sterilization and disinfection
DESCRIPTION
This is a series of lectures on microbiology useful for undergraduate medical and paramedical studentsTRANSCRIPT
STERILIZATION STERILIZATION AND DISINFECTIONAND DISINFECTION
Dr. Ashish JawarkarDr. Ashish JawarkarM.D.M.D.
Parul Sevashram HospitalParul Sevashram Hospital
INTRODUCTIONINTRODUCTION
Microorganisms are the agents of Microorganisms are the agents of contamination, infection, and decay.contamination, infection, and decay.
Hence it becomes necessary to remove Hence it becomes necessary to remove them from materials and areas.them from materials and areas.
Early civilization practiced salting, Early civilization practiced salting, smoking, pickling and exposure to smoking, pickling and exposure to sunlight .sunlight .
In mid 1800s Lister developed In mid 1800s Lister developed Aseptic techniquesAseptic techniques to prevent to prevent
contamination of surgical wounds. Prior contamination of surgical wounds. Prior to this development:to this development:• Nosocomial infections caused death in Nosocomial infections caused death in
10% of surgeries.10% of surgeries.• Up to 25% mothers delivering in Up to 25% mothers delivering in
hospitals died due to infection. hospitals died due to infection.
DEFINITIONDEFINITION
STERILIZATIONSTERILIZATION
The process of freeing an article from The process of freeing an article from microorganisms including their spores.microorganisms including their spores.
DISINFECTION:DISINFECTION: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases.
SepsisSepsis: : Comes from Greek for decay or Comes from Greek for decay or putrid. Indicates bacterial contamination.putrid. Indicates bacterial contamination.
AsepsisAsepsis: Absence of significant : Absence of significant contamination.contamination.
Aseptic techniquesAseptic techniques are used to prevent are used to prevent contamination of surgical instruments, contamination of surgical instruments, medical personnel, and the patient during medical personnel, and the patient during surgery. surgery.
Aseptic techniques are also used to Aseptic techniques are also used to prevent bacterial contamination in food prevent bacterial contamination in food industry.industry.
Bacteriostatic Agent: Bacteriostatic Agent:
An agent that An agent that inhibitsinhibits the growth of the growth of bacteria, but does not necessarily kill bacteria, but does not necessarily kill them. them.
BactericideBactericide: : An agent that kills bacteria. Most do not kill An agent that kills bacteria. Most do not kill
Endospores.Endospores.
SporicideSporicide:: An agent that kills spores.An agent that kills spores.
Methods of SterilisationMethods of Sterilisation
Microbial ControlMicrobial Control MethodsMethods
Physical Agents Chemical Agents Mechanical Removal Methods
Physical AgentsPhysical Agents
Heat Radiation
Dry Moist
Incineration
Dry Oven
Steam Under Pressure
Boiling Water/Hot Water Pasteurization
Sterilization
Ionizing Non Ionizing
X Ray, Cathode, Gamma
Disinfection
UV
Sterilization Disinfection
Chemical AgentChemical Agent
Gas Liquids
SterilizationDisinfectionAnimate Inanimate
ChemotherapyAntisepticsSterilizationDisinfection
Mechanical Removal Mechanical Removal MethodsMethods
Filtration
Air Liquids
Disinfection Sterilization
Physical Methods of SterilisationPhysical Methods of Sterilisation
Sterilisation By Dry Heat:Sterilisation By Dry Heat:
Hot Air Oven
• Kills by oxidation effects•The oven utilizes dry heat to sterilize articles • Operated between 50oC to 250/300oC.•A holding period of 160oC for 1 hr is desirable.• There is a thermostat controlling the temperature. •Double walled insulation keeps the heat in and conserves energy,
Uses:Uses:
To sterilise Forceps, Scissors, Scalpels, Swabs.To sterilise Forceps, Scissors, Scalpels, Swabs.
