Methods of Sterilization.pdf

Methods of Sterilization DR.ZAHIDA SHAIKH Assistant Professor in pathology department LUMHS. Terminology relating to the control of microbial Growth: CLEANING - It is a process which removes visible contamination but does not necessarily destroy micro organisms. ASEPSIS -...

Methods of Sterilization DR.ZAHIDA SHAIKH Assistant Professor in pathology department LUMHS. Terminology relating to the control of microbial Growth: CLEANING - It is a process which removes visible contamination but does not necessarily destroy micro organisms. ASEPSIS -Term used to describe methods which prevent contamination of wounds and other sites, by ensuring that only sterile object and fluids come into contact with them. Sanitizing - process that reduces microbial population on living surfaces to a safe level. Decontamination - process that removes pathogenic microorganisms from an object to make it safe to handle.  Terms used in sterilization Disinfection  Process that eliminates many or all pathogenic microorganisms, except bacterial spores, on inanimate objects. Antiseptics.  Chemical disinfectants which can be safely applied to skin or mucous membrane and are used to prevent infection by inhibiting the growth of bacteria. Bactericidal agents and germicides Agents which are able to kill bacteria. Bacteriostatic agents Agents which prevents the multiplication of bacteria ( doesn’t kill).  Sterilization  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. OR  The process by which an article, surface or medium is freed of all viable microorganisms either in the vegetative or spore state is known as sterilization.  Methods of sterilization Chemical agents Physical agents Modification of proteins 1.Chlorine 2.Iodine 1.Heat 3.Heavy metals 4.Hydrogen peroxide 2.Radiation Disruption of 3.filtration 5.formaldehyde& glutaraldehyde cell 6.Ethylene oxide membranes 7. Acids & alkalis 1.Alcohols 2. Detergents Modification of nucleic acids 3. Phenols 1.Crystal violet 2. Malachite green Methods of sterilization  Physical methods of Sterilization Sunlight Direct sunlight is a natural method of sterilization of water in tanks, rivers and lakes. Direct sunlight has an active germicidal effect due to the combined effect of ultraviolet and heat rays.  HEAT Most common and one of the most effective methods of sterilization. Factors influencing sterilization by heat are : - i. Nature of heat a. Dry b. Moist ii. Temperature & time iii. Number of organism present iv. Whether organism has sporing capacity v. Type of material from which organism is to be eradicated  DRY HEAT Killing is due to : - Dehydration and oxidation of organisms - Protein denaturation - Toxic effects of elevated levels of electrolytes 1. Red Heat : It is used to sterilize metallic objects by holding them in flame till they are red hot. Example : inoculating wires, needles, forceps etc. 2. Flaming : The article is passed over flame without allowing it to become red hot. Example : Glass plates, Cotton wool plays and glass slides.  HOT AIR OVEN :  First introduced by Louis Pasteur  An electrically heated chamber that has a thermostat which maintains the chamber’s air constantly at desired temp.  used to sterilize items, which do not get damaged by high temp: Used for sterilization of All metallic instruments Scissors, Forceps Glass ware eg,. Petri dishes, test tube Cotton swabs Syringes , Powders, , oils etc Temp. & Time: The sterilization is complete if these two factors are achieved throughout the load. Temperature Time(Min) 140oC 180 150oC 150 160oC 60 170oC 45 180oC 18 Sterilization by dry heat requires temperatures in the 190oC 7.5 range of 180°C for 2 hours. This process is used primarily for glassware  Moist heat:  Bacteria are more susceptible to moist heat.  Steam has more penetrating power.  Steam has more sterilizing power as more heat is given up during condensation.  BOILING : At 100 OC For 5 to 10 minutes is sufficient to kill all non- spore forming bacteria. used for sterilization of , syringes , needles and rubber articles Quite common especially in domestic circumstances Tyndallization:  Heat labile media like those containing sugar, milk, gelatin  Steaming at 100°C is done in steam sterilizer for 20 minutes followed by incubation at 37°C overnight.  This procedure is repeated for another 2 successive days. That is 'steaming' is done for 3 successive days. Spores, if any, germinate to vegetative bacteria during incubation and are destroyed during steaming on second and third day.  Pasteurization  primarily for milk  heating the milk to 62°C for 30 minutes followed by rapid cooling.  ("Flash" pasteurization at 72°C for 15 seconds is often used)  This is sufficient to kill the vegetative cells of the milk-borne pathogens, e.g.,  Ultrahigh-temperature Mycobacterium bovis, Salmonella, (UHT) pasteurization – Streptococcus, Listeria, and Brucella, but 134ºC for 1 second. not to sterilize the milk.  Inspissation.  Done between 75°C to 80°C for for 30 min for 3 successive days. It is done in 'Serum Inspissator'.  