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Sterilization Sterilization Refers the Complete Removal Or Elimination of Microorganisms from an Environment

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Sterilization Sterilization Refers the Complete Removal Or Elimination of Microorganisms from an Environment AGM151 2006-07 Sterilization Sterilization refers the complete removal or elimination of microorganisms from an environment. Physical and chemical agents are available to remove the microorganisms from the environment. The heat, irradiation and filtration are the three physical agents and various chemicals are as chemical agents available for sterilization. Terms related to control of microorganisms Cide or cidal means to kill the microorganism (Ex. Bactericidal refers the killing of bacteria) Static or stasis means inactivation or inhibition, but not killing. They prevent multiplication of the organism. (Ex. Fungistatic refers the inhibition of growth of fungi.) Sepsis refers break down of a living tissue by organisms and is accompanied by inflammation and pus formation. Antispetic is an agent applied externally on living tissues to kill or inhibit the growth of the organisms. Disinfectant is an agent applied externally on inanimate objects (not living tissues) to destroy harmful pathogens in their vegetative stage. (Antiseptics are milder than disinfectants because to avoid the side effects) Sanitation refers removal of organisms from a location by cleaning but not by sterilization. 1. Heat Principle : At high temperature, all the macro molecules lose their structure and ability to function called as denaturation. Similarly, the heat coagulates the protein and oxidize the elements lead for lethality. The heat susceptibility of the organism should know before the heat treatment to be applied. For this, the thermal death time and thermal death points are essential. Thermal death time refers the time to kill a suspension of organisms at a specific temperature. Thermal death point refers the lowest temperature to kill a suspension in a given time. Decimal reduction time (D time) – Time to kill 90% or 1 log unit of a population at a given temperature Based on the above parameters, the time and temperature to kill the microorganism from an environment is derived. Pasteurization Refers removal of undesired microorganisms without affecting the beneficial microorganisms, odour and taste. The pasteurization of milk is done to eliminate the pathogenic microorganisms which cause diseases like tuberculosis, brucellosis, Q fever, typhoid etc. In broad, the pasteurization is done to kill Salmonella and E. coli like organisms. Sterilization AGM151 2006-07 Flash pasteurization: holding the substance at 71°C for 15 seconds and rapid cooling referred as flash pasteurization Bulk pasteurization: holding the substance at 63-65°C for 30 min and slow cooling referred as bulk pasteurization Tyndallization Refers intermittent or fractional sterilization. The subsequent cooling and heating by steam for 3 days will remove the germs and their spores refers the tyndallization. Ex Soil which contains diversified microbes with spores. The tyndallization will remove all the vegetative cells and also the spores. The normal sterilization by autoclave will eliminate the vegetative cells only and not spores. Tyndallization > sterilization (by autoclave) > pasteurization is the order of degree of strength in terms of removal of microorganisms. 4 or 5 different ways of sterilization are available to kill the microorganisms using heat. 1. Direct flaming – showing the objects or equipments directly to the flame leads the sterilization. This is also called as incineration. The inoculation needles, spread rods, forceps can be sterilized using this technique. 2. Boiling – The liquids boiling also possible. In this method, only the vegetative forms are killed in few minutes whereas the spores are not. 3. Dry heat – Causes oxidation of cells. By using electrical coils, heat is generated and used for sterilization. Normally high temperature and long time are required for complete sterilization using this method. 160-180°C for 2- 3 hours are required for complete sterilization. Instrument: Hot air oven. A double walled (inner and outer walls) instrument protected with asbestos sheets to avoid heat loss. Thermostat is present to control the temperature and timer is also present to turn off automatically after particular time. The glasswares like conical flasks, beakers, petri dishes, pipettes can be sterilized using this technique. 