sterilization and disinfection.pptx

Sterilization and Disinfection Why we need Sterilization? ■ ■ ■ Microorganisms capable of causing infection are constantly present in the external environment and on the human body Microorganisms are responsible for contamination and infection The aim of sterilization is to remove or destroy th...

Sterilization and Disinfection Why we need Sterilization? ■ ■ ■ Microorganisms capable of causing infection are constantly present in the external environment and on the human body Microorganisms are responsible for contamination and infection The aim of sterilization is to remove or destroy them from materials or from surfaces Definitions: Sterilization : – It is a process by which an article, surface or medium is made free of all microorganisms either in vegetative or spore form Disinfection : – Destruction of all pathogens or organisms capable of producing infections but not – necessarily spores. All organisms may not be killed but the number is reduced to a level that is no longer harmful to health. Antiseptics : ■ Chemical disinfectants which can safely applied to living tissues and are used to prevent infection by inhibiting the growth of microorganisms Asepsis : ■ Technique by which the occurrence of infection into an uninfected tissue is prevented. Instrumen t reprocessi ng cycle Categories of Patient-Care Items Methods 1. Physical methods 2. Chemical methods 5 Physical methods: ■ Physical methods: 1. Sunlight 2. Drying 3. Heat 1. Dry heat 2. Moist heat 4. Filtration 5. Radiation 6 Chemical methods • Chemical methods: 1. 3. 2. 4. 5. 6. 7. 8. 9. Alcohols Phenols Aldehydes Halogens Oxidizing agents Salts Surface active agents Dyes Vapor phase disinfectants Physical methods 1. Sunlight Sunlight possesses appreciable bactericidal activity Due to its content of ultraviolet rays & heat rays Under natural conditions, its sterilising power varies according to circumstances Natural method of sterilization of water in tanks, rivers and lakes 2. Drying Moisture is essential for the growth of bacteria Drying in air has a deleterious effect on many 3. Heat Most reliable method of sterilization The factors influencing sterilization by heat are: ❖ Nature of heat ❖ Temperature and time ❖ Number of microorganisms present ❖ Characteristics of the organisms, such as species, strain and sporing capacity ❖ Type of material from which the organisms have to be eradicated Mechanism of action Dry heat Kills organisms by protein denaturation, oxidative damage and toxic effects of elevated levels of electrolytes Moist heat Kills microorganisms by coagulation and denaturation of their enzymes and structural proteins ■ Dry heat: 1. Red heat 2. Flaming 3. Incineration 4. Hot air oven Red heat Materials are held in the flame of a bunsen burner till they become red hot. ■ Inoculating wires or loops ■ Tips of forceps ■ Surface of searing ■ spatulae Needles Flaming Materials are passed through the flame of a bunsen burner without allowing them to become red hot. ■ Glass slides ■ scalpels ■ Mouths of culture tubes and bottles Incineration: ■ Materials are reduced to ashes by burning. ■ Instrument used was incinerator. ■Soiled dressings ■Animal carcasses ■Bedding ■Pathological Hot air oven ➢ Most widely used method of sterilization by dry heat ➢ It is used to process materials which can withstand high temperatures, but which are likely to be affected by contact with steam ➢ It is a method of choice for sterilization of glassware, forceps, scissors, scalpels, swab sticks packed in test tubes ➢ Materials such as oils, jellies and powders which are impervious to steam are sterilized by hot air oven ➢ Hot air oven is electrically heated and is fitted with a termostat that maintains the chamber air at a Hot air oven Holding temperature & time General purpose Temperature and time: 1600C for 2 hours 1700C for 1 hour 1800C for 30 minutes Cutting instruments such as those used in ophthalmic surgery, Should be sterilized at 1500C for 2 hours Oils, glycerol and dusting powder should be 0 Precautions 1. 2. 3. 4. 5. 6. 