STERILIZATION AND DISINFECTION Definition • Sterilization: Process by which all living microorganisms including viable spores, are either destroyed or removed from an article, body surface or medium. o Reduction of at least 106 log CFU of microorganisms. • Disinfection- Process that destroys or removes most if not all pathogenic organisms but not bacterial spores. o Reduction of at least 103 log CFU of microorganism. Definition • Asepsis- Process where the chemical agents (called antiseptics) applied to body surfaces (skin) will kill or inhibit the pathogenic microorganisms (and also commensals) present on skin. • Decontamination (or sanitization)- Reduction of pathogenic microbial population to a level at which items are considered as safe to handle without protective attire. Factors influencing efficacy of sterilant/ disinfectant 1. Organism load: Larger microbial population requires a longer time to be destroyed. 2. Nature of organisms: oPrions (highest resistance) > Cryptosporidium oocysts > Bacterial spores > Mycobacteria > Other parasite cysts (e.g. Giardia) > Small non-enveloped viruses > Trophozoites > Gram-negative bacteria > Fungi > Large non-enveloped viruses > Gram-positive bacteria > Enveloped viruses. Factors influencing efficacy of sterilant/ disinfectant 3. Concentration of the chemical agent or the temperature of heat sterilization. 4. Nature of the sterilant /disinfectant 5. Duration of exposure 6. Temperature Factors influencing efficacy of sterilant/ disinfectant 7. Local environment o Heat kills more readily at an acidic pH. o Biofilm are also a good example that prevents the entry of disinfectants to act on the microorganisms that are embedded inside the biofilm. Classification of sterilization methods 1.Sunlight 3.Heat 2.Drying Dry heat- Flaming, Incineration, Hot air oven Moist heat• Temperature below 1000C- pasteurization, water bath and inspissation • Temperature at 1000C- e.g. boiling, steaming and tyndallisation • Temperature above 1000C- e.g. autoclave 4.Filtration – depth filters and membrane filters 5.Radiation Ionizing radiation-γ rays, X-rays and cosmic rays Non-ionizing radiation- Ultraviolet (UV) and infrared rays 6. Ultrasonic vibration Classification of disinfection methods 1. Alcohols- Ethyl alcohol, isopropyl alcohol 2. Aldehydes- Formaldehyde, glutaraldehyde, Ortho-phthalaldehyde 3. Phenolic compounds- Cresol, lysol, chlorhexidine, chloroxylenol, hexachlorophene 4. Biguanide: Chlorhexidine gluconate 5. Halogens- Chlorine, iodine, iodophors 6. Oxidising agents- Hydrogen peroxide , Peracetic acid 7. Heavy metal salts- Mercuric chloride, copper salts 8. Surface active agents- Quaternary ammonium compounds and soaps 9. Dyes- aniline dyes and acridine dyes 10. Gas sterilization• Low temperature steam formaldehyde • Ethylene oxide (ETO) • Betapropiolactone (BPL) • Plasma sterilization PHYSICAL AGENTS OF STERILIZATION/ DISINFECTION Method Principle Sunlight Active microbicidal effect due to its content of ultraviolet rays. Drying 70-80% of the weight of the bacterial cell is due to water. Drying, therefore has a deleterious effect on many bacteria. Dry heat Kills the organisms by charring, denaturation of bacterial protein, oxidative damage and by the toxic effect of elevated levels of electrolytes. Moist heat Kills the microorganisms by denaturation and coagulation of proteins. Dry heat sterilization Flaming Items are held in the flame of a Bunsen burner either for long time or short time. o Longer time exposure - for inoculating wires or loops, tips of forceps. o For shorter period - mouth of test tubes. Incineration Used for the disposal of waste materials. o It burns (sterilizes) the anatomical waste and microbiology waste by providing a very high temperature 870 to 1,200°C - converting the waste into ash, flue gas, and heat. Hot air oven (Dry heat Sterilizer) • Most widely used method of sterilization by dry heat. • It is electrically heated and is fitted with a fan to ensure adequate and even distribution of hot air in the chamber. • It is also fitted with a thermostat which maintains the chamber air at a chosen temperature. • Holding temperature and time of 160°C for 2 hours. Hot air oven (Dry heat Sterilizer) • o o o Materials sterilized: Glassware - syringes, petri dishes, flasks, pipettes and test tubes. Surgical instruments - scalpels, forceps, etc. Chemicals such as liquid paraffin, fats, glycerol, oil, and glove powder, etc. Hot air oven - precautions • Overloading of hot air oven should be avoided. • Material should be arranged in a manner so that free circulation of air is maintained. • Material to be sterilized should be dried completely. • Cotton plugs should be used to close the mouths of test tubes, flasks, etc. • Paper wrapping of the items should be done. • Any inflammable material like rubber (except silicone rubber) should not be kept inside the oven. • Oven must be allowed to cool for two hours before opening the doors, since the glassware may crack by sudden cooling. Hot air oven - Sterilization control • Biological indicator: Spores (106) o Bacillus atrophaeus - to check the effectiveness of sterilization by dry heat. Earlier, non-toxigenic strain of Clostridium tetani was used. o Spores should be destroyed if the sterilization is done properly. • Physical control: For example, digital displays on the equipment displaying temperature and time. Moist Heat sterilization Below 100°C Pasteurization • Method used for control of microorganisms from beverages like fruit and vegetable, juices, beer, and dairy products, such as milk. o Holder method (63°C for 30 mins). o Flash method (72°C for 20 seconds followed by rapid cooling to 13° or lower). o All nonsporing pathogens, including mycobacteria, brucellae and salmonellae are killed except Coxiella burnetii which being relatively heat resistant may survive in holder method. Moist Heat sterilization Below 100°C Water bath • Used for disinfection of serum, body fluids and vaccines. • Bacterial vaccines are disinfected at 60°C for 1 hour. • Serum or heat labile body fluids can be disinfected at 56°C for one hour. Moist Heat sterilization Below 100°C Inspissation (fractional sterilization) • Process of heating an article on 3 successive days at 80–85Ë C for 30 minutes. • Working principle: o first exposure - kills all the vegetative forms. o Intervals between the heating the remaining spores germinate into vegetative forms which are then killed on subsequent heating. • Uses: Sterilization egg-based medium – Lowenstein Jensen medium and Dorset’s egg medium; serum-based media— Loeffler’s serum slope. Moist Heat sterilization Moist heat at a temperature at 100°C Boiling • Koch’s or Arnold’s steam sterilizer • useful for those media which are decomposed at high temperature of autoclave. • Articles exposed to steam (100°C) at atmospheric pressure for 90 minutes. Most of the vegetative forms are killed by this method except thermophiles and spores. Boiling of the items in water for 15 minutes kills most of the vegetative forms. • But do not kill the spores - not suitable for sterilization of surgical instruments. Moist Heat sterilization at 100°C Tyndallisation or intermittent sterilization (named after John Tyndall) • Involves steaming at 1000C for 20minutes for 3 consecutive days. • Principle is similar to that of inspissation, except that here, the temperature provided is 1000C, instead of 800C. • Used for sterilization of gelatin and egg, serum or sugar containing media which are damaged at higher temperature of autoclave. Moist Heat sterilization above 100°C • Principle of Autoclave: o Autoclave functions similar to a pressure cooker and follows the general laws of gas. o Water boils when its vapour pressure equals that of the surrounding atmosphere. o When the atmospheric pressure is raised, the boiling temperature is also raised. o At normal pressure, water boils at 100°C but when pressure inside a closed vessel increases, the temperature at which water boils also increases. Components of Autoclave • Autoclave comprises of three parts  Pressure chamber Lid Electrical heater. Horizontal autoclave and Vertical autoclave Sterilization Conditions • Autoclave can be set to provide higher temperatures by adjusting the pressure provided to the vessel. o 121⁰C for 15 minutes at pressure of 15 lbs psi. o 126 ⁰ C for 10 minutes at pressure of 20 psi. o 133 ⁰ C for 3 minutes at pressure of 30 psi. Uses of Autoclave • • • • • • • Surgical instruments Culture media Autoclavable plastic containers Plastic tubes and pipette tips Solutions and water Biohazardous waste Glassware (autoclave resistible). Precautions to be taken • Autoclave should