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Sterlization & Disinfection
Dr Rehma Dar Assistant professor Pathology, KEMU
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Learning Objectives What is Sterlization? What is Disinfection?
Chemical and physical methods of disinfection and sterilization Principles and application of each method Common disinfectants and antiseptics used in healthcare setting Quality control of sterlization techniques
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Historical background
The scientific use of disinfection and sterilization methods originated more than 100 years ago Ignatz Semmelweis( ) and Joseph Lister( ) - important pioneers for the promotion of infection control
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More than 100 years ago, Semmelweis demostrated that routine handwashing can prevent the spread of disease He worked in a hospital in Vienna when maternity patients were dying an alarming rate He recognized that medical students worked on cadavers during an anatomy class and afterwords they went to the maternity ward. Students did not wash their hands between touching the dead and the living!!! After administrating handwashing before examining the maternity patients the mortality rate decreased
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In 1867, Lister introduced British surgery to hand washing and the use of phenol as antimicrobial agent for surgical wound dressings • His principles were gradually and adopted in Britain and later in US • This was the beginning of infection control
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Sterilization destruction of ALL forms of life, including the bacterial spores, viruses, prions no degrees of sterilization: an all-or-nothing process Physical or Chemical methods
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Disinfection A process that eliminates a defined scope of microrganisms, except most spores, viruses and prions Purpose- prevent transmission of certain microorganisms with objects, hands or skin and prevent spreading the infection Physical or Chemical methods Most disinfectants are chemical agents applied to inanimate objects !
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Commonly used terms Aseptic techniques - prevent microbial contamination of materials or wounds Antisepsis - disinfection of living tissues(e.g., in a wound) Antiseptics are applied (do not kill spores) to reduce or eliminate the number of bacteria from the skin and mucus membranes Sanitizer- an agent, usually a detergent, that reduces the numbers of bacteria to a safe level
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Factors that influence the degree of killing
Types of organisms Number of organisms Concentration of disinfecting agent Presence of organic material (e.g., serum, blood) Nature (composition) of surface to be disinfected Contact time Temperature pH Biofilms Compatibility of disinfectants and sterilants
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Rate of killing of microorganisms
N α 1/CT where N = No of survivors C = Concentration of agent T= Time of application of agent *CT referred to as dose
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Types of organisms Organisms vary in their ability to withstand chemical and physical treatment e.g., Spores – have coats rich in proteins, lipids and carbohydrates Mycobacteria – cell walls are rich in lipids Biofilms - microorganisms living together in communities Prions – the most resistant known organisms to the action of heat, chemicals, and radiation !!!
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Different types of organisms and their resistance to killing agents
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Number of organisms The total number of organisms determine the exposure time of killing agent Composed of organisms with varying degrees of susceptibility to killing agents the death curve is logarithmic!!! higher numbers of organisms require longer exposure
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The effect of exposure time versus number of organisms
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Concentration of disinfecting agent
Proper concentration of disinfecting agents ensure the activation of target organisms, e.g., Povidone-iodine should be diluted with water before use because there is not enough free iodine to kill microorganisms in concentrated solution
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Presence of organic material
such as blood, mucus, pus affects killing activity by inactivating the disinfecting agent, e.g, by coating the surface to be treated, prevents full contact between object and agent (Glutaraldehyde) easily inactivate bleach (Sodium hypochlorite) For optimal killing activity, instruments and surfaces should be cleansed of excess organic material before disinfection !!!