7. Should not be overloaded Arranged in a manner which allows free circulation of air Material to be sterilized should be perfectly dry. Test tubes, flasks etc. should be fitted with cotton plugs. petridishes and pipetts should be wrapped in paper. Rubber materials and inflammable materials should not be kept inside. The oven must be allowed to cool for two hours before opening, since glass ware may crack by sudden cooling. Uses of Hot Air Oven ■ Sterilization 1. 2. 3. of Glassware like glass syringes, petri dishes, pipettes and test tubes. Surgical instruments like scalpels, scissors, forceps etc. Chemicals like liquid paraffin, fats etc. Sterilization controls 1. Spores of Bacillus subtilis subsp. Niger 2. Thermocouples 3. Browne’s tube (Tube containing red colour solution is inserted in each load and a colour change from red to green indicates proper Moist heat Moist heat is divided into three forms 1.Temperature below 1000C 2. At a temperature of 1000C 3.Temperature above 1000C Temperatures below 1000C 3. Pasteurization 4. Vaccine bath 5. Water bath Pasteurizati on ➢ Milk is sterilised by this method; Two methods 1.Holder method (630C for 30 min followed by rapid cooling to 130C or lower) 2. Flash method (720C for 15-20 seconds followed by rapid cooling to 130C or lower) ➢ The dairy industry sometimes uses ultrahightemperature (UHT) sterilization (140 to 1500C for 1-3 seconds followed by rapid cooling to 130C or lower) ➢ All nonsporing pathogens such as mycobacteria, brucellae and salmonellae are destroyed by these processes ➢ Coxiella burnetii is relatively heat resistant and may Principle of Pasteurization 03/09/1 4 2 4 Dr.T.V.Rao MD Vaccine bath Vaccines prepared from nonsporing bacteria may be inactivated in a water bath at 600C for 1 hour Water bath Serum or body fluids containing coagulable proteins can be sterilized by heating for 1 hour at 560C on several successive days Inspissati on Media containing egg or serum such as LowensteinJensen and Loeffler’s serum slope are rendered sterile by heating at 80-850C for 30 min on three successive days This process is called inspissation and instrument used is called inspissator LTSF sterilization Used for sterilizing items which cannot withstand the temperatue of 1000C In this method steam at 750C with formaldehyde vapor Inspissator Water bath Temperature at 100°C 1. Boiling 2. Tyndallisation 3. Steam sterilisation Boiling at 1000C ❑ Boiling at 1000C for 10-30 min kills all vegetative bacteria and some bacterial spores ❑ Sporing bacteria required prolonged periods of boiling ❑ Therefore, it is not recommended for sterilization of surgical instruments ❑ Addition of 2% sodium bicarbonate may promote sterilization Uses ❑ For the disinfection of medical and surgical equipment – when sterility is not essential in emergency or under Free steam at 1000C ❖ Steam at normal atmosheric pressure is at 1000C ❖ Used to sterilize heat-labile culture media ❖ A Koch or Arnold steam sterilizer is used ❖ It consists of a vertical metal cylinder with a removable conical lid ❖ Single exposure to steam for 90 min ensures complete sterilization Tyndallisat ion An exposure of steam1000C for 20 min on three consecutive days is known as Tyndallization or intermittent sterilisation The instrument used is Koch or Arnold steam sterilizer Principle First exposure kills all the vegetative forms, and in the intervals between the heatings the remaining spores germinate into vegetative forms which are killed on subsequent heating Uses Koch or Arnold steam sterilizer Temperature above 1000C Steam under pressure Saturated steam is more efficient sterilizing agent than hot air because 1. It provides greater lethal action of moist heat 2. It is quicker in heating up the exposed articles 3. It can easily penetrate porous material 4. When the steam meets the cooler surface of the article, it condenses into a small volume of water and liberates considerable latent heat Autocla ve Principle ➢ Water boils when its vapor pressure equals that of the surrounding atmosphere ➢ When pressure inside a closed vessel increases, the temperature at which water boils also increases ➢ Saturated steam has penetrative power and is a better sterilizing agent than dry heat ➢ Steam condenses to water and gives up its latent heat to the surface when it comes into contact with a cooler surface ➢ The large reduction in volume, sucks in more steam to the area and the