not be used for sterilizing waterproof materials • Materials are loaded in such a way that it allows efficient steam penetration (do not overfill the chamber) • Material should not touch the sides or top of the chamber • Clean items and the wastes should be autoclaved separately • olyethylene trays should not be used as they may melt and cause damage to the autoclave. Types of Autoclaves • Gravity displacement type autoclave: most commonly used. • Positive pressure displacement type autoclave • Negative pressure (vacuum) displacement type. Sterilization control • Biological indicator: Spores of Geobacillus stearothermophilus (formerly called Bacillus stearothermophilus). Spores are killed in 12 minutes at 121°C • Chemical indicators o Class I - external pack control, (e.g. autoclave tape) o Class II - equipment control (Bowie-Dick test) o Class IV/V - internal pack control. • Physical control: For example, digital displays on the equipment displaying temperature, time and pressure. Filtration • Filtration is an excellent way to remove the microbial population in solutions of heat-labile materials like vaccine, antibiotics, toxin, serum and sugar solution. • Types of Filters 1. Depth Filters. 2. Membrane Filters Depth Filters • Depth filters are porous filters composed of random mats of metallic, polymeric, or inorganic materials. • Advantages - Retain a large mass of particles before becoming clogged, flow rate of the fluid is high and low cost • Disadvantages- Not suitable for filtration of solution containing bacteria. • Depth filters - candle filters (Berkefeld filters), unglazed porcelain (Chamberlain filters),asbestos filters (Seitz and Sterimat filters) and sintered glass filters Membrane Filters • • • • Most widely used filters for bacterial filtration. Porous; retain all the particles on the surface that are smaller than their pore size. Composed of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials Pore size: o 0.22 µm - Most commonly used (removes most of the bacteria, allowing the viruses to pass through) o 0.45 µm - Used to retain coliforms. o 0.8 µm - Used to remove airborne microorganisms in clean rooms. Filtration of Liquid • To sterilize sera, sugar and antibiotic solutions. • Separation of toxins and bacteriophages from bacteria. • To obtain bacteria free filtrates of clinical samples for virus isolation • Purification of water—e.g. testing of water samples for Vibrio cholera or typhoid bacilli. Filtration of Air • Simple e.g. - surgical masks (that allow air in but keep microorganisms out). • Two important air filters that are used in biological safety cabinets and laminar airflow systems): o HEPA filters (High-efficiency particulate air filters) removes 99.97% of particles that have a size of 0.3 µm or more. o ULPA filters (Ultra-low particulate/penetration) removes from the air at least 99.999% of dust, pollen, mold, bacteria and any airborne particles with a size of 0.12 µm or larger. Sterilization Control • The sterilization control of membrane filters includes Brevundimonas diminuta and Serratia marcescens. Ionizing radiation (cold sterilization) • • • • • Include, X-rays, gamma rays (from Cobalt 60 source), and cosmic rays. Mechanism: It causes breakage of DNA without temperature rise. Uses o Disposable rubber or plastic syringes, infusion sets and catheters. o Catgut sutures, bone and tissue grafts and adhesive dressings as well as antibiotics and hormones. o Irradiation of food (permitted in some countries). Advantages of ionizing radiation: o High penetrating power, Rapidity of action and Temperature is not raised Sterilization control: Efficacy of ionizing radiation is tested by using Bacillus pumilus. Non-ionizing Radiation • Includes infrared and ultraviolet radiations. • Lethal but do not penetrate glass, dirt films, water; hence their use is restricted. • Recommended dose - 250–300 nm wavelength of UV rays, for 30 minutes • Uses – For disinfection of clean surfaces in operation theaters, laminar flow hoods as well as for water treatment. • Because UV radiation burns the skin and damages eyes, hence the area should be closed and UV lamps must be switched off immediately after use. Ultrasound (Ultrasonic) Waves • High-frequency ultrasonic and sonic sound waves disrupt bacterial cells. • Not reliable, hence is not used now a days.