Pharmaceuticals products like Liquid paraffin, Pharmaceuticals products like Liquid paraffin, dusting powder, fats and grease.dusting powder, fats and grease.
FLAMINGFLAMING
Inoculation loop Inoculation loop or Wire, the tip of or Wire, the tip of Forceps and Forceps and spatulas are held spatulas are held in a bunsen flame in a bunsen flame till they are red till they are red hothot..
Flaming Flaming
INCINERATIONINCINERATION
This is an excellent method of This is an excellent method of destroying materials such as destroying materials such as contaminated cloth, animal contaminated cloth, animal carcasses and pathological carcasses and pathological materials.materials.
IncinerationIncineration
Dry heat - HOT AIR OVENDry heat - HOT AIR OVEN Holding temp & time: 160Holding temp & time: 16000C for 1 hrC for 1 hr Used for glassware, forceps, swabs, water Used for glassware, forceps, swabs, water
impermeable oils, waxes & powdersimpermeable oils, waxes & powders Before placing in hot air oven Before placing in hot air oven
Dry glassware completelyDry glassware completely Plug test tubes with cotton woolPlug test tubes with cotton wool Wrap glassware in Kraft papersWrap glassware in Kraft papers
Don’t over load the ovenDon’t over load the oven Allow free circulation of air between the material Allow free circulation of air between the material
Dry heat - HOT AIR OVENDry heat - HOT AIR OVEN Sterilization controls: to check whether the Sterilization controls: to check whether the
equipment is working properlyequipment is working properly Chemical controls: Browne’s tubesChemical controls: Browne’s tubes
Color change from red to greenColor change from red to green ThermocouplesThermocouples Biological controls: paper strips containing10Biological controls: paper strips containing1066 spores spores
of Clostridium tetaniof Clostridium tetani Place strips in oven along with other material for the Place strips in oven along with other material for the
sterilization sterilization Later culture the strips in thioglycollate broth or RCM at 37Later culture the strips in thioglycollate broth or RCM at 3700C C
for 5 daysfor 5 days Growth in medium indicates failure of sterilizationGrowth in medium indicates failure of sterilization
Dry heat – Hot air ovenDry heat – Hot air oven
MOIST HEAT STERILIZATIONMOIST HEAT STERILIZATION
Kills microorganisms byKills microorganisms by coagulatingcoagulating their their proteins. proteins.
MOIST HEAT STERILISATION IS CARRIED OUT MOIST HEAT STERILISATION IS CARRIED OUT WITH FOLLOWING METHODSWITH FOLLOWING METHODS
Temp below 100Temp below 100ooC: C: “Pasteurisation”, Inspissator.“Pasteurisation”, Inspissator.
Temperature at 100Temperature at 100ooC: C: Boiling.Boiling.
Steam at atmospheric pressure: Steam at atmospheric pressure: Koch/Arnold’s Koch/Arnold’s steamer.steamer.
Steam under pressure: Steam under pressure: Autoclave.Autoclave.
PasteurisationPasteurisation Process of killing of pathogens in the milk but Process of killing of pathogens in the milk but
does not sterilize it .does not sterilize it .
Milk is heated at 63Milk is heated at 63ooC for 30 mins.C for 30 mins. (HOLDER METHOD)(HOLDER METHOD)
At 72At 72ooC for 15-20 Sec. Rapid cooling to 13C for 15-20 Sec. Rapid cooling to 13ooCC(FLASH PROCESS)(FLASH PROCESS)
Water BathWater Bath
HOT WATER BATHHOT WATER BATHTo inactivate non sporing bacteria for the To inactivate non sporing bacteria for the
preparation of vaccines - preparation of vaccines - Special vaccineSpecial vaccine bathbath at 60 at 60ooCC for one hour is usedfor one hour is used
Serum or body fluids containing Serum or body fluids containing coagulable proteins can be sterilized by coagulable proteins can be sterilized by heating for 1 hr at 56heating for 1 hr at 56ooC in a water bath for C in a water bath for several successive days.several successive days.