Used for sterilization of media containing eggs. Day: Temperature: Time: Purpose: Drying of the medium and killing 1 85 °C 60 minutes the organisms in their vegetative form Growth of vegetative forms from Time in between overnight incubation spores killing the organisms in their 2 75 to 80 °C 20 minutes vegetative form Growth of vegetative forms from Time in between overnight incubation any spores remaining killing the organisms in their 3 75 to 85 °C 20 minutes vegetative form as well as the leftover spores  AUTOCLAVING:  Autoclave is a container in which water is boiled above normal steam temp.  This is done by placing pressure upon the steam, so when pressure i-e 15 lb /15min /inches squares is applied the temp reaches 121 OC  At this temp all microorganism with their spores are completely destroyed.  USES:  Standard sterilization method in hospitals  Highly resistant spores of Cl. botulinum are completely destroyed  Sterilize culture media.  Glass ware  Surgical instruments like scalpel, forceps and dressings.  Q.control: By Geobacillus stearothermophilus  Advantages: Most commonly used, effective method of sterilization.  Sterilization cycle time is shorter than with dry heat or chemical sterilants.  Disadvantages:  Requires a continuous source of heat.  Requires equipment (steam sterilizer), which must be expertly maintained to keep it in working condition.  Requires strict adherence to time, temperature and pressure settings.  Plastic items cannot withstand high temperatures. Filteration: Filtration helps to remove bacteria from large volumes of fluid, especially fluid containing heat- labile components such as sera, solution of sugars and antibiotics. physically trapping particles larger than the pore size via electrostatic attraction of the particles to the filters A pore size of 0.22 micrometer is effective because filters act not only mechanically but by electrostatic adsorption of particles to their surface.(nitrocellulose) The filters are of two types (deep and membrane)  FILTERS DEEP FILTERS Made up of Examples Comments and Uses Candle filters 1.Diatomaceous earth Berkefeld filters They are available in different porosity. 2.Unglazed porcelain Chamberlain filters Mostly used for the purification of drinking water and industrial uses. Asbestos filters Asbestos (magnesium Seitz and Sterimat filters They have high adsorbing capacity and silicate) tend to alkalinize the filtered fluid. These are disposable , single use discs available in different grades. Asbestos is carcinogenic hence its use is discouraged. Sintered glass Fusing finely powdered They are expensive and brittle. filters glass particles They have low absorptive property. MEMBRANE Cellulose esters They are routinely used for water FILTERS Other polymers purification and analysis, sterilization, sterility testing and solutions of parenteral use. Wide range of average pore diameters(APD) are available but 0.2mm size is widely used.  Radiation:  ultraviolet (UV) light and x-rays.  UV light at 250-260 nm.  Formation of thymine dimers.  DNA replication is inhibited and the organism cannot grow.  can damage the cornea and skin  used in hospitals to kill airborne organisms, especially in operating rooms when they are not in use  Spores resistant  X-rays  higher energy and penetrating power than UV radiation.  production of free radicals, e.g., production of hydroxyl radicals by the hydrolysis of water.  Break covalent bonds in DNA.  X-rays are used in medicine for sterilization of :  heat-sensitive items, such as sutures and surgical gloves, and plastic items, such as syringes  RADIATION Non Ionisizing radiation: Types Produced by Mode of action Uses Comments Low energy (Infrared UV lamps DNA damage Infrared rays: Use of UV light is and ultraviolet rays) Sterilization of prepacked items limited by such as syringes and catheters. penetration and Ultraviolet rays: hazardous. Used for disinfecting operation theaters and laboratories. Ionisizing radiation: High energy ionizing Cobalt-60 based DNA damage For the sterilization of They are expensive. type (Gamma rays instruments antibiotics, hormones and They are very effective and high energy other pre packed disposable due to high penetrative electrons such as X- items such as catheters, gloves, power. rays and cosmic syringes, infusion sets, oils, rays) animal feeds, etc.  CHEMICAL METHODS. Mechanism of action of chemical disinfectants : The mechanism of action of most of the chemicals are nonspecific and complex but most of them effect microorganisms by one of the following mechanisms. 1. Cell membrane injury. 2. Coagulation and Denaturation. 3. Interactions with functional groups of proteins.  Modes of action Disruption of free sulfhydryl Modification of Nucleic acids. groups Disruption of Denaturation of cell Proteins. membranes.  DISRUPTION OF CELL MEMBRANES Alcohol:-  clean the skin before immunization or venipuncture.  Act by denaturing bacterial proteins.  Solutions of 70% ethanol are more effective than higher concentrations, as the presence of water speeds up the process of protein denaturation.  Frequently used for skin antisepsis prior to needle puncture.  Isopropyl alcohol is preferred as it is a better fat solvent, more bactericidal and less volatile.  