Sterilization methods and principles
- 1. BY -: NITESH KUMAR B.PHARM VII SEM JAIPUR COLLEGE OF PHARMACY, JAIPUR
- 2. CONTENTS Introduction Pharmaceutical Importance Methods of Sterilization Heat Sterilization Gaseous Sterilization Liquid Sterilization Radiation Sterilization Filtration Sterilization Tests for Sterility Method of Membrane Filtration Method of Direct Transfer Evaluation of Sterilization Method
- 3. Introduction Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses and prions) from a surface, equipment, foods, medications, or biological culture medium. In practice sterility is achieved by exposure of the object to be sterilized to chemical or physical agent for a specified time. Various agents used as steriliants are: elevated temperature, ionizing radiation, chemical liquids or gases etc
- 4. Pharmaceutical Importance of Sterilization Moist heat sterilization - It is used for: Surgical dressings, Sheets, Surgical and diagnostic equipment, Containers, Closures, Aqueous injections, Ophthalmic preparations and Irrigation fluids etc. Dry heat sterilization can only be used for thermo stable, moisture sensitive or moisture impermeable pharmaceutical and medicinal. These include products like; Dry powdered drugs, Suspensions of drug in non aqueous solvents, Oils, fats waxes, soft hard paraffin silicone, Oily injections, implants, ophthalmic ointments and ointment bases etc. Gaseous sterilization is used for sterilizing thermo labile substances like; hormones, proteins, various heat sensitive drugs etc. U.V light is perhaps the most lethal component in ordinary sunlight used in sanitation of garments or utensils.
- 5. Gamma-rays from Cobalt 60 are used to sterilize antibiotic, hormones, sutures, plastics and catheters etc. Filtration sterilizations are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products, air and other gases for supply to aseptic areas. Variables that affect sterilization include: 1. The dryness of devices to be processed 2. The temperature and humidity of the processing area 3. Whether or not the devices were properly prepared and loaded into the sterilizer 4. Whether or not the sterilizing agent is properly delivered into the system 5. The sterilizer’s condition and maintenance protocol 6. Whether or not the correct sterilization method and cycle were used
- 6. Expression of resistance D-value = D-value is indicative of the resistance of any organism to a sterilizing agent. For radiation and heat treatment, D-value is the time taken at a fixed temperature or the radiation dose required to achieve a 90% reduction in viable count. Z-value = Z-value represents the increase in temperature needed to reduce the D-value of an organism by 90%. Methods of Sterilization The various methods of sterilization are: 1. Physical Method a. Thermal (Heat) methods b. Radiation method c. Filtration method 2. Chemical Method a. Gaseous method
- 7. 1. Heat Sterilization Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents. The process is more effective in hydrated state where under conditions of high humidity, hydrolysis and denaturation occur, thus lower heat input is required. Under dry state, oxidative changes take place, and higher heat input is required. This method of sterilization can be applied only to the thermostable products, but it can be used for moisture- sensitive materials for which dry heat (160-1800 C) sterilization, and for moistureresistant materials for which moist heat (121-1340 C) sterilization is used
- 8. a. Dry Heat Sterilization: Examples of Dry heat sterilization are: 1. Incineration 2. Red heat 3. Flaming 4. Hot air oven It employs higher temperatures in the range of 160-180 degree Celsius and requires exposures time up to 2 hours, depending upon the temperature employed. The benefit of dry heat includes good penetrability and non- corrosive nature which makes it applicable for sterilizing glasswares and metal surgical instruments. It is also used for sterilizing non-aqueous thermostable liquids and thermostable powders. Dry heat destroys bacterial endotoxins (or pyrogens) which are difficult to eliminate by other means and this property makes it applicable for sterilizing glass bottles which are to be filled aseptically.
- 9. b. Moist Heat Sterilization: Moist heat may be used in three forms to achieve microbial inactivation 1. Dry saturated steam – Autoclaving 2. Boiling water/ steam at atmospheric pressure 3. Hot water below boiling point Moist heat sterilization involves the use of steam in the range of 121-134 degree Celsius. Steam under pressure is used to generate high temperature needed for sterilization. Autoclaves use pressurized steam to destroy microorganisms, and are the most dependable systems available for the decontamination of laboratory waste and the sterilization of laboratory glassware, media, and reagents.
- 10. Gaseous Sterilization The chemically reactive gases such as formaldehyde, (methanol, H.CHO) and ethylene oxide (CH2)2O possess biocidal activity. Ethylene oxide is a colorless, odorless, and flammable gas. The mechanism of antimicrobial action of the two gases is assumed to be through alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and amino groups of nucleic acids. Both of these gases being alkylating agents are potentially mutagenic and carcinogenic. a. Ethylene oxide sterilizer : Ethylene oxide gas has been used widely to process heat-sensitive devices, but the aeration times needed at the end of the cycle to eliminate the gas made this method slow.