Inspissator
INSPISSATORINSPISSATORSterilizes by heating at 80-85Sterilizes by heating at 80-85ooC for half an C for half an
hour for 3 successive dayshour for 3 successive days
Used to sterilize media such as Used to sterilize media such as Lowenstein-Jensen & Loefller’s serumLowenstein-Jensen & Loefller’s serum
TEMPERATURE AT 100TEMPERATURE AT 100OOCCBoiling:Boiling: Kills vegetative forms of bacterial Kills vegetative forms of bacterial
pathogens. pathogens. Hepatitis virusHepatitis virus: Can survive up to 30 : Can survive up to 30
minutes of boiling.minutes of boiling. EndosporesEndospores: Can survive up to 20 hours or : Can survive up to 20 hours or
more of boilingmore of boiling
Steam Sterilizer
STEAM AT ATMOSPHERIC PRESSURESTEAM AT ATMOSPHERIC PRESSURE
Steam is generated using a steamer Steam is generated using a steamer (Koch/ Arnold)(Koch/ Arnold)
Consists of a Tin cabinet Consists of a Tin cabinet Has a conical lid to enable the drainage of Has a conical lid to enable the drainage of
condensed steamcondensed steamPerforated tray above ensures materials Perforated tray above ensures materials
are surrounded by steam.are surrounded by steam.For routine sterilization exposure of 90 For routine sterilization exposure of 90
mins is usedmins is used
For media containing sugar and gelatin For media containing sugar and gelatin exposure of 100exposure of 100ooC for 20 min for 3 C for 20 min for 3 successive days is usedsuccessive days is used
The process is termed as The process is termed as Tyndallisation /Intermittent SterilizationTyndallisation /Intermittent Sterilization
STEAM UNDER PRESSURE - STEAM UNDER PRESSURE - AUTOCLAVEAUTOCLAVE
Works on the principle of Works on the principle of Steam under Steam under pressurepressure
Invented by Charles Chamberland in 1879. Invented by Charles Chamberland in 1879.
AUTOCLAVE
Autoclave consists of a vertical or a Autoclave consists of a vertical or a horizontal cylinder.horizontal cylinder.
One end has an opening which is meant One end has an opening which is meant for keeping materials to be sterilised.for keeping materials to be sterilised.
The lid is provided with a Pressure gauge, The lid is provided with a Pressure gauge, to measure the pressureto measure the pressure
A safety valve is present to permit the A safety valve is present to permit the escape of steam from the chamberescape of steam from the chamber
Articles to be sterilised are placed in the Articles to be sterilised are placed in the basket providedbasket provided
Sterilisation is carried out under pressure Sterilisation is carried out under pressure at 121at 121ºº for 15 mnts. for 15 mnts.
CHEMICAL AGENTSCHEMICAL AGENTS
Chemical agents act byChemical agents act by Protein coagulationProtein coagulationDisruption of the cell membraneDisruption of the cell membraneRemoval of Sulphydryl groupsRemoval of Sulphydryl groupsSubstrate competitionSubstrate competition
ALCOHOLSALCOHOLSEthanol /Isopropyl alcohol are frequently Ethanol /Isopropyl alcohol are frequently
usedusedNo action on sporesNo action on sporesConcentration recommended 60-90% in Concentration recommended 60-90% in
waterwaterUsesUsesDisinfection of clinical thermometer.Disinfection of clinical thermometer.Disinfection of the skin – Venupuncture Disinfection of the skin – Venupuncture
ALDEHYDESALDEHYDESFormaldehyde & Glutaraldehyde are Formaldehyde & Glutaraldehyde are
frequently usedfrequently usedFormaldehyde is bactericidal, sporicidal & Formaldehyde is bactericidal, sporicidal &
has a lethal effect on viruses.has a lethal effect on viruses.Glutaraldehyde is effective against Glutaraldehyde is effective against
Tubercle bacilli, fungi and virusesTubercle bacilli, fungi and viruses
USESUSES
FORMALDEHYDEFORMALDEHYDETo preserve anatomical specimens To preserve anatomical specimens Destroying Anthrax spores in hair and Destroying Anthrax spores in hair and
