11133 - 3.pptx
- 1. According to ISO 11133 Preparation, production, storage and performance testing of culture media
- 2. Selection of test organisms Preservation and maintenance of test organisms Microorganisms for performance testing Microbiological quality control General requirements microbiological performance testing Performance evaluation and interpretation of results Confirmation media and reagents
- 3. Methods for quantitative tests Quantitative method for solid culture media Testing of culture media used for membrane filtration Methods for qualitative tests Quantitative tube method for performance testing of liquid enrichment media Qualitative tube method for performance testing of selective liquid media Qualitative single tube method (turbidity) for performance testing of liquid media
- 4. Method for quantitative testing of diluents Method for testing transport medium
- 5. • Selection of test organisms A set of test organisms should contain a stable characteristics representative of their performance on the prepared medium. The test organisms of each media may contain a variety of strains. It is preferable to use strains which have originated from food and water.
- 6. • Preservation and maintenance of test organisms There are a several method for the successful maintenance of all microorganisms. The performance testing shall be handled in a way that minimize the opportunity of cross- contamination. The number of transfers of test organisms should be documented to prevent excessive subculturing.
- 7. Suspension in nutrient broth Isolate on solid suitable medium, on sheep blood 5% or TSA (Streak method) Verify purity and characteristics properties of strain Incubate at 37°C for 24h. Resuspension in TSB supplemented with 10:15% glycerol using a loop Using a reference strain Freeze at a temp. below -70°C to extend storage period. Reference stock
- 8. • Microorganisms for performance testing Incubate Preparation of stock culture Using a reference solid medium to achieve single colonies. It can be used for 14days at an appropriate conditions. Incubate most aerobic bacteria at 37°C for 18h to 24h.
- 9. Preparation of working culture • Microorganisms for performance testing Transfer to a non-selective broth (TSB) Mix using vortex Take 1 or 2 colonies with a loop Using a reference stock Storage and use for maximum 3days at appropriate Working culture Incubate at 37°C for 18h to 24h.
- 10. Preparation of inocula (suspensions) Prepare serial dilutions in a suitable diluent. Use the inocula up to 2h at room temp. or within 24h if stored at 5 °C Determine the most suitable dilution. The volume of the inoculum shall be usually 10% of the medium. • Microorganisms for performance testing
- 11. • Microorganisms for performance testing Inoculum level Inoculum level for productivity testing Quantitative testing A level around 10^2 cfu (80: 120 cfu) is necessary to achieve precision. (with a minimum 50 cfu) Qualitative testing
- 12. Inoculum level for selectivity testing • Microorganisms for performance testing Inoculum level Use a suspension of the non-target microorganism containing 10^4 to 10^6 cfu Inoculum level for specificity testing Use an inoculum level of 10^3 to 10^4 cfu
- 13. The quality of culture media Quality assessment by visual inspection shall ensure that each culture medium conforms to stated recommendations. The quality of the ingredients Quality of preparation procedures Appropriate packaging and storage conditions
- 14. Microbiological performance test shall be carried out on a representative sample of a batch of end product. Shall be consistent of high quality Use quantitative RM containing well-defined number of organisms Use a defined production process Follow the appropriate procedures for assuring the quality Use a large rang of test organisms Use the same manufacturer • Microbiological quality control Reference medium Shall be consistent of high quality Use quantitative RM containing well-defined number of organisms
- 15. Evaluate a batch of culture medium appropriately assessed by either qualitative or quantitative methods. Inoculate the medium with an appropriate volume. Quantitative method shall be performed for enumeration purposes and when a new medium or a new manufacturer is being evaluated. Using solid medium is preferable to evaluate the successful growth. • General requirements microbiological performance testing
- 16. Media prepared form basic individual components • General requirements microbiological performance testing Periodic checks shall be carried out to ensure the quality of media has been maintained during transport. For read-to-use finished medium which supplemented have been added at least qualitative test is recommended. For incomplete media which supplements are added by the user, checking the production records or performing a qualitative test. Ready-to-use media Media prepared form commercially available dehydrated formulations Media which contain indicators or selective agents, strains which demonstrate the function of the indicator should be utilized. Quantitative testing shall be performed to evaluate the quality of basic material and productivity.
- 17. • Performance evaluation and interpretation of results Agar medium fails to solidify Incorrect pH Abnormal color Formation of precipitates Medium inhibitory/Low productivity Poor selectivity/ Specificity Contamination
- 18. • Confirmation media and reagents The confirmation tests shall be performed on culture media before use, using appropriate positive or negative test organisms. The biochemical characteristic shall be performed on reagents and gram stain solutions before use, using appropriate positive or negative test organisms.
