STERILIZATION STERILIZATION TECHNIQUESTECHNIQUES

COURSE : B.SC. (MICRO)COURSE : B.SC. (MICRO)

SUBJECT : ELEMENTARY MICROBIOLOGYSUBJECT : ELEMENTARY MICROBIOLOGY

UNIT 4.2UNIT 4.2

INTRODUCTIONINTRODUCTION

Microorganisms are the agents of Microorganisms are the agents of contamination, infection, and decay.contamination, infection, and decay.

Hence it becomes necessary to Hence it becomes necessary to remove them from materials and remove them from materials and areas.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. contamination of surgical wounds. Prior to this development:Prior to this development:• Nosocomial infections caused Nosocomial infections caused

death in 10% of surgeries.death in 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 microorganisms including their spores.spores.

AsepsisAsepsis: Absence of significant : Absence of significant contamination.contamination.

Aseptic techniques are used to prevent Aseptic techniques are used to prevent contamination of surgical instruments, contamination of surgical instruments, medical personnel, and the patient medical personnel, and the patient during surgery. during 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.

� Bactericide: Bactericide: An agent that kills bacteria. Most do not kill An agent that kills bacteria. Most do not kill

Endospores.Endospores.

� Sporicide:Sporicide:

An agent that kills spores.An agent that kills spores.

Methods of SterilisationMethods of Sterilisation

Microbial ControlMicrobial Control MethodsMethods

Physical AgentsPhysical Agents Chemical AgentsChemical AgentsMechanical RemovalMechanical Removal

Methods 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

1.Physical agents1.Physical agentsI.I. SunlightSunlight

II.II. Heat Heat

III.III. RadiationRadiation

sunlightsunlight

The bactericidal activity of sunlight is mainly The bactericidal activity of sunlight is mainly due to the presence of ultra violet rays.due to the presence of ultra violet rays.

It is responsible for spontaneous sterilization It is responsible for spontaneous sterilization in natural conditions.in natural conditions.

the sunlight is more effective in killing germs the sunlight is more effective in killing germs due to combination of ultraviolet rays and due to combination of ultraviolet rays and heat.heat.

Heat Heat

Heat acts by oxidative effects as well as Heat acts by oxidative effects as well as denaturation and coagulation of proteins.denaturation and coagulation of proteins.

Heat is considered to be most trustful Heat is considered to be most trustful method of sterilization for which articles that method of sterilization for which articles that can withstand heat.can withstand heat.

Those articles that cannot withstand high Those articles that cannot withstand high temperatures can still be sterilized at lower temperatures can still be sterilized at lower temperature by prolonging the duration of temperature by prolonging the duration of exposure.exposure.

Dry heatDry heat

Dry heat acts by protein denaturation, Dry heat acts by protein denaturation, oxidative damage and toxic effects of oxidative damage and toxic effects of elevated levels of electrolytes.elevated levels of electrolytes.

Dry heatDry heat

Flaming:Flaming: Inoculation loop Inoculation loop

or Wire, the tip or Wire, the tip of Forceps and of Forceps and spatulas are spatulas are held in a held in a bunsen flame till bunsen flame till they are red hotthey are red hot..

Dry heatDry heat

INCINERATIONI:INCINERATIONI:This is an excellent method This is an excellent method

of destroying materials such of destroying materials such as contaminated as contaminated cloth, cloth, animal carcasses animal carcasses and and pathologicalpathological materials. materials.

Moist heatMoist heat

Moist heat acts by coagulation and Moist heat acts by coagulation and denaturation of proteins.denaturation of 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”“Pasteurisation”

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.

Temp below 100Temp below 100ooCC

Pasteurisation:Pasteurisation: originally employed by Louis Pasteur.originally employed by Louis Pasteur. Use in food and dairy industry.Use in food and dairy industry. Process of killing of pathogens in the milk Process of killing of pathogens in the milk

but does not sterilize it .but does not sterilize it . There are two methods of pasteurization:- the There are two methods of pasteurization:- the

holder method (heated at 63oC for 30 holder method (heated at 63oC for 30 minutes) and flash method (heated at 72oC minutes) and flash method (heated at 72oC for 15 seconds) followed by quickly cooling for 15 seconds) followed by quickly cooling to 13oC.to 13oC.