woolwool10% Formalin+0.5% Sodium tetra borate 10% Formalin+0.5% Sodium tetra borate
is used to sterilise metal instrumentsis used to sterilise metal instruments
USESUSES
GLUTARALDEHYDEGLUTARALDEHYDEUsed to treat corrugated rubber Used to treat corrugated rubber
anesthetic tubes, Face masks, Plastic anesthetic tubes, Face masks, Plastic endotracheal tubes, Metal instruments and endotracheal tubes, Metal instruments and polythene tubingpolythene tubing
DYESDYES
Two groups of dyes are usedTwo groups of dyes are used
Aniline dyesAniline dyes
Acridine dyesAcridine dyes
ANILINE DYESANILINE DYES
Are Brilliant green, Malachite green & Are Brilliant green, Malachite green & Crystal violetCrystal violet
Active against Gram positive bacteriaActive against Gram positive bacteria
No activity against tubercle bacilliNo activity against tubercle bacilli
ACRIDINE DYESACRIDINE DYES
Acridine dyes in use are orange in colourAcridine dyes in use are orange in colour
Effective against Gram positive than Gram Effective against Gram positive than Gram negativenegative
Important dyes are Proflavine, Important dyes are Proflavine, Acriflavine,Euflavine Acriflavine,Euflavine
HALOGENSHALOGENS IodineIodine in aqueous and alcoholic solution in aqueous and alcoholic solution
has been used widely as a skin has been used widely as a skin disinfectantdisinfectant
Actively bactericidal with moderate against Actively bactericidal with moderate against spores spores
ChlorineChlorine and its compounds have been and its compounds have been used as disinfectants in water supplies & used as disinfectants in water supplies & swimming poolsswimming pools
PHENOLSPHENOLSObtained by distillation of coal tarObtained by distillation of coal tarPhenols are powerful microbicidal Phenols are powerful microbicidal
substancessubstancesPhenolic derivatives have been widely Phenolic derivatives have been widely
used as disinfectants for various purposes used as disinfectants for various purposes in hospitalsin hospitals
Eg: Lysol, cresolEg: Lysol, cresol
USESUSES
Various combinations are used in the Various combinations are used in the control of pyogenic cocci in surgical & control of pyogenic cocci in surgical & neonatal units in hospitals.neonatal units in hospitals.
Aqueous solutions are used in treatment Aqueous solutions are used in treatment of woundsof wounds
GASESGASES
Ethylene OxideEthylene OxideColourless ,Highly penetrating gas Colourless ,Highly penetrating gas
with a sweet ethereal smell.with a sweet ethereal smell.Effective against all types of Effective against all types of
microorganisms including viruses microorganisms including viruses and sporesand spores
USESUSESSpecially used for sterilising heart-lung Specially used for sterilising heart-lung
machines,respirators,sutures,dental machines,respirators,sutures,dental equipments, books and clothing.equipments, books and clothing.
Also used to sterilise Glass, metal and Also used to sterilise Glass, metal and paper surfaces ,plastics, oil,some foods paper surfaces ,plastics, oil,some foods and tobacco.and tobacco.
FORMALDEHYDE GASFORMALDEHYDE GAS
Widely employed for fumigation of Widely employed for fumigation of operation theatres and other roomsoperation theatres and other rooms
BETA PROPIOLACTONEBETA PROPIOLACTONEUsed in fumigationUsed in fumigation
For sterilisation 0.2% BPL is usedFor sterilisation 0.2% BPL is used
Has a rapid biocidal activityHas a rapid biocidal activity
Very effective against virusesVery effective against viruses
SURFACE ACTIVE AGENTSSURFACE ACTIVE AGENTS AND AND METALLIC SALTSMETALLIC SALTS
Substances which reduce the surface Substances which reduce the surface tension – tension –
Surface active agentsSurface active agents
Cations are widely used in the form of Cations are widely used in the form of quaternary ammonium compounds.quaternary ammonium compounds.
