Animal Biotech Sterilization Methods PDF

WHAT IS STERILIZATION? Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses etc) from a surface, equipment, foods, medications, or biological culture medium. METHODS OF STERILIZATION  The various meth...

WHAT IS STERILIZATION? Sterilization can be defined as any process that effectively kills or eliminates transmissible agents (such as fungi, bacteria, viruses etc) from a surface, equipment, foods, medications, or biological culture medium. 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 PHYSICAL METHODS: 1. HEAT STERILIZATION:  Heat sterilization is the most widely used and reliable method of sterilization, involving destruction of enzymes and other essential cell constituents.  This method of sterilization can be applied only to the THERMO STABLE PRODUCTS, but it can be used for MOISTURE-SENSITIVE MATERIALS. i)Dry Heat (160-1800˚C) Sterilization for thermo stable products ii)Moist heat (121-1340 ˚C) sterilization is used for moisture- resistant materials.  The efficiency with which heat is able to inactivate microorganisms is dependent upon i) the degree of heat, the exposure time and ii) the presence of water.  The action of heat will be due to induction of lethal chemical events mediated through the action of water and oxygen.  In the presence of water much lower temperature time exposures are required to kill microbe than in the absence of water.  THERMAL (HEAT) METHODS Thermal methods includes: i)Dry Heat Sterilization Ex:1. Flaming 2. Incineration 3. Hot air oven ii)Moist Heat Sterilization 1.Boiling water/ steam at atmospheric pressure 2.Hot medium below boiling point (Pasteurization) 3.Dry saturated steam – Autoclaving Dry Heat Sterilization  It employs higher temperatures in the range of 160-180˚C 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 glass wares and metal surgical instruments. It is also used for sterilizing non-aqueous thermo stable liquids and thermo stable 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 Moist Heat Sterilization  Moist heat sterilization involves the use of steam in the range of 121-134˚C. Steam under pressure is used to generate high temperature needed for sterilization.  Saturated steam acts as an effective sterilizing agent.  Autoclave  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. For efficient heat transfer, steam must flush the air out of the autoclave chamber.  Generally the conditions employed are Temperature upto121-134˚C for 15-20 min under 15 psi pressure, based on type of metiral used.  Radiation There Sterilization are 2 general types of radiation used for sterilization, ionizing radiation and non-ionizing radiation. Ionizing radiation is the use of short wavelength, high- intensity radiation to destroy microorganisms. This radiation can come in the form of gamma or X-rays that react with DNA resulting in a damaged cell. Non-ionizing radiation uses longer wavelength and lower energy. As a result, non-ionizing radiation loses the ability to penetrate substances, and can only be used for sterilizing surfaces. The most common form of non-ionizing radiation is ultraviolet light, which is used in a variety of manners throughout industry.  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. Advantages:  No degradation of media during sterilization, thus it can be used for thermally labile media  Leaves no chemical residue  Administration of precise dosage and uniform dosage distribution  Immediate availability of the media after sterilization Disadvantages:  This method is a more costly alternative to heat sterilization  Requires highly specialized equipment LAMINAR AIR FLOW CHAMBER  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.  Ex:HEPA FILTERS  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 fermentors, centrifuges, autoclaves and freeze driers.  There are two types of filters used in filtration sterilization: (a)Depth filters: Depth filters are the variety of filters that use a porous filtration medium to retain particles throughout the medium (b)Membrane filters: These are porous membrane about 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, and polyvinylidene fluoride, or some other synthetic material. CHEMICAL STERILIZATION METHOD GASEOUS METHOD 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 alkylations of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and amino groups of nucleic acids.  The concentration ranges (weight of gas per unit chamber volume) are usually in range of 800- 1200 mg/L for ethylene oxide and 15-100 mg/L for formaldehyde with operating temperatures of 45-63°C and 70-75°C respectively.  Both of these gases being alkylating agents are potentially mutagenic and carcinogenic. They also produce acute toxicity including irritation of the skin, conjunctiva and nasal mucosa  Liquid sterilization Liquid sterilization is the process of sterilization which involves the submerging of equipment in the liquid sterilant to kill all viable microorganisms and their spores. Although liquid sterilization is not as effective as gaseous sterilization, it is appropriate in conditions where a low level of contamination is present. MERITS, DEMERITS AND APPLICATIONS OF DIFFERENT METHODS OF STERILIZATION S.no METHOD MERITS DEMERITS APPLICATIONS MECHANISM Most widely Can be Dry heat is used and applied only applicable 1 Heat Destroys reliable to the for sterilizing glass sterilization bacterial method of thermo wares and metal endo toxins sterilization, stable surgical instruments involving and moist heat is products destruction of the most enzymes and dependable other method for essential cell decontamination of constituents laboratory waste and the sterilization of laboratory glassware, media, and reagents. S.no METHOD MERITS DEMERITS APPLICATIONS MECHANIS M 1 Gaseous Alkylation Penetrating Gases being Ethylene oxide gas has sterilization ability of gases. alkylating agents been used widely to are potentially process heat-sensitive mutagenic and devices. carcinogenic. It is a useful Undesirable Radiation sterilization is method for the changes occur in Radiation generally applied to articles Ionization of industrial irradiated 2 sterilization in the dry state; including nucleic acids sterilization of products,an surgical instruments, heat sensitive example is sutures, prostheses, unit products aqueous solution dose ointments, plastics where radiolysis of water occurs. S.n METHOD MERITS DEMERITS APPLICATIONS o MECHANISM 1 Filtration Does not destroy It is used for both Does not This method is Sterilizing sterilization but removes the the clarification and differentiate grade filters are used in microorganisms sterilization of between viable the treatment of heat liquids and gases as and non viable sensitive injections and it is capable of particles ophthalmic solutions, preventing the biological products and passage of both air and other gases for viable and non supply to aseptic areas viable particles  Importance of Sterilization  Moist heat sterilization is the most efficient biocidal agent. Generally 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 thermolabile 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.  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.  They are also used in industry as part of the venting systems on fermentors, centrifuges, autoclaves and freeze driers. Membrane filters are used for sterility testing.