- 19. • Methods for quantitative tests Productivity Determine the productivity ratio, 𝑃𝑅 𝑃𝑅 = 𝑁𝑆 𝑁𝑂 𝑁𝑆 is total count of colonies obtained in the culture medium under test. 𝑁𝑂 is total count of colonies obtained in the reference culture medium. 𝑃𝑅 shall be ≥ 0.5 for comparison of a selective medium with non-selective reference medium 𝑃𝑅 shall be ≥ 0.7 for comparison of a non-selective medium with non- selective reference medium
- 20. • Methods for quantitative tests Selectivity Inoculate selective culture media and non-selective reference media with different dilutions of non-target organism. Determine the selectivity factor, 𝑆𝐹 𝑆𝐹 = 𝐷0 - 𝐷𝑆 𝐷0 is the highest dilution showing growth on the non-selective reference medium. 𝐷𝑆 is the highest dilution showing growth on the selective culture test medium. The 𝑆𝐹 of non-target microorganisms is at least 2
- 21. Quantitative method for solid culture media Use an appropriate concentration of a target strain, the recovery of the new batch of culture medium will be compared of non-selective culture medium (reference medium) Inoculate test medium and reference medium by spreading or using membrane filter Incubate An appropriate concentration of a target strain Count the colonies present on the plates, assess the size and appearance of the colonies on the test medium and compare with the recovery on non-selective culture medium
- 22. • Testing of culture media used for membrane filtration Evaluate the quality of membrane filter to demonstrate their stability. Filter the liquid and place the membrane on the surface of the test medium. Repeat with a new membrane and place the second membrane on the surface of the reference medium. Spread the inoculum on the test medium and reference medium when needing to evaluate the influence of the membrane.
- 23. • Methods for qualitative tests Qualitative streaking method For productivity and specificity, use a plate of test medium and streak each test microorganism in a way to obtain separate colonies. For selectivity, Streak microorganism in parallel straight lines using 1µl loop on the surface of the medium Interpretation of results: 0 = zero growth 1= weak growth (Reduction in amount of growth or colony size) 2= good growth (Target microorganism have typical appearance, size and morphology)
- 24. Quantitative tube method for performance testing of liquid enrichment media Generally, used for non-selective or selective liquid medium. Use suitable dilution to achieve absence of organisms in the highest dilutions (1/5 or ½ dilution steps are suitable) Count the number of colonies at the lowest dilution containing up to 150 colonies and record the number of the higher dilution than this For the target microorganisms use 10^5 to 10^8 dilutions For the non-target microorganisms use 10^1 to 10^4 dilutions
- 25. Inoculate a tube of test medium and a plate of solid reference medium (non-selective) Incubate Dilutions of target or non-target microorganisms Count the colonies present on the plates which containing up to 150 colonies Separate 10µl loop from the incubated tubes to non-selective agar medium Incubate Examine the presence or absence of growth
- 26. Qualitative tube method for performance testing of selective liquid media Target organism (An inoculum containing ≤ 𝟏𝟎𝟐 cfu) Non-target organism (An inoculum containing ≥ 𝟏𝟎𝟑 cfu) Target and non-target organism (An inoculum containing ≤ 𝟏𝟎𝟐 cfu of target organism and 𝟏𝟎𝟑 cfu of non- target organism) Incubate Remove 10µl and streak on non-selective medium In mixed culture streak on a plate of the specific medium of target organism Incubate Growth of target organism should reach at least 10 colonies Growth of non-target organism should be less than 10 colonies Growth of target organism should reach at least 10 colonies, Growth of non-target organism should be less than 10 colonies
- 27. Qualitative single tube method (turbidity) for performance testing of liquid media This method is suitable for performance testing of non-selective liquid culture media and selective media used for confirmation testing. Interpretation of results 0 = no visual turbidity 1 = very light turbidity 2 = good turbidity Other characteristics such as gas production, color change can be assessed. Sometimes the growth can only be observed as a cell aggregation at the base of the tube.
- 28. Method for quantitative testing of diluents This method determines the ability of the diluent to make a recovery of microorganism without multiplication or reduction before plating on agar or broth. 1- Inoculate 9 ml of diluent with 1ml of test organism. 2- Mix and transfer 0.1ml on the surface of non-selective agar. 3- Remove the same volume on a plate of reference medium. 4- Incubate at 30℃ for 72h 5- Count the colonies, the number of organisms on the reference medium shall be within ±30% of the number on non-selective medium.
- 29. Method for testing transport medium This method determines the ability of the transport medium to maintain the viability microorganism during the period of transport. Transport systems are tested at 5℃ ± 3℃ for the normal transport duration.
- 30. Method for quantitative testing of liquid transport media Method for testing transport medium 1- Inoculate 10 ml of liquid transport media with test organism. 2- Mix and transfer 0.1ml on the surface of non-selective agar (TSA) 3- Remove the same volume on a plate of reference medium. 4- Incubate at 25℃ for 5days 5- Count the colonies, the number of organisms on the reference medium shall be within ±30% of the number on non-selective medium. The inoculum level is 103 to 104 cell into 10 ml tube. Method for quantitative testing of solid transport media The inoculum level is 104 to 106 cell into 10 ml tube.