- 11. b. Low temperature steam formaldehyde (LTSF) sterilizer: An LTSF sterilizer operates with sub atmospheric pressure steam. At first, air is removed by evacuation and steam is admitted to the chamber. Liquid Sterilization a. Peracetic Acid liquid sterilization - It disrupts bonds in proteins and enzymes and may also interfere with cell membrane transportation through the rupture of cell walls and may oxidize essential enzymes and impair vital biochemical pathways. b. Hydrogen Peroxide Sterilization - The temperature of this sterilization method is maintained in the 40-50°C range, which makes it particularly well-suited for use with heat- sensitive and moisture-sensitive medical devices
- 12. Radiation Sterilization Many types of radiation are used for sterilization like electromagnetic radiation (e.g. gamma rays and UV light), particulate radiation (e.g. accelerated electrons). The major target for these radiation is microbial DNA. Gamma rays and electrons cause ionization and free radical production while UV light causes excitation. Radiation sterilization is generally applied to articles in the dry state; including surgical instruments, sutures, prostheses, unit dose ointments, plastic syringes and dry pharmaceutical products. UV light, with its much lower energy, and poor penetrability finds uses in the sterilization of air, for surface sterilization of aseptic work areas, for treatment of manufacturing grade water, but is not suitable for sterilization of pharmaceutical dosage forms.
- 13. Gamma ray Sterilizer: Gamma rays for sterilization are usually derived from cobalt-60 source, the isotope is held as pellets packed in metal rods, each rod carefully arranged within the source and containing 20 KCi of activity. This source is housed within a reinforced concrete building with 2 m thick walls Articles being sterilized are passed through the irradiation chamber on a conveyor belt and move around the raised source. Ultraviolet Irradiation: The optimum wavelength for UV sterilization is 260 nm. A mercury lamp giving peak emission at 254 nm is the suitable source of UV light in this region.
- 14. Filtration Sterilization Filtration process does not destroy but removes the microorganisms. It is used for both the clarification and sterilization of liquids and gases as it is capable of preventing the passage of both viable and non viable particles. The major mechanisms of filtration are sieving, adsorption and trapping within the matrix of the filter material. Sterilizing grade filters are used in the treatment of heat sensitive injections and ophthalmic solutions, biological products and air and other gases for supply to aseptic areas. They are also used in industry as part of the venting systems on fomenters, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing.
- 15. Application of filtration for sterilization of gases: HEPA (High efficiency particulate air) filters can remove up to 99.97% of particles >0.3 micrometer in diameter. Air is first passed through prefilters to remove larger particles and then passed through HEPA filters. There are two types of filters used in filtration sterilization : (a) Depth filters (b) Membrane filters Application of filtration for sterilization of liquids Membrane filters of 0.22 micrometer nominal pore diameter are generally used, but sintered filters are used for corrosive liquids, viscous fluids and organic solvents.
- 18. Tests for Sterility Tests for sterility are carried out by two methods: (a) Membrane Filtration Method (b) Direct Transfer / Inoculation Method. The Membrane Filtration Method is used as the method of choice wherever feasible. Media used in Sterility Testing 1. Fluid Thioglycollate Medium (Medium 1) 2. Soybean-Casein Digest Medium (Medium 2)
- 19. Medium 1 (Fluid Thioglycollate Medium) Composition Pancreatic Digest of Casein----15.0 g Yeast Extract (water-soluble)----5.0 g Glucose monohydrate/anhydrous-----5.5 g/5.0 g Sodium chloride------2.5 g L- Cystine-------0.5 g Sodium thioglycollate------ 0.5 g 0.1% Resazurin Sodium Solution (freshly prepared)---1.0 mL Granulated Agar (moisture not more than 15%) ----0.75 g Purified Water------1000 mL Polysorbate 80-----5.0 mL pH after sterilization (measured at room temperature): 7.1± 0.2
- 20. Method of Preparation: The pancreatic digest of casein, yeast extract, glucose, sodium chloride, L- cystine, agar and water are mixed in the proportions given above and heat until dissolved. Sodium thioglycollate is dissolved in the solution. The specified quantity of Polysorbate 80 is added if this ingredient is to be included. If necessary, 1 M sodium hydroxide or 1 M hydrochloric acid is added so that after the solution is sterilized its pH will be 7.1± 0.2. If the solution is not clear, mixture is heated to boiling and filtered while hot through moistened filter paper. Resazurin sodium solution is added and mix.
