Quality Assurance - mikrobiyoloji.org

1 CONFIDENTIALQ uality Assurance and Performance Testing of Culture Media Implementation of ISO Standard 11133 Part 1 and Part 2 CONFIDENTIALC ontents of Quality Control Lecture1. Introduction of new ISO Standard 11133-12. Practices for Quality control of culture media3. Introduction of new ISO Standard 11133-24. SummaryCONFIDENTIALWhy was ISO 11133 developed ? International Standard exist in which the Quality Control Specifications ofcommercial Culture Media are Culture Media Manufacturer has establishedit s own QC of Culture Media have no information which Growth limits are used by the new ISO 11133 describes now for the first timewhich limits for Productivity and Selectivity eachCulture Medium MUST fulfil,CONFIDENTIALISO 11133-1 (2000-06-01)Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture media ISO 11133-2 (2003-12-15)Microbiology of food and animal feeding stuffs Practical guidelines on performance testing of culture media This new standard describe for the first time in history ofMicrobiology the definition of Quality control for commercialculture media.

2 Quantitative testing and reporting of results ( CoA )with recovery rates is the new preferred testing methodISO 11133 part 1 and 2 CONFIDENTIALM icrobiology of food and animal feeding stuffs Guidelines on preparation and production of culture mediaPart 1:General guidelines on Quality Assurance for the preparation of culture media in the laboratory Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture mediaPart 1:General guidelines on Quality Assurance for the preparation of culture media in the laboratory ISO 11133 Part 1 CONFIDENTIALP ractices for Quality control of culture mediaCONFIDENTIAL Any performance evaluation and test organism used Technical data sheet Quality -control certificate Safety and/or hazard data where neededDocumentation CONFIDENTIALD ocumentation - ISO 11133 part 1 Check list by the laboratory Laboratory checks upon receipt of the medium name of the medium and batch code date of receipt expiry date condition and integrity of packagingCONFIDENTIAL SterilisationSterilisation Dehydrated media are not free of contaminants besides heat-resistant microoganisms Be sure that no residual media covers the wall of container Autoclave only 15 min.

3 At 121 C Sterilisation duration depends on the size of load and containers Avoid excessive autoclaving to prevent Maillard reactions degradation and breakdown of nutrient constituents Sterilise heat labile substrates by filtration Add heat labile supplements with aseptic precautions to the cooled medium (45 C)Practices for Quality Control CONFIDENTIALS terikon plusBioindicator for checking steamsterilization efficiencyacc. to USP and Ph. EURThe performance of the autoclave should be monitoredPractices for Quality Control CONFIDENTIALT hese media have the highest risk of overheating-Brilliant green-Bile salts-Iron saltsPreparation of heat sensitive mediaXLD Agar is most criticalPractices for Quality Control should not be autoclavedMedia for Enterobacteriaceaecontaining:CONFIDENTIA LHot plate has the high risk that the Culture Medium can be overheatedWather bath versus Hot plateMelting of agar culture mediaCONFIDENTIALP reparation of media in Petri dishes Pour the molten agar culture medium into Petri dishes so as to obtain a thickness of at least 2 mm ( for 90 mm diameter dishes, 15 ml of agar are normally required) or as specified in the International Standard.

4 Allow the agar to cool and solidify by placing the plates with lids in place on a cool, horizontal surface. During incubation, a loss of moisture of the agar media will occur. A loss of water content can affect the growth of microorganisms in some circumstances. CONFIDENTIALS torage of media in Petri dishes Use the solidified medium immediately or store under conditions which prevent its composition from being modified, in the dark and / or in the refrigerator at 5 C 3 C in sealed bags Label the plates on the base with date of preparation and/or expiry date and identity In general, for the surface inoculation of a solid culture medium, dry the plates, preferably with the lids removed and with the agar surface facing downwardsCONFIDENTIALS helf life of prepared media The shelf-life of media varies.

5 Specific International or National Standards may stipulate specific conditions and shelf life. It is generally recommended not to exceed 2 to 4 weeks of storage for plates and 3 to 6 months for bottles and tubes, unless otherwise specified in specific standards or results of the laboratory shelf life validation indicate a longer shelf life. A validated expiry date for stored media should be established. Observe any colour change, sign of evaporation/dehydration or microbial growth. Batches of media showing such changes should not be life of prepared VRB Agar (200 ml in a bottle)100 %100 %100 %100 %100 %100 %100 %96 %97 %83 %100 %0 %0 %0 %100 %97 %99 %100 %0 %0 %0 %TSAVRB AgarTest strainsEscherichia coliEscherichia coliEnterobacter cloacaeSalmonella typhimuriumStaphylococcus aureusEnterococcus faecalisBacillus cereusATCCATCCATCCATCCATCCATCCATCC873925 922130471402865381943311778 Internal validation study (Recovery %)8 months at RT+ 2 weeks at 35 CCONFIDENTIALM icrobiology of food and animal feeding stuffs Guidelines on preparation and production of culture mediaPart 2:Practical guidelines on performance testing of culture media Microbiology of food and animal feeding stuffs Guidelines on preparation and production of culture mediaPart 2.