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Nature (composition) of surface to be disinfected
Some medical instrumentsare manufactured of biomaterials that exclude the use of certain disinfection and sterlization methods because of possible damage, e.g., endoscopic instruments cannot be sterilized by the heat in an autoclave
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Contact time The amount of time a disinfectant or sterilant is in contact with the object is critical! e.g., The spores of bacteria and fungi need a much longer time Determine whether it is disinfecting or sterilizing the object
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Temperature Disinfectants are generally used at room temp. (20-25 ̊C)
Activity is increased by an increased temp. Decreased by a drop in temp
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pH The pH of the material to be desinfected or sterilized can have an effect on the activity of disinfecting or sterilizing agent
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Biofilms Communities of microorganisms
can be on a surface of either inanimate or animate objects, e.g., catheters (criticial place!), pipes that carry water and dionizing columsused to make processed water make disinfection more difficult Concentration of the disinfectant and the contact time need to be increased
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Compatibility of disinfectants
A common mistake is to belive that two disinfectants are better than one ! some of them may inactivate other, e.g., the bleach and quaternary ammonium compound together negate the activity of both
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Classes of Medical materials
Medical materials are categorized into Criticial materials Semicriticial materials Noncriticial materials
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Criticial materials Invade sterile tissues or enter the vascular system most likely to produce infection if contaminated and therefore require sterilization
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Semicriticial materials
have contact with mucos membranes require high-level disinfection agents
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Non-criticial materials
have contact with intact skin require intermediate- level to low- level disinfection
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Types of disinfectants
High-level disinfectants activity against bacterial spores Intermediate-level disinfectants tuberculocidal activity but not sporocidal Low-level disinfectants a wide range of activity against microorganisms but no sporocidal or tuberculocidal activity
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Classification of Disinfectants
Physical methods Chemical methods
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Physical methods Heat moist heat dry heat pasteurization boiling
Filtration Radiaton
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Heat The most common method used for elimination of microorganisms !
reliable effects ease of use economical
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Heat Moist heat (Boiling/Autoclaving) Dry heat Pasteurisation
Mechanism: denaturation of proteins, membrane damage and cleavage of DNA.
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Moist heat (Autoclave)
(heat under steam pressure) destroy ALL microorganisms including spores steam under 15lb/inch2 of pressure, at 121 ̊C temp., minutes of exposure in autoclaves or 2 atm./ 134 ̊C / 3 min. destroy the prions at higher temperatures, ̊C for at least 1hour under 2 atm Application: method of choice for heat-stable objects
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Autoclaves
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Dry heat (Hot air oven) requires much longer exposure times and higher temperatures than moist heat 2 hours at 180 ̊̊C Application: Heat stable substances that are not penetrated by moist heat e.g glassware or surgical instruments
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Dry heat oven
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Boiling & Pasteurisation
Boiling and pasteurisation achieve disinfection but not sterilisation (do not eliminates spores)!!! Boiling kills most microorganisms in 10 min. at 10 ̊C Pasteurisation 62 ̊C for 30 minutes Flash pasteurisation 72 ̊C for 15 seconds Application: Milk borne pathogens Mycobacterium bovis, Salmonells, Streptococcus, Listeria, Brucella
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Filtration The membrane filters composed of plastic polymers or cellulose esters containing pores of certain size Mechanism: Liquid is pulled (vacum) or pushed (pressure) through the filter matrix, organisms larger than the size of the pores are retained and small particles are retained by electrostatic attraction of particles to filters
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Commonly used : Nitrocellulose with pore size of 0.22 μm
Application: Sterlization of solutions witth heat sensitive components.
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Filtration of Air filters remove microorganisms larger than 0.3 μm
the high efficiency particulate air (HEPA) filters Application: In laboratory hoods and in rooms of immunocompromised patients
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Radiation Two forms: Ionizing Non-ionizing
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Ionizing radiation gamma rays, X rays or electron beams short wavelenght and high energy Mechanism: formation of free radicals- damage DNA Application: sterilizationof disposable supplies (syringes,sututres, bandages, catheters and gloves) and heat-sensitive pharmaceuticals
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Non-ionizing Ultraviolet rays (UV) : nm Long wavelenght and low energy poor penetrabilty limited use Mechanism: Thymine dimers – inhibiting DNA replication
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Application: Disinfect smooth surfaces with UV lamps and to reduce airborn pathogens (surgical theaters)
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Chemical methods Physical methods are used mainly to achieve sterilization whereas Chemical agents are used mainly as disinfectants But some chemical agents may be used to sterilize (chemosterilizers)
