Downloaded 955 times
Uploaded byMohammed Fawzy
Sterility test and modern microbiological methods
This document provides an overview of sterility testing and rapid microbiological methods. It discusses sterility testing, including definitions, common media used, methods for preparing different types of test products, incubation periods, growth promotion tests, and interpreting results. It also briefly introduces some rapid microbiological methods like ATP bioluminescence, colorimetric growth detection, and cytometry systems. The key purpose of sterility testing is to detect any viable microorganisms in pharmaceutical products or medical devices labeled as sterile.
More Related Content
Similar to Sterility test and modern microbiological methods
Sterility test and modern microbiological methods
- 1. Sterility test andRapid microbiological methods BY/ MOHAMMED FAWZY MICROBIOLOGIST AT NODCAR
- 2. Agenda Sterility test Introduction Definition Media insterility test Methods Preparation of test products Incubation period Growth promotion tests Interpretation of results Rapid microbiological methods Introduction ATP-bioluminescence Colorimetric growth detection Autofluorescence detection Cytometry systems
- 3. Sterility test introduction The testis applied to substances, preparations, or articles which, according to the Pharmacopeia, are required to be sterile. However, a satisfactory result only indicates that no contaminating microorganism has been found in the sample examined under the conditions of the test Sterility test is applied only for sterile, non-pyrogenic products Example as parenteral, ophthalmic products, Pulmonary drug delivery system, and sterile medical devices as ocular inserts.
- 4. Introduction All products labeledsterile must be pass sterility test This test is suitable to reveal the presence of viable form of bacteria, fungi and yeasts in pharmaceutical and medical devices Sterile product must be free from Microorganisms Spores Pyrogens Pathogens
- 5. Definition Sterility is defineas freedom from the presence of viable microorganisms, is a strict, uncompromising requirement of an injectable dosage form. Sterility test is define as: Microbiological test applied to sterile product to show are products manufactured and processed under specification guided by cGMP. Or to confirm the products either sterile or non. sterility test is a destructive test thus, it is impossible to test every item for sterility.
- 6. Media in sterilitytest USP and EP describe two primary types of culture media to be used in the sterility testing of parenteral products. Fluid thioglycollate media (FTM) Soybean casein digest broth FTM used for detection of aerobic and anaerobic bacteria. SCDB used for detection of molds and molds.
- 7. Media in sterilitytest FTM 1. Detect aerobic and anaerobic bacteria. 2. Contain O2 sensitive substance (resazurin sodium) 3. The top part should not consume more than 1/3 of tube. 4. Neutralize the bacteriostatic properties of mercuric compounds. SCDB 1-Detect molds and yeasts 2-possesses a higher pH 3- a better nutrient for fungal contaminants 4-Suitable for aerobic bacteria
- 8. FTM CONPONANTS FUNCTION L-Cysteine 0.5 g Antioxidant Agar,granulated (moisture 0.75 g Nutrient and viscosity content _15%) Sodium chloride 2.5 g inducer Isotonic agent Dextrose 5.5 g Nutrient Yeast extract 5.0 g Pancreatic digest of casein 15.0 g Nutrient Nutrient Sodium thioglycollate or 0.5 g thioglycollic acid 0.3 ml Resazurin sodium solution 1.0 ml (1:1000), freshly prepared Purified water QS 1000 ml Antioxidant Nutrient Oxidation indicator pH after sterilization 7.1 _ 0.2.
