Published on March 9, 2014
Sterilisation and Disinfection: Sterilisation and Disinfection Dr. Pendru Raghunath Reddy Why we need Sterilization?: 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 sterilisation is to remove or destroy them from materials or from surfaces Definitions:: Definitions: Sterilisation : 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. PowerPoint Presentation: 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. Methods : Methods Physical methods Chemical methods 5 Physical methods: : Physical methods: Physical methods: Sunlight Drying Heat Dry heat Moist heat Filtration Radiation 6 Chemical methods: Chemical methods Chemical methods: Alcohols Aldehydes Phenols Halogens Oxidizing agents Salts Surface active agents Dyes Vapor phase disinfectants PowerPoint Presentation: 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 sterilisation 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 bacteria This method is unreliable and is only of theoretical interest PowerPoint Presentation: 3. Heat Most reliable method of sterilisation The factors influencing sterilisation 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 PowerPoint Presentation: 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 PowerPoint Presentation: Dry heat: Red heat Flaming Incineration Hot air oven PowerPoint Presentation: 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 PowerPoint Presentation: 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: : Incineration: Materials are reduced to ashes by burning. Instrument used was incinerator. Soiled dressings Animal carcasses Bedding Pathological material PowerPoint Presentation: Hot air oven Most widely used method of sterilisation 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 sterilisation of glassware, forceps, scissors, scalpels, swab sticks packed in test tubes Materials such as oils, jellies and powders which are impervious to steam are sterilised by hot air oven Hot air oven is electrically heated and is fitted with a termostat that maintains the chamber air at a chosen temperature Fitted with a fan that distributes hot air in the chamber PowerPoint Presentation: Hot air oven PowerPoint Presentation: Holding temperature & time General purpose Temperature and time: 160 0 C for 2 hours 170 0 C for 1 hour 180 0 C for 30 minutes Cutting instruments such as those used in ophthalmic surgery, Should be sterilized at 150 0 C for 2 hours Oils, glycerol and dusting powder should be sterilised at 150 0 C for 1 hour PowerPoint Presentation: Precautions 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: Uses of Hot Air Oven Sterilisation of Glassware like glass syringes, petri dishes, pipettes and test tubes. Surgical instruments like scalpels, scissors, forceps etc. Chemicals like liquid paraffin, fats etc. Sterilisation controls : Sterilisation controls Spores of Bacillus subtilis subsp. Niger Thermocouples Browne’s tube (Tube containing red colour solution is inserted in each load and a colour change from red to green indicates proper sterilization) PowerPoint Presentation: Moist heat Moist heat is divided into three forms Temperature below 100 0 C At a temperature of 100 0 C Temperature above 100 0 C Temperatures below 100 0 C 1. Pasteurization 2. Vaccine bath 3. Water bath 4. Inspissation 5. Low temperature steam-formaldehyde (LTSF) sterilization PowerPoint Presentation: Pasteurization Milk is sterilised by this method; Two methods Holder method (63 0 C for 30 min followed by rapid cooling to 13 0 C or lower) 2. Flash method (72 0 C for 15-20 seconds followed by rapid cooling to 13 0 C or lower) The dairy industry sometimes uses ultrahightemperature (UHT) sterilization (140 to 150 0 C for 1-3 seconds followed by rapid cooling to 13 0 C or lower) All nonsporing pathogens such as mycobacteria , brucellae and salmonellae are destroyed by these processes Coxiella burnetii is relatively heat resistant and may survive the holder method Principle of Pasteurization: Principle of Pasteurization 3/9/2014 Dr.T.V.Rao MD 24 PowerPoint Presentation: Vaccine bath Vaccines prepared from nonsporing bacteria may be inactivated in a water bath at 60 0 C for 1 hour Water bath Serum or body fluids containing coagulable proteins can be sterilized by heating for 1 hour at 56 0 C on several successive days PowerPoint Presentation: Inspissation Media containing egg or serum such as Lowenstein-Jensen and Loeffler’s serum slope are rendered sterile by heating at 80-85 0 C 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 100 0 C In this method steam at 75 0 C with formaldehyde vapor is used PowerPoint Presentation: Inspissator Water bath Temperature at 100°C : Temperature at 100°C Boiling Tyndallisation Steam sterilisation PowerPoint Presentation: Boiling at 100 0 C Boiling at 100 0 C 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 field conditions PowerPoint Presentation: Free steam at 100 0 C Steam at normal atmosheric pressure is at 100 0 C 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 PowerPoint Presentation: Tyndallisation An exposure of steam100 0 C 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 Used for sterilisation of egg, serum or sugar containing media PowerPoint Presentation: Koch or Arnold steam sterilizer PowerPoint Presentation: Temperature above 100 0 C 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 PowerPoint Presentation: Autoclave 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 temperature of that surface is raised to that of the steam Components of autoclave: : 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 121 0 C for 15 min (15 lbs or psi