6 Practical guidelines on performance testing of culture media ISO 11133 Part 2 CONFIDENTIAL The Quality of culture media depends on the Quality of the basicingredients, correct formulation, Quality of preparation procedures, elimination of contaminant microbial agents and appropriate packaging and storage conditions. Quantitative, semi-quantitative or qualitative evaluations shall be performed by the methods described in this Standard or by another generally accepted technique. For interpretation of the results of testing, it is necessary tocompare the amount of growth on the test medium with that on a reference medium. The use of a specific reference medium is therefore mandatory for quantitative methods General Quality Criteria CONFIDENTIALQ uality of Basic Media Components peptones and meat or yeast extracts variable in their nutritive properties agar variable in its gelling properties buffering substances bile salts, bile extract and deoxycholate, antibacterial dyes, depending on their selective properties antibiotics depending on their activityGeneral Quality Criteria Culture media described in the International Standards were judged satisfactory.

7 However, due to the variability of their Quality , it may be acceptable for media manufacturers to modify the concentration of some basic biological ingredients, as listed below:CONFIDENTIAL Growth-quantitative-semi quantitative-qualitative method Productivity Selectivity Biochemical Characteristics Performance Evaluation and Interpretation of ResultGeneral Quality Criteria BAIRD-PARKER AGAR S. aureusATCC 6538 CONFIDENTIAL Solid, semi solid or liquid culture medias shall be inoculated with an appropriate inoculum of the working culture of each of the defined test microoganisms using an appropriate device spiral plater Productivity has to reach a fixed limitProductivityCONFIDENTIAL To asses selectivity quantitatively, selective culture media and a reference medium are inoculated with an appropriate inoculum of test trains.

8 Selectivity has to reach defined For isolation and enumeration media, at least semi-quantitative testing should be performed. For characterization test media, qualitative testing may be sufficient. Quantitative tests will give greater Assurance of media Quality . For those media which contain no indicators or selective agents the use of a single positive test strain is appropriate. For those media which do contain indicators or selective agents, strains which demonstrate the function of the indicator(s) and selectivity must be utilized. For complex media, with added supplements, each batch should be verified with strains with It is a general method suitable for most solid culture media- A spiral plater can be used- The pour plate method for enumeration can also be used in this way It may not suitable for testing types of mouldsMethods for Solid Culture Media Quantitative methodCONFIDENTIAL Pre-culture the test strain in CASO Broth or (Candida) at 35 C for 24 h Dilute the pre - culture with 1/4 Ringer - solution Inoculate the test medium, an older reference lot and a reference medium with the Spiral Plater Count the colonies in comparison to an older reference batch and to a reference mediumQuantitative method (TSA)GOLD StandardUsed only for internal controlNEW batchBROLACIN (CLED) AgarCalculation.

9 85 / 100 = 85 % recoveryQuantitative method - (TSA)NEW batchCEREUS Selective Agar (MYP)Calculation : 84 / 123 = 68 % recovery rateQuantitative method InterpretationGOLD StandardUsed only for internal controlCONFIDENTIAL> 70% for non selective media > 50% for selective mediaMost important change !ProductivityThese values are generally achievable, however less rigorous criteria can be accepted for certain combinations of media and test media which are not specified in Annex B, those described in local standards, these criteria may not be for Solid Culture Media CONFIDENTIALE numeration Media Selective MediaEnumeration Media Non Selective MediaEnrichment Media Non Selective MediaPerformance criteria (Annex B) CONFIDENTIALC ulture Media in ISO Food Norms with a recovery rate > 50% ISO 6888-1 Baird Parker AgarStaphylococcus aureus ISO 7954 OGY AgarYeast & Moulds ISO 15214 MRS AgarLactic acidbacteria ISO 7932 MYP AgarBacillus cereus ISO 11290 Oxford AgarL.

10 Monocytogenes ISO 11290 PALCAM AgarL. monocytogenes ISO 7938 TSC AgarClostridium perfringens ISO 7402 ISO 8523 ISO 4832 VRB AgarColiforms ISO 16654CT SMAC AgarEnterobacteriaceaeCONFIDENTIALC ertificate of Analysis Tryptic Soy Agarall test strains must have > 70 %otherwise batchis rejectedCONFIDENTIALC ertificate of Analysis VRB AgarCONFIDENTIALC ertificate of Analysis Baird Parker AgarCONFIDENTIAL304050607080901001101206 43 806541 906476 306370 506291 Rates VRB Agar%Batch NoCONFIDENTIALQ ualitative streaking method The method is suitable for supplementary performance testing of solid culture media. The method is only qualitative and scores are therefore only growth on the plates after incubation is assessed as: 0 corresponds to zero growth 1 corresponds to weak growth 2 corresponds to good growthQualitative method InterpretationTarget microorganisms shall score 2 and have typical appearance,size and colony morphology.