- 9. SCDB Components Function Pancreatic digest ofcasein 17.0 g Nutrient Papaic digest of soybean meal 3.0 g Nutrient Sodium chloride 5.0 g Isotonic agent Dibasic potassium phosphate 2.5 g Buffer Dextrose 2.5 g Nutrient Purified watera QS 1000 ml Solvent pH after sterilization 7.3 _ 0.2. ---------------------
- 10. Minimum Quantity tobe used for Each Medium Minimum Quantity to be Used (unless otherwise justified and authorized) Quantity per Container Liquids (other than antibiotics) Less than 1 Ml The whole contents of each container 1–40 Ml Half the contents of each container, but not less than 1 mL Greater than 40 mL, and not greater than 100 mL 20 mL Greater than 100 Ml 10% of the contents of the container, but not less than 20 mL Antibiotic liquids 1 mL Other preparations soluble in water or in isopropyl myristate The whole contents of each container to provide not less than 200 mg Insoluble preparations, creams, and ointments to be suspended or emulsified Use the contents of each container to provide not less than 200 mg Solids Less than 50 mg The whole contents of each container 50 mg or more, but less than 300 mg Half the contents of each container, but not less than 50 mg 300 mg–5 g 150 mg Greater than 5 g 500 mg Devices Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long) Surgical dressing/cotton/gauze (in packages) 100 mg per package Sutures and other individually packaged single-use material The whole device Other medical devices The whole device, cut into pieces or disassembled
- 11. Methods I. Direct transfer method Itis most usable method and carried out by transfer the test product after preparation to the medium and incubate for not less than 14 days. II. Membrane filtration method It is usually used in sterility test of antimicrobial agents as antibiotic and other. It is carried out by using a membrane filter having a pore size about (0.45) micrometer that retain most of bacteria, after filtration of the test product, the membrane is aseptically transfer and washed with a diluting fluid such as fluid A,D and K. then aseptically transfer to the medium and incubate for not less than 14 days.
- 12. Preparation of testproducts a- Oily liquid Added the tween 80 as 10gm.to 1000 ml of media for emulsification and to be easily dispersion to media. b- Ointment and creams: Preparing by taking about 1 gm. of test product and aseptically transfer to 10 ml of diluting fluid as fluid A, then transfer to a medium not contain an emulsifying agent. And incubate for not less than 14 day. Observe the cultures several times during the incubation period. Shake cultures containing oily products gently each day. However, when thioglycollate medium or other similar medium is used for the detection of anaerobic microorganisms, keep shaking or mixing to a minimum in order to maintain anaerobic conditions.
- 13. Preparation of testproducts c- devices with pathways labeled sterile Aseptically pass not less than 10 pathway volumes of Fluid D through each device tested. Collect the fluids in an appropriate sterile vessel, and proceed as directed for Aqueous Solutions or Oils and Oily Solutions, whichever applies. d- solids: Transfer the quantity as described before in the previous table either directly in a dray form or after making a suspension by adding sterile diluent to the immediate container.
- 14. Preparation of testproducts e-Sterile devices: Articles can be immersed intact or disassembled. To ensure that device pathways are also in contact with the media, immerse the appropriate number of units per medium in a volume of medium sufficient to immerse the device completely, and proceed as directed above. For extremely large devices, immerse those portions of the device that are to come into contact with the patient in a volume of medium sufficient to achieve complete immersion of those portions.
- 15. Preparation of testproducts f-For catheters: where the inside lumen and outside are required to be sterile, either cut them into pieces such that the medium is in contact with the entire lumen or fill the lumen with medium, and then immerse the intact unit. g-Surgical dressing: Three sample each of about 1 gm. or 10 cm are taken from each dressing after opening the wrappers. They are chosen from different places including regions where the contamination is more likely (center and outside). If dressing is less than 1 gm. Or 10 cm2, it is used entire or divided in to equal pieces for inoculation in the bacterial and fungal medium. OR each sample is shaken for 10 minutes in 50 ml. of sterile broth and then the liquid is pass through a membrane filter, the membrane is washed with diluting fluid and then incubated in to medium.
- 16. Incubation period For bacteria: Temp Time For 30-35°C. NLT 14 days. molds and yeasts: Temp Time 20-25°C NLT 14 days.
- 17. Growth promotion tests Aerobicbacteria Bacillus subtilis Staphylococcus aureus Pseudomonas aeruginosa Anaerobic bacterium Clostridium sporogenes Fungi Candida albicans Aspergillus brasiliensis (Aspergillus Niger)
- 18. Growth promotion Inoculate media Notmore than 100 CFU Incubate Bacteria Fungi Not more than 3 days Not more than 5 days Clear growth of microorganism: Suitable for use
- 19. Interpretation of result examinethe media for macroscopic evidence of microbial growth. If the material being tested renders the medium turbid so that the presence or absence of microbial growth cannot be readily determined by visual examination 14 days after the beginning of incubation transfer portions (each not less than 1 mL) of the medium to fresh vessels of the same medium, and then incubate the original and transfer vessels for not less than 4 days.