Markedly bactericidal, active against Gram Markedly bactericidal, active against Gram positive organisms.positive organisms.
No action on spores, tubercle bacilli, No action on spores, tubercle bacilli, virusesviruses
METALLIC SALTSMETALLIC SALTS
The salts of silver, copper and mercury are The salts of silver, copper and mercury are used as disinfectants.used as disinfectants.
Act by coagulating proteinsAct by coagulating proteins
Marked bacteriostatic, weak bactericidal Marked bacteriostatic, weak bactericidal and limited fungicidal activityand limited fungicidal activity
Sterilisation by filtrationSterilisation by filtration Filtration helps to remove bacteria from heat Filtration helps to remove bacteria from heat
labile liquids such as sera and solutions of labile liquids such as sera and solutions of sugar, Antibioticssugar, Antibiotics..
The following filters are usedThe following filters are usedCandle filtersCandle filtersAsbestos filtersAsbestos filtersSintered glass filterSintered glass filterMembrane filtersMembrane filters
CANDLE FILTER
CANDLE FILTERSCANDLE FILTERSWidely used for purification of waterWidely used for purification of water
Two typesTwo types(a) Unglazed ceramic filter – Chamberland (a) Unglazed ceramic filter – Chamberland filterfilter
(b) Diatomaceous earth filters – Berkefeld (b) Diatomaceous earth filters – Berkefeld filterfilter
SEITZ FILTER
ASBESTOS DISCS
ASBESTOS FILTERASBESTOS FILTERDisposable single use discsDisposable single use discs
High adsorbing tendencyHigh adsorbing tendency
Carcinogenic Carcinogenic Eg: Seitz filterEg: Seitz filter
SINTERED GLASS FILTER
SINTERED GLASS FILTERSINTERED GLASS FILTER
Prepared by heat fusing powdered glass Prepared by heat fusing powdered glass particles of graded sizeparticles of graded size
Cleaned easily, brittle, expensive.Cleaned easily, brittle, expensive.
MEMBRANE FILTER
MEMBRANE FILTERSMEMBRANE FILTERSMade of cellulose esters or other polymersMade of cellulose esters or other polymers
Uses Uses Water purification & analysisWater purification & analysisSterilization & sterility testing Sterilization & sterility testing Preparation of solutions for parenteral usePreparation of solutions for parenteral use
RADIATIONRADIATION
Two types of radiations are usedTwo types of radiations are used
NON –IONISINGNON –IONISINGIONISINGIONISING
Non- Ionising radiationNon- Ionising radiation::Electromagnetic rays with longer wavelength Electromagnetic rays with longer wavelength
Absorbed as heatAbsorbed as heat
Can be considered as hot air sterilisationCan be considered as hot air sterilisation
Used in rapid mass sterilisation of prepacked Used in rapid mass sterilisation of prepacked Syringes and cathetersSyringes and catheters
Eg: UV raysEg: UV rays
IONISING RADIATIONSIONISING RADIATIONS
X- rays, gamma rays & cosmic rays.X- rays, gamma rays & cosmic rays.
High penetrative powerHigh penetrative power
No appreciable increase in the No appreciable increase in the temperature – temperature – COLD STERILISATIONCOLD STERILISATION
Sterilise plastics Syringes, catheters, Sterilise plastics Syringes, catheters, grease fabrics metal foilsgrease fabrics metal foils
ULTRASONIC AND SONIC VIBRATIONULTRASONIC AND SONIC VIBRATION
Bactericidal Bactericidal
Microorganisms vary in their sensitivity, Microorganisms vary in their sensitivity, hence no practical value in sterilisation hence no practical value in sterilisation and disinfectionand disinfection
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