of execution and/or automation, analytical attributes (specificity, limitof detection, limit of determination, repeatability, reproducibility, accuracy), extent towhich the parameter of interest is expressed, and reduction of costs. Adequate oper-ation of a routine method involves the application of quality assurance principles.The below example of the use of a rapid microbiological method for routine work(3) that has been in use in Slovenia since 1995 (4) illustrates the evaluation of aroutine method with a reference method for the determination of the bacteriologicalquality of milk based on automatic epifluorescent microscopy using the BactoScan8000 instrument.When introducing a routine method in order to comply with national and inter-national regulations or standards, a number of issues is to be addressed: which criteria are essential for a routine method; what influencing factors should be considered when evaluating a routine method; how should a routine method replace a reference method; which constructive measures should be taken to improve the relationship be-tween a reference and a routine method.64 Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67In most regulations for determination of the bacteriological quality of milk thereference method is the colony count at 30 C (5). However, many routine methodsare applied, often with different methodological principles. The difference between thereference and routine values is expected to be less pronounced when the two meth-ods are based on the same test principle.The routine method must be evaluated as an estimate of the reference methodaccording to general principles (6), either in-house or elsewhere.In case of an expected deviation from the method of evaluation (e.g. milk com-position, milk of other species, sample preservation, sample pre-treatment), the rou-tine method must be validated additionally. The results of additional validation mustbe archived.The laboratory must follow the following sets of control procedures: daily operatingprotocols, middle term controls, and long term controls. The daily protocol includes: start-up procedure for the instrument/equipment; start-up controls including limits; sample identification; sample storage and pre-treatment; sample checks (temperature, visible condition); sample analysis; routine checks during measurement (instrument settings, visual checks, temper-ature control, blanks, pilot samples); cleaning in between samples; shut-down procedure; truncation of results at the lower and upper limit of detection; evaluation and authorisation of resultsMiddle-term controls include: instrument checks; volume controls; carry-over controls; repeatability checks; within-lab reproducibility.Long-term controls include: reproducibility; interlaboratory comparisons.The purpose of interlaboratory comparisons is to establish that the precisioncharacteristics of the laboratory’s procedure are still in line with those recognised asfeasible and achieved elsewhere and that the measured level is in agreement with thatof the other participants. Conversion characteristics must express results in units ofanother method.The results of interlaboratory comparisons are statistically evaluated. Graphicalinterpretations are frequently used. Figure 1 shows an evaluation of performance oflaboratories that participated in international proficiency testing using the BactoScaninstruments. It is presented as the Euclidian distance between accuracy and repeat-ability, which gives information about the performance characteristics of the instru-ments (7). Figure 2 illustrates a deviation of laboratory results from the total average.The figure has been taken from the report on cell count collaborative study organisedby Milk Standard Service, Wangen, Germany.65Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67Figure 2 Cell count proficiency testing – difference (%) from the mean of control milk samplesFigure 1 Evaluation of performance of individual laboratories using the BactoScan instruments403020100-40 -20 0 20 40AccurancyRepeatabilityIn order to make comparable the use of routine methods the International Stan-dards Organization and AWI are preparing a new standard proposal »Milk-Quantitativedetermination of bacteriological quality – Protocol for establishing a conversion rela-tionship between routine method result and anchor method results and its verifica-tion« (8).66 Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67CONCLUSIONSIn the assessment of microbiological examination the choice and the evaluation of amethod, and the use of a quality assurance system are very important. This is whyevery laboratory should rely on good laboratory practice, protocols, and standards.Our experiences suggests that it is equally important to perform quality control of thereference plate count method as a prerequisite for a reliable evaluation of an alterna-tive microbiological method. The performance of test methods is evaluated throughparticipation in interlaboratory proficiency studies.REFERENCES1. Microbiology of food and animal feeding stuffs – Protocol for the validation of alternativemethods. European/International Standard ISOprEN 16140.2. General requirements for the competence of testing and calibration laboratories. European/International Standard EN-ISO/IEC 17025; 2000.3. International Dairy Federation (IDF). Milk – quantitative determination of bacteriological quality– guidance on evaluation of routine methods. IDF-Standard 161 A; 1995.4. Golc Teger S. Primjernost automatske epifluorescentne mikroskopije za odre|ivanje bakteriolo{kekakvo}e sirovog mlijeka [Study of automated epifluorescent microscopy for evaluation of bac-teriological quality of raw milk, in Croatian]. Mljekarstvo 1995;45:3–9.5. International Dairy Federation (IDF). Milk and milk products – enumeration of microorganisms– Colony count technique at 30 °C. IDF-Standard 100B; 1991.6. Milk – Definition and evaluation of the overall accuracy of indirect methods of milk analysis –Part 1: Analytical attributes of indirect methods. International Standard ISO/FDIS 8196-1;2000.7. Bühlmann G, Sinani-Gashi E. Proficiency-Test »Hüfner-Kontrolmilch« Januar bis Mai 2000.FAM-Information 2000; 403W: 2-27.8. Milk – Quantitative determination of bacteriological quality – Protocol for establishing a con-version relationship between routine method results and anchor method results and its verifi-cation. International Standard ISO/AWI 21187; 2000.67Golc Teger S. MICROBIOLOGY AND PROFICIENCY TESTING IN DAIRY LABORATORIESArh Hig Rada Toksikol 2001:52:61–67Sa`etakMIKROBIOLO[KO ISPITIVANJE I PROSUDBA VRSNOSTILABORATORIJA ZA MLIJE^NE PROIZVODEU ovome su ~lanku prikazani glavni ~imbenici u procjeni mikrobiolo{kog ispitivanja hrane. Prikazano je nekolikoto~aka validacije kvantitativnih i kvalitativnih mikrobiolo{kih metoda. Osim toga, definiraju se izrazi »sukladnost« i»istovjetnost« referentne metode s »internom« metodom u smislu akreditacija metoda. ^lanak opisuje kakoevaluirati rutinsku metodu u odnosu na referentne/slu`bene metode s obzirom na rezultate dobivene brojenjemautomatskim epifluorescentnim mikroskopom s pomo}u ure|aja BactoScan 8000 namijenjenog za utvr|ivanjebakteriološke kakvo}e mlijeka te daje op}e upute za usporedbu navedenih metoda. Tu je i pregled aspekataosiguranja kakvo}e koji se uzimaju u obzir u primjeni rutinske metode te primjer me|ulaboratorijske prosudbevrsnosti uz pomo} epifluorescentne mikroskopske metode koja je uobi~ajena u laboratorijima za mlije~neproizvode.Klju~ne rije~i:BactoScan 8000, kontrola mlijeka, osiguranje kakvo}eRequests for reprints:Slavica Golc Teger, Ph. D.University of Ljubljana, Biotechnical FacultyZootechnical Department, Institute of DairyingGroblje 3, SI-1230 Dom`ale, SloveniaE-mail: slavica.golc@bfro.uni-lj.si

Mikrobiološko ispitivanje i prosudba vrsnosti laboratorija za mliječne proizvode

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