iso 11133:2014

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(1)Quality Assurance and Performance Testing of Culture Media Implementation of ISO Standard 11133 Part 1 and Part 2. CONFIDENTIAL.

(2) Contents of Quality Control Lecture 1. Introduction of new ISO Standard 11133-1 2. Practices for quality control of culture media 3. Introduction of new ISO Standard 11133-2 4. Summary. CONFIDENTIAL.

(3) Why was ISO 11133 developed ? 1. No International Standard exist in which the Quality Control Specifications of commercial Culture Media are described 2. Each Culture Media Manufacturer has established it´s own QC Specification 3. User of Culture Media have no information which Growth limits are used by the Manufacturer 4. The new ISO 11133 describes now for the first time which limits for Productivity and Selectivity each Culture Medium MUST fulfil, CONFIDENTIAL.

(4) ISO 11133 part 1 and 2 ISO 11133-1 (2000-06-01). ISO 11133-2 (2003-12-15). Microbiology of food and animal feeding stuffs —. Microbiology of food and animal feeding stuffs –. Guidelines on preparation and production of culture media. Practical guidelines on performance testing of culture media. This new standard describe for the first time in history of Microbiology the definition of quality control for commercial culture media. Quantitative testing and reporting of results ( CoA ) with recovery rates is the new preferred testing method CONFIDENTIAL.

(5) ISO 11133 – Part 1 Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media Part 1: General guidelines on quality assurance for the preparation of culture media in the laboratory CONFIDENTIAL.

(6) Practices for quality control of culture media. CONFIDENTIAL.

(7) Documentation Any performance evaluation and test organism used Technical data sheet Quality-control certificate Safety and/or hazard data where needed. CONFIDENTIAL.

(8) Documentation - 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 packaging. CONFIDENTIAL.

(9) Practices for Quality Control Sterilisation 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. 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) CONFIDENTIAL.

(10) Practices for Quality Control The performance of the autoclave should be monitored. Sterikon® plus Bioindicator for checking steam sterilization efficiency acc. to USP and Ph. EUR. CONFIDENTIAL.

(11) Practices for Quality Control Preparation of heat sensitive media Media for e.g. Enterobacteriaceae containing: - Brilliant green - Bile salts - Iron salts should not be autoclaved. XLD Agar is most critical. These media have the highest risk of overheating CONFIDENTIAL.

(12) Melting of agar culture media Wather bath versus Hot plate. Hot plate has the high risk that the Culture Medium can be overheated CONFIDENTIAL.

(13) Preparation 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 (e.g. for 90 mm diameter dishes, 15 ml of agar are normally required) or as specified in the International Standard. 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. CONFIDENTIAL.

(14) Storage of media in Petri dishes. Use the solidified medium immediately or store under conditions which prevent its composition from being modified, i.e. 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 downwards. CONFIDENTIAL.

(15) Shelf life of prepared media The shelf-life of media varies. 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 used. CONFIDENTIAL.

(16) Shelf life of prepared VRB – Agar (200 ml in a bottle). Internal validation study (Recovery %). VRB – Agar Test strains. Escherichia coli Escherichia coli Enterobacter cloacae Salmonella typhimurium Staphylococcus aureus Enterococcus faecalis Bacillus cereus. ATCC 8739 ATCC 25922 ATCC 13047 ATCC 14028 ATCC 6538 ATCC 19433 ATCC 11778. TSA. 8 months at RT. + 2 weeks at 35°C. 100 % 100 % 100 % 100 % 100 % 100 % 100 %. 100 % 97 % 99 % 100 % 0% 0% 0%. 96 % 97 % 83 % 100 % 0% 0% 0% CONFIDENTIAL.

(17) ISO 11133 – Part 2 Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media Part 2: Practical guidelines on performance testing of culture media. CONFIDENTIAL.

(18) General Quality Criteria . The quality of culture media depends on the quality of the basic ingredients, 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 to compare 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 CONFIDENTIAL.

(19) General Quality Criteria Quality of Basic Media Components Culture media described in the International Standards were judged satisfactory; 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: 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 activity. CONFIDENTIAL.

(20) General Quality Criteria Growth - quantitative - semi quantitative - qualitative method. Productivity. BAIRD-PARKER AGAR S. aureus ATCC 6538. Selectivity Biochemical Characteristics Performance Evaluation and Interpretation of Result. CONFIDENTIAL.

(21) Productivity 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 e.g. spiral plater Productivity has to reach a fixed limit. CONFIDENTIAL.