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Chemical agents Alcohols Aldehydes Halogens Phenols Surfactants
Heavy metals Dyes Gases (ethylene oxide, oxidants)
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Mechanism of action Reaction with components of the cytoplasmic membrane (sufactant compounds, alcohols) Denaturation of cellular proteins (alcohols, phenols, aldehydes, oxidants) Reaction with the thiol (-SH) groups of enzymes (heavy metals) Damage of RNA and DNA (aldehydes, oxidants, dyes) The agent can exert one or a combination of actions on microorganisms
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Classes of chemical agents according to action
Disruption of Cell membranes Alcohol, Detergents Modification of Proteins Halogens, Heavy metals, H2O2, Aldehydes. Ethylene oxide, Acids & alkalis Modification of Nucleic acids Dyes
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Alcohols Ethanol 70%, isopropanol 70%, propanol 60%
Mechanism: Inactivate microorganisms by disorganize lipids in membrane and denaturing proteins but not sporocidal! The most effective concentrations are between 60%-90% (water is needed in chemical reactions) Application: Ethanol – clean skin before venipuncture and surgical disinfection of hands
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Detergents/ Surfactants
also known as surface-active agents surfactants e.g quaternary ammonium compounds Mechanism: disrupt cell membrane by interacting lipid within it. moderate bactericidal effect (good against Gram(+) but not sporecidal, tuberculocidal Application: disinfectionof noncriticial surfaces (bench tops, floors) and skin antisepsis
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Phenols Mechanism: not only damage membranes but also denaturate proteins broad-spectrum, but not sporocidal or virucidal Application: disinfection of hospital and household environment
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Halogens Chlorine, Iodine, and their derivatives
Mechanism: oxidizing agent that crosslinks sulfhydryl groups in enzymes to form inactive disulfide Chlorine acts after dissolution of chloride ions with water and also used in the form of hypochlorite (household bleach) Application: disinfection of water and swimming pool
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Iodine Mechanism: inactivates sulfhydryl containing enzymes and binds to tyrosine in proteins Application: skin antiseptic Tincture Iodine (2% iodine solution and potassium iodide in ethanol) used to prepare skin prior to blood culture. Iodophors complex of iodine with detergent used to prepare skin prior to surgery.
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Heavy metals Mercury and silver have greatest antibacterial activity.
Mechanism: Inactivate enzymes by binding to the sulfhydryl groups of enzymes Application: Silver nitrate(1% eyedrop solution) used in the prevention of eyes infections Silver sulfadiazine used to prevent infection of burn wounds
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Hydrogen peroxide oxidizing agent – effectiveness limited by organism’s ability to produce catalase. Bubbles produced on wounds as a result of O2 produced by tissue catalase on H2O2 Mechanism: inactivates sulfhydryl containing enzymes Application: antiseptic to clean wounds and disinfect contact lenses.
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Aldehydes Formaldehyde - the most imporatant & glutaraldehyde (disinfectant and sterilizer!!!) Mechanism: denaturate proteins and nucleic acids Application: disinfection of surfaces and objects (plastic and rubber items) Glutaraldehyde ( Cidex) – the sterilizer of choice for heat-sensitive medical equipment e.g endoscopes
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Gases Ethylene oxide - the most commonly used for sterilization
Mechanism: alkylates proteins and nucleic acids Application: widely in hospitals for heat sensitive materials like surgical instruments and
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Dyes acridine dyes, brilliant green, crystal violet
Mechanism interact with bacterial nucleic acids Application: used topically as antiseptics to treat e.g. mild burns
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Quality Control of Sterilization
To ensure that potentially infectious agents are destroyed by adequate sterilization regimes Three levels: Physical Chemical Biological
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Physical: includes measuring device control temperature, time and pressure
Chemical: substances that undergo a colour change or have melting points within the sterilizing range e.g Browne's tubes, Bowie Dick tape give an immediate indication of successful sterilization
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Biological Bacillus stearothermophilus spores survives steam heat at 121 ̊C for 5 min. and is killed at 121 ̊C in 13 min. validate the adequacy of steam sterilization Bacillus subtilis spores validate the adequacy of dry heat sterilization
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Chemical indicators Browne's tubes are glass tubes that contain heat sensitive dyes. These change colour after sufficient time at the desired temperature. Before heat exposure, the contents of the tube appear red. As heating progresses, the colour changes to green indicating adequate sterlization.
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Bowie Dick tape is applied to articles being autoclaved
Bowie Dick tape is applied to articles being autoclaved. Before heat exposure, the tape is uniformly buff in colour. After adequate heating, the tape develops dark brown stripes. The pack on the left has been properly sterilised; that on the right has not.
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Sterilization methods
Physical methods Moist heat in autoclaves Dry-heat in ovens Gamma irradiaton Filtration Chemical agents Ethylene oxide Glutaraldehye (high concentration) Disinfection methods Physical methods Boiling and pasteurisation Ultraviolet radiation Chemical agents Alcohols Aldehydes Halogens Phenols Surfactants Heavy metals Dyes Oxidants
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Thank you
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