Used for disinfection of clinical thermometer. Detergents:- * "surface-active" agents composed of a long-chain, lipid- soluble, hydrophobic portion and a polar hydrophilic group, which can be a cation, an anion, or a nonionic group. * interact with the lipid in the cell membrane through their hydrophobic chain and with the surrounding water through their polar group and thus disrupt the membrane. Quaternary ammonium compounds, e.g., benzalkonium chloride, are cationic detergents widely used for skin antisepsis Phenols * Phenol was the first disinfectant used in the operating room (by Lister in the 1860s), but it is rarely used as a disinfectant today because it is too caustic. * Hexachlorophene → germicidal soaps, (neurotoxicity has limited its use.) * Chlorohexidene is nontoxic, hence used as skin antiseptic (surgical scrub) which is more effective against Gram positive organisms than Gram negative organisms. * Phenols not only damage membranes but also denature proteins.  MODIFICATION OF PROTEINS Chlorine:- * disinfectant to purify the water supply and to treat swimming pools. * Active component of hypochlorite (bleach, Clorox), which is used as a disinfectant in the home and in hospitals. * Chlorine is a powerful oxidizing agent that kills by cross-linking essential sulfhydryl groups in enzymes to form the inactive disulfide.. Iodine: * most effective skin antiseptic used in medical practice and should be used prior to obtaining a blood culture and installing intravenous catheters because contamination with skin flora such as Staphylococcus epiderrnidis can be a problem. * Iodine is supplied in two forms: (1) Tincture of iodine (2% solution of iodine and potassium iodide in ethanol) : used to prepare the skin prior to blood culture.(should be removed with alcohol) (2) lodophors are complexes of iodine with detergents that are frequently used to prepare the skin prior to surgery because they are less irritating than tincture of iodine. Heavy Metals: * Metals Mercury and silver : greatest antibacterial activity & most widely used in medicine. * Thimerosal (Merthiolate) and merbromin (Mercurochrome), which contain mercury, are used as skin antiseptics. * Silver nitrate drops are useful in preventing gonococcal ophthalmia neonatorum. * Silver sulfadiazine is used to prevent infection of burn wounds. * They act by binding to sulfhydryl groups, thereby blocking enzymatic activity.  Brass is an alloy of copper and zinc.  storage of river water in brass containers as a way to prevent disease.  The river water may have up to 1 million fecal bacteria per ml. That count could be reduced to undetectable by 2 days of storage in a brass container! 4. Hydrogen Peroxide: * Hydrogen peroxide is used as an antiseptic to clean wounds and to disinfect contact lenses. * Its effectiveness is limited by the organism's ability to produce catalase, an enzyme that degrades H202. * The bubbles produced when peroxide is used on wounds are formed by oxygen arising from the breakdown of H202 by tissue catalase. * Hydrogen peroxide is an oxidizing agent that attacks sulflnydryl groups, thereby inhibiting enzymatic activity.  5. Formaldehyde & Glutaraldehyde: * Formaldehyde, which is available as a 37% solution in water (Formalin), denatures proteins and nucleic acids.  A broad-spectrum antimicrobial agent, used for disinfection, has limited sporicidal activity.  Hazardous substance, inflammable and irritant to the eye, skin and respiratory tract. * Glutaraldehyde, is 10 times more effective than formaldehyde and is less toxic. * In hospitals, it is used to sterilize respiratory therapy equipment. 6. Ethylene Oxide: * Ethylene oxide gas is used extensively in hospitals for the sterilization of heat-sensitive materials such as surgical instruments and plastics. *Mechanism of action: It destroys micro-organisms by alkylation and cause denaturation of nucleic acids of micro-organisms. * Highly penetrating gas with sweet ethereal smell. * Highly inflammable & in conc. greater than 3%, highly explosive.  MODIFICATION OF NUCLEIC ACIDS  A variety of dyes not only stain microorganisms but also inhibit their growth.  One of these is crystal violet (gentian violet), which is used as a skin antiseptic.  Its action is based on binding of the positively charged dye molecule to the negatively charged phosphate groups of the nucleic acids.  Malachite green, a triphenylamine dye like crystal violet, is a component of Lowenstein- Jensen's medium, which is used to grow M. tuberculosis.  The dye inhibits the growth of unwanted organisms in the sputum during the 6-week incubation period. ss Clinical Use: ss Commonly used disinfectant or sterilization methods: Disinfect surgeon’s hands prior to surgery Chlorohexidine Disinfect surgical site prior to surgery Iodophor Disinfect skin prior to venipuncture 70% ethanol Disinfect skin prior to blood culture Tincture of iodine followed by 70% ethanol, chlorohexidine, iodophor Cleanse wounds Chlorohexidine,Thimerosal,hydrogen peroxide Cleans burn wounds Silver sulfadiazine Sterilize non heat sensitive materials(eg. Drapes, surgical Autoclave gowns) Sterilize intravenous solutions Filtration Disinfect air in operating room (when not in use) Ultraviolet light Disinfect floor of operating use Benzalkonium chloride (Lysol) Disinfect stethoscope 70% ethanol Preservative in vaccines Thimerosal Clean up of blood spill from a patient with hepatitis B or C ( Hypochlorite (bleach, clorox) disinfect area)  RULES FOR HANDLING CORROSIVE MATERIALS Use proper eye, hand and body protection Prevent exposure to corrosive fumes and vapours PRECAUTIONARY Always add acid to water, MEASURES never reverse Pour Chemicals properly Infection Control Precautions and Practices