(22) Selectivity. To asses selectivity quantitatively, selective culture media and a reference medium are inoculated with an appropriate inoculum of test trains. Selectivity has to reach defined values.. CONFIDENTIAL.

(23) General For isolation and enumeration media, at least semiquantitative 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, i.e. with added supplements, each batch should be verified with strains with characteristics.. CONFIDENTIAL.

(24) Methods for Solid Culture Media Quantitative method 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 moulds CONFIDENTIAL.

(25) Quantitative method – Procedure Pre-culture the test strain in CASO Broth or Sab.-2%-Broth (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 medium. CONFIDENTIAL.

(26) Quantitative method - Interpretation BROLACIN (CLED) Agar GOLD Standard Used only for internal control. Ref.Medium (TSA). NEW batch. Calculation : 85 / 100 = 85 % recovery CONFIDENTIAL.

(27) Quantitative method – Interpretation CEREUS Selective Agar (MYP) GOLD Standard Used only for internal control. Ref.Medium (TSA). NEW batch. Calculation : 84 / 123 = 68 % recovery rate CONFIDENTIAL.

(28) Methods for Solid Culture Media Productivity. > 70% for non selective media > 50% for selective media. Most important change ! These values are generally achievable, however less rigorous criteria can be accepted for certain combinations of media and test microorganisms. For media which are not specified in Annex B, e.g. those described in local standards, these criteria may not be applicable. CONFIDENTIAL.

(29) Performance criteria (Annex B) Enumeration Media – Selective Media. Enumeration Media – Non Selective Media. Enrichment Media – Non Selective Media. CONFIDENTIAL.

(30) Culture Media in ISO Food Norms with a recovery rate > 50% . ISO 6888-1. Baird Parker Agar. Staphylococcus aureus. . ISO 7954. OGY Agar. Yeast & Moulds. . ISO 15214. MRS Agar. Lactic acid bacteria. . ISO 7932. MYP Agar. Bacillus cereus. . ISO 11290. Oxford Agar. L. monocytogenes. . ISO 11290. PALCAM Agar. L. monocytogenes. . ISO 7938. TSC Agar. Clostridium perfringens. . ISO 7402 ISO 8523. VRBD Agar. Enterobacteriaceae. . ISO 4832. VRB Agar. Coliforms. . ISO 16654. CT SMAC Agar. E.coli 0157. CONFIDENTIAL.

(31) Certificate of Analysis Tryptic Soy – Agar. all test strains must have > 70 % otherwise batch is rejected. CONFIDENTIAL.

(32) Certificate of Analysis VRB – Agar. CONFIDENTIAL.

(33) Certificate of Analysis Baird Parker – Agar. CONFIDENTIAL.

(34) Recovery Rates – VRB Agar 120 110 100 90. %. 80 70 60 50 40 30 643 806. E.coli. 541 906. 476 306. S.typhimurium. 370 506. 291 306. Batch No. Enterob.cloaceae CONFIDENTIAL.

(35) Qualitative streaking method. General The method is suitable for supplementary performance testing of solid culture media. The method is only qualitative and scores are therefore only indicative. CONFIDENTIAL.

(36) Qualitative method – Interpretation The growth on the plates after incubation is assessed as: 0 corresponds to zero growth 1 corresponds to weak growth 2 corresponds to good growth Target microorganisms shall score 2 and have typical appearance, size and colony morphology. The growth of non-target microorganisms shall be partly or completely inhibited (0 or 1). CONFIDENTIAL.

(37) Methods for Liquid Media Quantitative dilution method for target and non – target microorganisms To determine the productivity of a liquid medium an appropriate inoculum shall be used. The method is also appropriate for evaluation of new culture media, broths or diluents.. CONFIDENTIAL.

(38) Methods for Liquid Media Quantitative method – Procedure . Select an appropriate number of tubes or 10 ml portions of each batch of liquid medium to be tested.. . Inoculate test broth and reference broth for each test organism with a small number (e.g. 10 cfu to 100 cfu).. . Inoculate test broth and reference broth for each test organism with a higher number (>1000 cfu).. . For testing mixed cultures in selective media inoculate test broth and reference broth with a small number of target microorganisms and in the same tube with a higher number of non-target microorganisms. CONFIDENTIAL.