process continues till the Components of autoclave: ■ Consists of vertical or horizontal cylinder of gunmetal or stainless steel. ■ Lid is fastened by screw clamps and rendered air tight by an asbestos washer. ■ Lid bears a discharge tap for air and steam, a pressure gauge and a safety valve. Holding period 1210C for 15 min (15 lbs or psi pressure) Precautio ns 1. All the air must be removed from the autoclave chamber ✓The admixture of air with steam results in low temperature being achieved ✓Air hinders penetration of steam ✓The air being denser forms a cooler layer in the lower part of the autoclave 2. Materials should be arranged in such a manner which ensures free circulation of steam inside the chamber 3. Lid should not open until inside pressure Uses : 1. 2. 3. To sterilize culture media, rubber material, gowns, dressings, gloves, instruments and pharmaceutical products For all materials that are water containing, permeable or wettable and not liable to be damaged by the process Useful for materials which cannot withstand the high temperature of hot air oven Sterilization controls 1. 2. Thermocouples Bacterial spores- B a c i l u s st earothermophil us 3. 4. Browne’s tube Autoclave tapes Filtration Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents Used for the sterilization of heat labile materials such as sera, sugar solutions, and antibiotics Types of Filters 1.Earthenware filters (Candle filters) 2. Asbestos disc (Seitz) filters 3.Sintered glass filters 4.Membrane Earthenware filters ➢ Manufactured in several different grades of porosity ➢ Used widely for purification of water for industrial and drinking purposes ➢ They are of two types 1. Unglazed ceramic filters eg: Chamberland and Doulton filters 2. Compressed diatomaceous earth filters eg: Berkefeld and Mandler filters Earthenware (Candle) filters Asbestos filters ➢ Made up of a disc of asbestos (magnesium trisilicate) ➢ Discs are available with different grades of porosity ➢ It is supported on a perforated metal disc within a metal funnel ➢ It is then fitted onto a sterile flask through a silicone rubber bung ➢ The fluid to be sterilized is put into the funnel and flask connected to the exhaust pump through its side tap Asbestos Filter holder Sintered glass filters ➢ Prepared by fusing finely powdered glass particles ➢ Available in different pore sizes ➢ Pore size can be controlled by the general particle size of the glass powder ➢ The filters are easily cleaned, have low absorption properties and do not shed particles ➢ But they are fragile and relatively expensive Sintered glass filter Membrane filters ❑ Made of variety of polymeric materials such as cellulose nitrate, cellulose diacetate, polycarbonate and polyester ❑ Membrane filters are available in pore sizes of 0.015 to 12 µm ❑ The 0.22 µm filter is most commonly used because the pore size is smaller than that of bacteria ❑ These are routinely used in water analysis, bacterial counts of water, sterility testing, and for the preparation of solutions for parenteral use Membrane filters Syringe filters ✓Syringes fitted with membrane filters of different pore sizes are available ✓For sterilization, the fluid is forced through the the disc (membrane) by pressing the piston of the syringe Air filters ➢ Air can also be sterilized by filtration ➢ Large volumes of air may be rapidly freed from infection by passage through high efficiency particulate air (HEPA) filters ➢ They are used in laminar air flow system in microbiology laboratories ➢ HEPA filters can remove particles of 0.3 µm or larger Radiation Two types of radiations are used for sterilization 1. Nonionising 2.Ionising Nonionising radiatio ns ➢ These inculde ➢ It acts by denaturation of bacterial protein and interference with DNA replication (produces thymine dimers) ➢ They can penetrate only a few mm into liquids and not at all into solids ➢ UV radiation is used for disinfecting enclosed areas such as bacteriological laboratory, inoculation hoods, laminar flow and operation theatres ➢ Most vegetative bacteria are susceptible but spores are highly resistant ➢ Susceptibility of viruses is variable ➢ Source of UV radiations must be shielded Ionising radiations ❑ These include X-rays, Υ (gamma) rays and cosmic