- 21. Medium 2 (Soybean-Casein Digest Medium) Composition Pancreatic Digest of Casein----17.0 g Papain Digest of Soybean Meal----3.0 g Glucose monohydrate/anhydrous--2.5 g /2.3 g Sodium chloride----5.0 g Dipotassium hydrogen phosphate (K2HPO4) -----2.5 g Purified Water----1000 mL Polysorbate 80----5.0 mL pH after sterilization (measured at room temperature): 7.3±0.2
- 22. Method of Preparation: The ingredients are mixed in the proportions given above with slight warming. The solution is cooled to room temperature. The specified quantity of Polysorbate 80 is added if this ingredient is to be included. If necessary, sufficient 1 M sodium hydroxide or 1M hydrochloric acid so that after the solution is sterilized its pH will be 7.3± 0.2. If the solution is not clear it is filtered through moistened filter paper.
- 23. Method of Membrane Filtration Procedure The filter should be a membrane filter disc of cellulose esters or other suitable plastics, having a nominal average pore diameter not exceeding 0.45 µm. The membrane should be held firmly in a filtration unit which consists of a supporting base for the membrane, a receptacle for the fluid to be tested, a collecting reservoir for the filtered fluid, and the necessary tubes or connections. Cellulose nitrate filters are recommended for aqueous, oily and weakly alcoholic solutions and cellulose acetate filters for strongly alcoholic solutions. The entire unit should be sterilized by appropriate means with the membrane filter and sterile airways in place.
- 24. The method of sterilization should not be deleterious to the membrane, e.g., weaken it or change the nominal average pore diameter. After sterilization, the apparatus should be free of leaks to the atmosphere except through the sterile airways. Method of Direct Transfer Procedures Liquids and soluble or dispersible solids: Appropriate quantities of the preparation to be examined are added directly into Medium 1 and Medium 2. Approximately equal quantities of the preparation should be added to each vessel of medium. The test vessels of Medium 1 is incubated at 30 - 35°C and the vessels of Medium 2 is incubated at 20- 25°C
- 25. The volume of Medium 1 should be such that the air space above the medium in the container is minimized. The volume of Medium 2 should be such that sufficient air space is left above the medium to provide conditions that permit the growth of obligate aerobes. Place half the articles into Medium 1 and the remaining half into Medium 2. Incubate the test vessels of Medium 1 at 30 - 35°C and the vessels of Medium 2 at 20 - 25°C. Ointments and oily preparations: Ointments and oily preparations may be tested by the method of Direct Transfer if testing by the method of Membrane Filtration is not feasible, i.e. when a suitable solvent is not available
- 26. Incubation and examination of sterility tests: All test vessels of Medium 1 are incubated at 30 - 35°C. The vessels of Medium 2 are incubated at 20 - 25°C. All test and control vessels, other than the sub cultured vessels referred to below, must be incubated for at least 14 days unless microbial contamination is detected at an earlier time. If turbidity, precipitate, or other evidence of microbial growth during incubation is seen: the suspected growth is examined microscopically by Gram stain; attempts are made to grow single colonies using appropriate microbiological methods; colonies of each type of micro-organism present are examined for colonial morphology and cellular morphology by Gram stain; attempts are made to identify the isolates, as far as the genus, and preferably species.
- 27. Interpretation of the test results: If microbial growth is not evident in any of the vessels inoculated with the product, the sample tested complies with the test for sterility, if microbial growth is evident the product 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 being examined. If the test is declared to be invalid it may be repeated with the same number of units as in the original test. If there is no evidence of growth in any vessels inoculated with the product during the repeat test the product passes the test for sterility. This interpretation applies even if growth occurs in negative product control vessels. If there is evidence of growth in the test vessels the product fails the test for sterility.
- 28. Evaluation of Sterilization Method Sterile products possess several unique properties, such as freedom from microorganism, pyrogens, particulates and high standards of purity and quality . The principle involve in the evaluation of sterilization process are: i. To build sterility into product. ii. Perform a maximum level of probability. iii. Establish specification and performance characteristic. iv. To provide greater assurance of support of the result. v. Specific methodology, process and equipment. vi. Final product testing using validated analytical method and vii. Verification, calibration and maintenance of equipments used in the processes.
- 29. Evaluation of sterilization methods provides a high degree of assurance which indicates a specific process will consistently produce a product that will meets it predetermined specifications and quality assurance. Process of Microbial Destruction Microbial destruction methods such as heat, chemical, and radiation sterilization are used. Upon exposure of such treatment, microorganisms die according to logarithmic relationship between concentration or population of the living cells and the time exposure or radiation dose. The relationship between microbial population and time may be linear or non linear.
- 30. Evaluation and In Process Monitoring of Sterilization Procedures 1. Dry Heat Sterilization