- 20. Positive and negativecontrol Positive control For validation of nutrient medium. For testing the growth ability of media. Negative control For validation of nutrient medium. For testing the sterilization of media. Done by exposure to the same environmental condition as test exposure. Done by inoculation of NMT 100 CFU. Incubate at 30-35 for 14 days. Incubate at 30-35 for 14 days. If growth where obtain →used in sterility test. If growth where obtain →sterilization failed or improper storage of media
- 21. Interpretation of result If no evidence of microbial growth is found, the product to be examined complies with the test for sterility. If evidence of microbial growth is found, the product to be examined does not comply with the test for sterility, unless it can be clearly demonstrated that the test was invalid for causes unrelated to the product to be examined.
- 22. Interpretation of result Thetest may be considered invalid only if one or more of the following conditions are fulfilled: a. The data of the microbiological monitoring of the sterility testing facility show a fault. b. A review of the testing procedure used during the test in question reveals a fault. c. Microbial growth is found in the negative controls.
- 23. Interpretation of result Ifthe test is declared to be invalid, it is repeated with the same number of units as in the original test. If no evidence of microbial growth is found in the repeat test, the product examined complies with the test for sterility. If microbial growth is found in the repeat test, the product examined does not comply with the test for sterility
- 24. RMM Introduction Current harmonized compendialsterility test methods using either membrane filtration or direct inoculation require at least 14days of incubation. In cases where drug products either possess an intrinsic turbidity, or because of their formulation become opaque or cloudy during the incubation period, identification of microbial contamination based on visual confirmation of turbidity of growth media becomes difficult. In such instances, at the end of the 14-day incubation, a portion of the sample is sub-cultured into fresh medium for an additional 4–5 days to allow detection, further extending the incubation period.
- 25. Introduction The replacement orthe supplement of the conventional sterility test by a rapid microbiology test will have significant benefits. a test based on current technologies can increase throughput and allow better data handling. a greater assurance of product safety. A rapid method has the potential to produce test results much faster at enhanced sensitivity. Time saving. Characters of ideal method: simple economic relatively easy to implement.
- 26. ATP Bioluminescence Adenosine triphosphate(ATP) bioluminescence is a well established rapid method utilizing a specific substrate and enzyme combination, luciferin/luciferase, to break down microbial ATP from growing cells and produce visible light, which can be measured using a luminometer. Several commercial systems have been developed for a range of pharmaceutical test applications, including sterility testing, especially for filterable samples where non-microbial ATP in the sample is less of a concern. The test time can be reduced considerably because detection of microbial growth in culture media is accomplished by ATP-bioluminescence, rather than by visible turbidity. Typically, results equivalent to those of compendial tests are available within 7 days or less.
- 27. Celsis Rapid DetectionAdvance luminometer system, AkuScreen™ reagents which use the enzyme adenylate kinase to increase the quantity of microbial ATP produced and reduce detection times by 25-50%.
- 28. ATP Bioluminescence ATP (adenosinetriphosphate) act as energy source for all a live organisms That includes microorganisms, and it is for the detection of bacteria, yeasts, and molds that ATP bioluminescence has been applied most successfully. ATP bioluminescence is well-established technique in food industry Used for hygienic monitoring and finished product testing ATP bioluminescence today used in cosmetic industry and pharmaceutical industry
- 29. ATP Bioluminescence Bioluminescence isan enzymatically catalyzed reaction that generates light. based on a reaction that occurs naturally in the North American firefly, Photinus pyralis. The reaction was famously first described by McElroy 1947.
- 30. ATP Bioluminescence ATP bioluminescencereaction depend on production of light that can be detected by an instrument after magnification . The amount of emitted light is proportionally to amount of ATP. SO. that there is a linear relationship between the amount of ATP in a sample and the amount of light generated.
- 31. Working mechanism luciferin-based bioluminescencereaction makes use of the firefly enzyme luciferase (luciferin 4-monooxygenase, EC 1.13.12.7),which catalyzes the oxidation of D-luciferin in the presence of ATP, magnesium ions and molecular oxygen. The reaction yields a quantum of yellow light at 564 nm for each molecule of the luciferin substrate oxidized. ATP + Luciferin + O2 →AMP + Oxyluciferin + CO2 + PPi + Light (Bioluminescence). In presence of luciferase Light produced from the luciferin–luciferase reaction is proportional to the amount of ATP used and can be measured in Relative Light Units (RLU) by a luminometer.