rays ❑ Possess high penetrative power and are highly lethal to all cells including bacteria ❑ They damage DNA by various mechanisms ❑ Gamma radiations are used for sterilization of disposable items such as plastic syringes, swabs, culture plates, cannulas, catheters etc ❑ Since theres is no appreciable increase in the temperature, in this method it is known as “cold sterilization” ❑ Large commercial plants use gamma radiation emitted from a radioactive element, usually cobalt 60 ❑ The advantage of this method include speed, high penetrating power (it can sterilise materials through outer packages and wrappings) ❑ Bacillus pumilis used to test the efficacy of ionizing Chemical A methods variety of chemical agents are used as antiseptics and disinfectants. An ideal antiseptic or disinfectant should 1) Be fast acting in presence of organic substances 2) Be effective against all types of infectious agents without destroying tissue or acting as a poison if ingested 3) Easily penetrate material to be disinfected, without damaging/discoloring it 4) Be easy to prepare, stable when exposed to light, heat or other environ-mental factors 5) Be inexpensive, easy to obtain and use 6) Not have an unpleasant odor Chemical agents act in various ways. The main modes of action are 1. Protein coagulation 2. Distruption of cell membrane 3. Removal of free sulphydryl groups 4. Substrate competition Factors that determine the potency of disinfectants are a) Concentration of the substance b) Time of action c) pH of the medium d) Temperature e) Nature of organism f) Presence of organic matter Disinfectants can be divided into three groups 1.High level disinfectants (Glutaraldehyde, hydrogen peroxide, peracetic acid and chlorine compounds) 2. Intermediate level disinfectants (Alcohol, iodophores and phenolic compounds) 3. Low level disinfectants (Quarternary ammonium compounds) Alcohol s ➢ Ethanol and isopropanol are the most frequently used ➢ Used as skin antiseptics and act by denaturing bacterial proteins ➢ Rapidly kill bacteria including tubercle bacilli but they have no sporicidal or virucidal activity ➢ 60-70% is most effective ➢ Isopropyl alcohol is preferred to ethyl alcohol as it is a better fat solvent, more bactericidal and less volatile Aldehyde s Two aldehydes (formaldehyde and glutaraldehyde) are currently of considerable importance Formaldehyde ➢ Formaldehyde is active against the aminogroup in the protein molecules ➢ It is lethal to bacteria and their spores, viruses and fungi ➢ It is employed in the liquid and vapor states Uses ➢ To sterilise bacterial vaccines ➢ 10% formalin containing 0.5% sodium tetraborate is used to sterilize clean metal instruments ➢ Formaldehyde gas is used for sterilizing instruments, heat sensitive catheters and for fumigating wards, sick rooms and laboratories Glutaraldeh yde ➢ Action ➢ More similar to formaldehyde active and less toxic than formaldehyde ➢ It is used as 2% buffered solution ➢ It is available commercially as ‘cidex’ Uses ➢ For sterilization of cystoscopes, endoscopes and bronchoscopes ➢ To sterilize plastic endotracheal tubes, face masks, corrugated rubber anaesthetic tubes and metal Pheno ls ➢ Obtained by distillation of coal tar between temperatures of 1700C and 2700C ➢ Lethal effect is due to cell membrane damage ➢ Phenol (1%) has bactericidal action ➢ Phenol derivatives like cresol, chlorhexidine and hexachlorophane are commonly used as antiseptics Cresols ❑ Lysol is a solution of cresols in soap ❑ Most commonly used for sterilization of infected Chlorhexidine ➢ Savlon (Chlorhexidine and cetrimide) is widely used in wounds, pre-operative disinfection of skin ➢ More active against Gram positive than Gram negative bacteria ➢ No action against tubercle bacilli or spores and have very little activity against viruses ➢ Has a good fungicidal activity Haloge ns and iodine are two commonly used disinfectants ➢ Chlorine ➢ They are bactericidal and are effective against sporing bacteria and viruses ➢ Chlorine is used in water supplies, swimming pools, food and dairy industries ➢ Chlorine is used in the form of bleaching powder, sodium- hypochlorite and chloramine ➢ Hypochlorites have a bactericidal, fungicidal, virucidal and sporicidal action ➢ Bleaching