Vaccines(water bath):Vaccines(water bath): The contaminating bacteria in a vaccine The contaminating bacteria in a vaccine

preparation can be inactivated by heating in a preparation can be inactivated by heating in a water bath at 60oC for one hour. water bath at 60oC for one hour.

Only vegetative bacteria are killed and spores Only vegetative bacteria are killed and spores survive.survive.

Temp below 100Temp below 100ooCC

Temp below 100Temp below 100ooCC

Serum bath:Serum bath: The contaminating bacteria in a serum The contaminating bacteria in a serum

preparation can be inactivated by heating in a preparation can be inactivated by heating in a water bath at 56oC for one hour on several water bath at 56oC for one hour on several successive days.successive days.

Only vegetative bacteria are killed and spores Only vegetative bacteria are killed and spores survive.survive.

TEMPERATURE AT 100TEMPERATURE AT 100OOCC

Boiling: Boiling:

Kills vegetative forms of bacterial Kills vegetative forms of bacterial pathogens. pathogens.

Hepatitis virus: Can survive up to 30 Hepatitis virus: Can survive up to 30 minutes of boiling.minutes of boiling.

� Endospores: Can survive up to 20 hours Endospores: Can survive up to 20 hours or more of boilingor more of boiling

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 Has a conical lid to enable the drainage

of condensed steamof condensed steamPerforated tray above ensures Perforated tray above ensures

materials are surrounded by steam.materials 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

STEAM UNDER PRESSURE - STEAM UNDER PRESSURE - AUTOCLAVEAUTOCLAVE

Works on the principle of Works on the principle of Steam under Steam under pressurepressure

Invented by Invented by Charles ChamberlandCharles Chamberland in in 18791879. .

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 One end has an opening which is meant for keeping materials to be meant for keeping materials to be sterilised.sterilised.

The lid is provided with a Pressure The lid is provided with a Pressure gauge, to measure the pressuregauge, to 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 Articles to be sterilised are placed in the basket providedthe basket provided

Sterilisation is carried out under Sterilisation is carried out under pressure at 121pressure at 121ºº for 15 mnts. for 15 mnts.

1.Physical agents1.Physical agents

II.II. RadiationRadiation Two types of radiations are usedTwo types of radiations are used

a)a) Ionizing :-x ray , cathod , gammaIonizing :-x ray , cathod , gamma

- sterilization- sterilization

b)b) Non ionizing :-uvNon ionizing :-uv

-disinfection-disinfection

IONISING RADIATIONSIONISING RADIATIONS

X- rays, gamma rays & cosmic rays.X- rays, gamma rays & cosmic rays.

High effective strong powerHigh effective strong 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

Non- Ionising radiationNon- Ionising radiation::Electromagnetic rays with longer Electromagnetic rays with longer

wavelength 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 Used in rapid mass sterilisation of prepacked Syringes and cathetersprepacked Syringes and catheters

Eg: UV raysEg: UV rays

ULTRASONIC AND SONIC VIBRATIONULTRASONIC AND SONIC VIBRATION

Bactericidal Bactericidal

Microorganisms vary in their Microorganisms vary in their sensitivity, hence no practical value in sensitivity, hence no practical value in sterilisation.sterilisation.

Chemical AgentChemical Agent

Gas Liquids

Sterilization Disinfection Animate Inanimate

Chemotherapy Antiseptics Sterilization Disinfection

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 Ethanol /Isopropyl alcohol are

frequently usedfrequently usedNo action on sporesNo action on sporesConcentration recommended 60-90% in Concentration recommended 60-90% in

waterwater

UsesUsesDisinfection of clinical thermometer.Disinfection of clinical thermometer.Disinfection of the skin – Venupuncture Disinfection of the skin – Venupuncture

ALDEHYDESALDEHYDES

Formaldehyde & Glutaraldehyde are Formaldehyde & Glutaraldehyde are frequently usedfrequently used