4. Moist heat – Causes denaturation and coagulation of protein. To kill vegetative structure of bacteria, yeast and molds - 80°C for 5-10 min; To kill mold spores - 80°C for 30 min; bacterial spores - 121°C, 15 lbs/sq.inch pressure for 15 min. are the optimum moist heat. (The moist heat has more penetration power than dry heat and leads faster reduction in number of living organism and under pressure, the penetration of heat will be more in the autoclave) Instrument: The autoclave is used for sterilization using moist heat under pressure. Autoclave is made up of double walled steel plates with air tight lids. Electrical coil submerged in water is provided to generate the stream. To measure the pressure, a pressure gauge and for safety, a safety valve is also attached in the lid. If the steam pressure inside the closed vessel is increased to 15 lb/sq. inch, the temperature will raise Sterilization AGM151 2006-07 to 121.6°C. Keeping this condition for 15 – 20 min. will kill all the vegetative and spore structures of microbes. (Note : For soil like heavy solid material, the penetration of moist heat is not sufficient to kill all the spores and vegetative structures. So intermittent sterilization (tyndallization) is necessary) 2. Radiation Microwaves, ultra violet rays, gamma rays and electrons have the power to kill the microorganisms. Among them, UV light (non-ionizing radiation) and gamma rays (ionizing radiation) are commonly used for sterilization. UV radiation: The wave length considered to be between 220 – 300 nm is used to control the microorganisms. It has sufficient energy to form dimers (bridges) between two adjacent pyrimidines in the DNA and there by affect the DNA replication leads to death. This light is highly useful to disinfect surfaces, air, and water, which do not absorb the light. Instrument used: laminar air flow chamber – A clean bench used to handle the microbes in a microbe free environment. It has two methods of sterilization. Radiation by UV and filtration by HEPA filters (High efficiency particulate filters), a microbe free air will be present in side. Ionizing radiation : Ionizing radiation is electromagnetic radiation of sufficient energy to produce ions like electron, hydroxyl ions, protons, etc. which can alter the biopolymers such as DNA and protein causing break of polymers. γ-rays produced from 60Co and 137Cs are useful for sterilization of the plastic wares, gloves, heat labile plastic containers, poly bags, tissue grafts, plastic syringes etc. In few cases, food were also sterilized by γ-rays. 3. Filtration Heat is the most common and effective way of sterilization but some heat sensitive liquids and chemicals cannot be sterilized by heat. An alternate technique for the sterilization is by filters. A filter is too small for a passage of microorganisms but large enough to passage of liquids. There are three types of filters 1. Pyrex glass or sintered glass filters: Filter glass made with powdered glass disc with pore size of 0.5 to 2 µm are used as filters. These discs were fit into funnels and used for filtration. These were used by suction pumps for faster flow rate. 2. Seitz filters: The filter mat is made up of asbestos – cellulose mixture. 3. Membrane filters: They are composed of polymers with high tensile strength such as cellulose acetate, cellulose nitrate or polysulfonate, manufactured in such a way that they contain a large number of tiny holes. By adjusting the polymerization conditions during manufacture, the size of holes in the membrane can be precisely controlled. They trap the microbes on the surface and allow the microbe free solution to pass. Sterilization AGM151 2006-07 4. Chemical agents : Different chemicals like halogens, heavy metals, phenols, alcohols and ethanol are used for sterilization. The following are different classes of chemicals involved in sterilization. a) Phenol and Phenolics - 5% aqueous solutions kill vegetative forms of microorganisms. Some of their derivatives are lysol, dettol, cresol etc. They alter the selective permeability of the cytoplasmic membrane of organisms and cause death. b) Alcohols - They are bactericidal and fungicidal but not sporicidal. They denature proteins and are solvents of lipids. 60 – 90 % is the range for this activity. Water is required for the lethal effect. c) Halogens - Cause oxidation and direct halogenation of proteins thus inhibiting the activity of proteins e.g. hypochlorite, iodophores etc. d) Surfactants - Surface active agents, good wetting and solubilizing agents e.g. Soap, detergent etc. They have both hydrophilic and hydrophobic groups (ambivalent) to dissolve compounds. e) Alkylating agents - They substitute alkyl groups for hydrogen of reactive groups in nucleic acids and proteins, causing disruption of metabolic pathways e.g. Formaldehyde, glutaraldehyde, ethylene oxide (gaseous agent) f) Heavy metals - React with sulfhydryl group (SH) of enzyme and make them inactive. Mercury, copper and silver are commonly used heavy metals. g) Antibiotics - Antibiotics are low molecular weight molecules produced as secondary metabolites by microorganisms, which kill or inhibit the other microorganisms. The following table shows some examples of common antibiotics and their mode of action. S. Antibiotics Source Effective Mode of action
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