(39) Methods for Liquid Media Quantitative method – Reading. Remove an aliquot volume or if necessary a dilution from each broth after the incubation step and spread to a non-inhibitory agar plate. After incubation colonies of target and non-target microorganisms are counted, in the case of mixed cultures distinguishing the different types. Calculation and interpretation shall be done with respect to the aim of examination.. CONFIDENTIAL.

(40) Methods for Liquid Media Quantitative method – Calculation / Interpretation Target bacteria. 106 to 108 cfu per ml. Unwanted. below 104 cfu per ml. Extremely important to avoid false-negative results. CONFIDENTIAL.

(41) Certificate of Analysis FRASER - Broth. CONFIDENTIAL.

(42) Methods for Liquid Media Quantitative method – Mixed Culture RVS / TTn / Selenite Cystine broth Quality control. Test strains. Minimum level must be 105 cfu Inoculum. Growth after 24 hours. Escherichia coli. Approx. 99. %. < 10 %. ATCC 25922 Salmonella typhimurium ATCC 14028. Approx.. 1%. > 90 % CONFIDENTIAL.

(43) Methods for Liquid Media Quantitative method – Mixed Culture Results on CLED agar after selective enrichment in RVS broth. 0 hr. 24 hr. E. coli: Lactose (+) = yellow Salmonella typhimurium: Lactose (-) = blue CONFIDENTIAL.

(44) Methods for Liquid Media Qualitative method - Interpretation Qualitative evaluation shall be carried out visually by allocating growth scores, e.g. from 0 to 2. - 0 corresponds to zero turbidity - 1 corresponds to very light turbidity - 2 corresponds to good turbidity. The score of a target microorganism shall be 2. Other characteristics such as gas formation, colour change, etc. can also be assessed by this method. CONFIDENTIAL.

(45) Documentation of Test Results The manufacturer or supplier shall provide, on request, the specific microbiological characteristics and general information relating to the specific batch of culture medium. The use of control sheets for documenting and evaluating the results of the routine performance testing is recommended (see Annex A).. CONFIDENTIAL.

(46) Control Card (Annex A). CONFIDENTIAL.

(47) ISO 11133 – overview to actual parts ISO TS 11133 Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media Part 2 Amd 1: 2011 Test microorganisms for commonly used culture media contains tables with the media used for food microbiology contains WDCM numbers instead of ATCC numbers for all strains of microorganisms used for quality control Reason: ATCC did not allowed for future publications of ISO any impression of equivalence of strains from different culture collections compared to ATCC (planned as a table for revised ISO 11133); also only allowed to use ATCC numbers cited as ATCC™ using of ™ is only possible in ISO standards, if equivalent substance / material can also be used – but how can we show equivalence of the strains from different culture collections ?. ISO 11133 for quality control of culture media B. Gerten Merck KGaA. 09-2013. 47.

(48) ISO 11133 – overview to actual parts ISO TS 11133 Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media WDCM numbers are given as universal strain identifiers by the World Data Centre for Microorganisms (WDCM) WDCM is part of WFCC (World Federation of Culture Collections) they have initiated a system that will help users find local sources of the reference strains by citing all collections and providing contact details and the collection’s unique reference. ISO 11133 for quality control of culture media B. Gerten Merck KGaA. 09-2013. 48.

(49) ISO 11133 for quality control of culture media B. Gerten Merck KGaA. 09-2013. 49.

(50) ISO 11133 – overview to actual parts ISO TS 11133 Microbiology of food and animal feeding stuffs – Guidelines on preparation and production of culture media WDCM numbers on the new website of WDCM (www.wfcc.info) it is also possible to search by -. species name. -. WDCM strain number. -. strain accession number. -. number of standard (ISO and other standards). ISO 11133 for quality control of culture media B. Gerten Merck KGaA. 09-2013. 50.

(51) Summary Part 1 and Part 2 Demands to a reliable manufacturer of culture media: Media manufacturer must be certified according to at least ISO 9001 standards Media manufacturer should produce culture media in full compliance with ISO 11133 Quality control methods. Merck as manufacturer of culture media: Merck is certified as culture media manufacturer acc. various ISO norms. Merck has available a clear documentation about the performance of their culture media with values that exceeds ISO 11133. Customers: Customers labs which are certified acc. ISO standards need: - To use validated culture media - To follow validated enrichment procedures CONFIDENTIAL.

(52) ISO 11133 – overview to actual parts Availability of ISO standards: ANSI web store. ISO 11133 for quality control of culture media B. Gerten Merck KGaA. 09-2013. (http://webstore.ansi.org/). 52.

(53) CONFIDENTIAL.

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