Formaldehyde is bactericidal, Formaldehyde is bactericidal, sporicidal & has a lethal effect on sporicidal & has a lethal effect on viruses.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 10% Formalin+0.5% Sodium tetra

borate is used to sterilise metal borate is used to sterilise metal instrumentsinstruments

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 endotracheal tubes, Metal instruments and polythene tubingand polythene 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 Acridine dyes in use are orange in colourcolour

Effective against Gram positive than Effective against Gram positive than Gram negativeGram negative

Important dyes are Proflavine, Important dyes are Proflavine, Acriflavine,Euflavine Acriflavine,Euflavine

HALOGENSHALOGENS

IodineIodine in aqueous and alcoholic in aqueous and alcoholic solution has been used widely as a solution has been used widely as a skin disinfectantskin disinfectant

Actively bactericidal with moderate Actively bactericidal with moderate against sporesagainst 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 pools& swimming pools

PHENOLSPHENOLS

Obtained 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 used as disinfectants for various purposes in hospitalspurposes in 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 Aqueous solutions are used in treatment of woundstreatment of wounds

GASESGASES

Ethylene OxideEthylene OxideColourless ,Highly penetrating Colourless ,Highly penetrating

gas with a sweet ethereal smell.gas with a sweet ethereal smell.Effective against all types of Effective against all types of

microorganisms including microorganisms including viruses and sporesviruses and spores

USESUSES

Specially used for sterilising heart-Specially used for sterilising heart-lung 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 paper surfaces ,plastics, oil,some foods and tobacco.foods 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 PROPIOLACTONE

Used 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

Mechanical Removal Mechanical Removal MethodsMethods

Filtration

Air Liquids

Disinfection Sterilization

Sterilisation by filtrationSterilisation by filtration

Filtration helps to remove bacteria from Filtration helps to remove bacteria from heat labile liquids such as sera and heat labile liquids such as sera and solutions of sugar, Antibioticssolutions of sugar, Antibiotics ..

The following filters are usedThe following filters are usedCandle filtersCandle filtersAsbestos filtersAsbestos filtersSintered glass filterSintered glass filterMembrane filtersMembrane filters

CANDLE FILTER

CANDLE FILTERSCANDLE FILTERS

Widely used for purification of waterWidely used for purification of water

Two typesTwo types

(a) Unglazed ceramic filter – (a) Unglazed ceramic filter – Chamberland filterChamberland filter

(b) Diatomaceous earth filters – (b) Diatomaceous earth filters – Berkefeld filterBerkefeld filter

ASBESTOS FILTERASBESTOS FILTER

Disposable single use discsDisposable single use discs

High adsorbing tendencyHigh adsorbing tendency

Carcinogenic Carcinogenic

Eg: Seitz filterEg: Seitz filter

SINTERED GLASS FILTERSINTERED GLASS FILTER

Prepared by heat fusing powdered Prepared by heat fusing powdered glass particles of graded sizeglass particles of graded size

Cleaned easily, brittle, expensive.Cleaned easily, brittle, expensive.

MEMBRANE FILTERSMEMBRANE FILTERS

Made of cellulose esters or other Made of cellulose esters or other polymerspolymers

Uses Uses Water purification & analysisWater purification & analysisSterilization & sterility testing Sterilization & sterility testing Preparation of solutions for parenteral Preparation of solutions for parenteral

useuse

Reference:Reference:

1.1. Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig, Microbiology VI Edition, M.J. Pelczar, E.C.S. Chan and N.R. Kreig, Tata McGraw HillTata McGraw Hill

2. Brock Biology of Microorganisms (13th Edition) by Michael T. 2. Brock Biology of Microorganisms (13th Edition) by Michael T. Madigan, John M. Martinko, David Stahl.Madigan, John M. Martinko, David Stahl.

3. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R. 3. Microbiology by Gerard J. Tortora, Christine L Case, and Berdell R. FunkeFunke

4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne 4. Microbiology by Prescott, Harley, and Klein's Microbiology Joanne M. Willey, Linda MM. Willey, Linda M

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