pressure) PowerPoint Presentation: Precautions 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 reaches to the atmospheric pressure PowerPoint Presentation: Uses : 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 Sterilisation controls : Sterilisation controls Thermocouples Bacterial spores- Bacillus stearothermophilus Browne’s tube Autoclave tapes PowerPoint Presentation: 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: Types of Filters 1. Earthenware filters (Candle filters) 2. Asbestos disc (Seitz) filters 3. Sintered glass filters 4. Membrane filters 5. Syringe filters 6. Air filters PowerPoint Presentation: 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 PowerPoint Presentation: Earthenware (Candle) filters PowerPoint Presentation: 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 PowerPoint Presentation: Asbestos Filter holder PowerPoint Presentation: 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 PowerPoint Presentation: Sintered glass filter PowerPoint Presentation: 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: Membrane filters PowerPoint Presentation: 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 PowerPoint Presentation: 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 The roles of HEPA filters in biological flow safety cabinets: The roles of HEPA filters in biological flow safety cabinets Exhaust HEPA filter Blower Supply HEPA filter Light High-velocity air barrier Safety glass viewscreen 3/9/2014 Dr.T.V.Rao MD 53 PowerPoint Presentation: Radiation Two types of radiations are used for sterilization Non- ionising Ionising Non- ionising radiations These inculde infrared and ultraviolet (UV) radiations Infrared is used for rapid mass sterilisation of syringes and catheters UV radiation with wavelength of 240-280 nm has marked bactericidal activity PowerPoint Presentation: 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 otherwise causes damage to skin and eyes. PowerPoint Presentation: 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 PowerPoint Presentation: Since theres is no appreciable increase in the temperature, in this method it is known as “cold sterilisation ” 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 radiations PowerPoint Presentation: Chemical methods A 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 PowerPoint Presentation: 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 matte r PowerPoint Presentation: Disinfectants can be divided into three groups 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) PowerPoint Presentation: Alcohols 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 Methyl alcohol is effective against fungal spores PowerPoint Presentation: Aldehydes 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 A 10% aqueous solution is routinely used PowerPoint Presentation: 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 PowerPoint Presentation: Glutaraldehyde Action similar to formaldehyde More 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 instruments PowerPoint Presentation: Phenols Obtained by distillation of coal tar between temperatures of 170 0 C and 270 0 C Lethal effect is due to cell membrane damage Phenol (1%) has bactericidal action Phenol derivatives like cresol, chlorhexidine and hexachloro- phane are commonly used as antiseptics Cresols Lysol is a solution of cresols in soap Most commonly used for sterilization of infected glasswares, cleaning floors, disinfection of excreta PowerPoint Presentation: 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 PowerPoint Presentation: Halogens Chlorine and iodine are two commonly used disinfectants 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 PowerPoint Presentation: Iodine 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 PowerPoint Presentation: Dyes Aniline and acridine dyes are used extensively as skin and wound antiseptics Aniline dyes include crystal violet, brilliant green, and malachite green are more active against gram positive organisms 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 Acridine dyes affected very little by the presence of organic matter PowerPoint Presentation: 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 Copper salts are used as fungicides PowerPoint Presentation: Surface active agents Substances which alter energy relationships at interfaces, producing a reduction of surface tension; 4 types 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 4. Amphoteric ( Tego compounds) PowerPoint Presentation: 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 KMnO 4 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 vapours should be nullified by exposure to ammonia vapour PowerPoint Presentation: Ethylene oxide (ETO) Colourless liquid with a boiling point of 10.7 0 C 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 CO 2 , its explosive tendency can be eliminated PowerPoint Presentation: 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 PowerPoint Presentation: Betapropiolactone (BPL) Condensation product of ketane and formaldehyde Boiling point: 163 0 C 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 PowerPoint Presentation: 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 PowerPoint Presentation: Testing of disinfectants Minimum inhibitory concentration (MIC) Rideal Walker test Chick Martin test Capacity test (Kelsey and Sykes test) In-use test PowerPoint Presentation: Sterilisation of prions Dry heat 360 0 C for one hour Moist heat 134-138 0 C for 18 min Chemicals 25% sodium hypochlorite for one hour Sensitive to household bleach, phenol (90%) and iodine disinfectants Thank you !!!: Thank you !!!