powder or hypochlorite solution are the most widely used for HIV infected material ➢ Chloramines are used as antiseptics for dressing wounds Iodi ne ➢ Iodine in aqueous and alcoholic solution used as skin disinfectant ➢ Iodine often has been applied as tincture of iodine (2% iodine in a water-ethanol solution of potassium iodide) ➢ Actively bactericidal, moderate action against spores ➢ Also active against the tubercle bacteria and viruses ➢ Compounds of iodine with surface active agents known as iodophores ➢ Used in hospitals for preoperative skin degerming ➢ Povidine-iodine (Betadine) for wounds and Wescodyne for skin and laboratory disinfection are some examples of iodophores Dyes ➢ Aniline and acridine dyes are used extensively as skin and wound antiseptics ➢ Aniline dyes include crystal violet, brilliant green, and malachite green organisms are more active against gram positive ➢ No activity against tubercle bacilli ➢ They interfere with the synthesis of peptidoglycan of the cell wall ➢ Their activity is inhibited by organic material such as pus ➢ Acridine dyes also more active against gram positive organisms ➢ More important dyes are proflavine, acriflavine, euflavine and aminacrine ➢ They interfere with the synthesis of nucleic acids and proteins in bacterial cells Metallic salts ➢ Salts of silver, copper and mercury are used as disinfectants ➢ Protein coagulants and have the capacity to combine with free sulphydryl groups ➢ The organic compounds thiomersal, phenyl mercury nitrate and mercurochrome are less toxic and are used as mild antisepics Surface active agents Substances which alter energy relationships at interfaces, producing a reduction of surface tension; 4 types 1.Anionic Common soaps, have strong detergent but weak antimicrobial properties 2. Cationic Act on phosphate group of the cell membrane Eg: Quaternary ammonium compounds such as benzalkonium chloride and acetyl trimethyl ammonium bromide (cetrimide) 3. Nonionic Vapour Phase Disinfectants Formaldehyde gas ➢ Employed for fumigation of heat-sensitive equipment (anaesthetic machine and baby incubators), operation theatres, wards and laboratories etc ➢ Formaldehyde gas is generated by adding 150 gm of KMnO4 to 280 ml of formalin for 1000 cubic feet of room volume ➢ This reaction produces considerable heat and so heat resistant containers should be used ➢ Sterilisation is achieved by condensation of gas on exposed surfaces ➢ After completion of disinfection, the effect of irritant Ethylene oxide (ETO) ❑ Colourless liquid with a boiling point of 10.70C ❑ Highly lethal to all kinds of microbes including spores ❑Action is due to its alkylating the amino, carboxyl, hydroxyl and sulphydryl groups in protein molecules ❑ In addition it reacts with DNA and RNA ❑ Highly inflammable and in concentrations (>3%) highly explosive ❑ By mixing with inert gases such as CO2, its explosive Uses ❑ Used for sterilising plastic and rubber articles, respirators, heart-lung machines, sutures, dental equipments and clothing ❑ It is commercially used to sterilise disposable plastic syringes, petridishes etc ❑ It has a high penetrating power and thus can sterilise prepackaged materials ❑ Bacillus globigi ( a red pigmented variant of B. subtilis) has been used as a biological control fot testing of ETO sterilisers Betapropiolactone (BPL) ➢ Condensation product of ketane and formaldehyde ➢ Boiling point: 1630C ➢ Has low penetrating power but has a rapid action ➢ For sterilization of biological products 0.2% BPL is used ➢ Capable of killing all microorganisms and is very active against viruses Uses In the liquid form it has been used to sterilize vaccines and sera Recent vapor phase disinfectants Hydrogen peroxide ➢ Used to decontaminate biological safety cabinets Peracetic acid ➢ It ia an oxidising agent ➢ One of the high level disinfectants ➢ Used in plasma sterilizers Testing of disinfectants 1. Minimum inhibitory concentration (MIC) 2. Rideal Walker test 3. Chick Martin test 4. Capacity test (Kelsey and Sykes test) 5. In-use test Sterilisation of prions Dry heat 3600C for one hour Moist heat 134-1380C for 18 min Chemicals 25% sodium hypochlorite for one hour Sensitive to household bleach, phenol (90%) and iodine disinfectants Thank you !!!