- 32.
- 33. Milliflex Rapid Microbiology Detectionand Enumeration System The Milliflex® Rapid Microbiology Detection and Enumeration system from Millipore also uses ATP-bioluminescence to detect microbial cells and is designed specifically for monitoring microbial contamination in filterable samples. It is automated, employing image analysis technology to detect microcolonies growing directly on the surface of a membrane filter after the addition of bioluminescence reagents. The system is designed to be quantitative, but a method has been developed and validated to use it for a rapid sterility test with an incubation time of just five days
- 34. Milliflex Rapid Microbiology Detectionand Enumeration System prons cons Equivalent to compendial tests Results in 7 days Non-microbial detection ATP
- 35. Colorimetric Growth Detection Colorimetricgrowth detection methods rely on a colour change being produced in a growth medium as a result of microbial metabolism during growth, often as a result of CO2 production.
- 36. Colorimetric Growth Detection mechanism 1.Media (two different media are used) as SCDB supplemented with complex amino acids and carbohydrates and are designed both to support growth and to ensure optimal C02 production. 2. CO2 sensor :a C02 sensor is bonded to the bottom of each bottle and is separated from the broth medium by a semipermeable membrane. 3. The semipermeable membrane is permeable to CO2 and impermeable to H2 ion and most ion are exist in the media
- 37. Colorimetric Growth Detection mechanism 4.Carbon dioxide produced by growing organisms diffuses across the membrane into the sensor and dissolves in the water, thereby generating hydrogen ions according to the following equation: C02 + HO2 +H2CO3 -> H+ + HCO35. Free hydrogen ions can interact with the sensor, which is blue to dark green in the alkaline state. As C02 is produced and dissolves in the water, the concentration of hydrogen ions increases and the pH decreases. This causes the sensor to become lighter green and eventually yellow, which results in an increase of red light reflected by the sensor.
- 38. BacT/ALERT® 3D Dual-TMicrobial Detection System The system is automated and employs sensitive colour detection and analysis technology to produce a result in as little as three days. It can detect both aerobic and anaerobic bacteria, as well as yeasts and molds. 1-Results 3 days. 2-Aerobic/anaerobic bacteria. 3-Yeasts and molds.
- 39. Autofluorescence detection All livingcells produce a small amount of fluorescence (autofluorescence) and this can be used to detect microbial colonies growing on a solid surface long before they are visible to the naked eye. particularly useful for filterable samples. where a membrane filter can be incubated on a conventional nutrient medium and scanned using highly sensitive imaging systems to detect microcolonies. Automatic detection. uses a large area CCD imaging system without magnification to detect developing microcolonies. detect both aerobic and anaerobic bacteria, as well as yeasts and molds. produce a result in as little as three days. BacT/ALERT® 3D Dual-T Microbial Detection System from bioMerieux.
- 40. Cytometry systems Cytometry doesnot rely on microbial growth to detect contamination. but instead uses cell labelling techniques to detect viable microorganisms. detect a wide range of organisms, including yeasts and molds, within minutes. the cells are labelled using a fluorescent dye or a non-fluorescent substrate, which is converted to a fluorochrome in viable cells. Detection of the labelled cells occurs by laser scanning in either a flow cell (flow cytometry), or on a solid phase platform such as a membrane filter (solid phase cytometry). AES Chemunex has developed solid phase cytometry detection systems. The company’s Scan®RDI (also known as ChemScan RDI) system is capable of detecting 1 CFU per sample and has been evaluated as a possible RMM for sterility testing. The technology has been developed for the Stereal®-T sterility testing system.
- 41. Rapid Sterility Methods AES BD Chemunex Scan®RDI (ChemScan RDI) system Diagnostic Systems BD FACSMicroCount™ bioMérieux (industrial applications) BacT/ALERT® 3D Dual-T Microbial Detection System Celsis Rapid Detection Advance luminometer system, AkuScreen™ reagents
- 42. Rapid Sterility Methods Merck KGaA Milliflex® Rapid Microbiology Detection and Enumeration System Pall Life Sciences Pallchek™ Rapid Microbiology System Rapid Micro Biosystems Growth Direct™ System