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Sterilization : 

Sterilization By Dr. Ashish Tharoor Jitendranath First year Post Graduate Dept of Microbiology

Why do we need sterilization : 

Why do we need sterilization

Need for Sterilization : 

Need for Sterilization For sterilization of culture media, reagents and equipments. Effective decontamination of spillage. Sterilization of surfaces and equipments after use. Sterilization of critical medical devices. Sterilization of food

Sterilization of Medical devices : 

Sterilization of Medical devices In 1968 Earle.H.Spaulding created a classification for sterilization of medical devices

Definition : 

Definition Standard: Closely monitored validated process used to render a product free of all forms of viable microorganism including all bacterial endospores. Technical: Greater than 106 log CFU reduction of most resistant spores for the sterilization process studies and achieved at the half time of regular cycle

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Monitoring of Sterilization : 

Monitoring of Sterilization For validating the process of sterilization. Methods of monitoring using indicator: Mechanical/Physical: Thermometer and thermocouple. Chemical: Bowie Dick test, Browne tube. Biological:

Dry Heat : 

Dry Heat Killing effect due to Protein denaturation, oxidative damage and toxic effect of elevated levels of electrolytes. For glassware instruments, water impermeable oil, waxes, & paper wrapped articles. Red heat: Hot air sterilizer: Incinerator:

Red Heat : 

Red Heat Main application is on Inoculation wires and loops

Hot Air Sterilizer : 

Hot Air Sterilizer Useful for sterilizing loads that cannot be readily penetrated by steam and can tolerate high temp. Presence of port for thermocouple.

Thermocouple : 

Thermocouple A thermocouple or thermocouple thermometer is a junction between two different metals that produces a voltage related to a temperature difference. Thermocouples are a widely used type of temperature sensor for measurement and control[1] and can also be used to convert heat into electric power. They are inexpensive[2] and interchangeable, are supplied fitted with standard connectors, and can measure a wide range of temperatures. The main limitation is accuracy: system errors of less than one kelvin (K) can be difficult to achieve.

Hot air sterilizer { Contd} : 

Hot air sterilizer { Contd} Safety features: Cut off above 200o C. Door interlocks Disadvantage: Long exposure time. Temperature >185o C it resinfy paraffin and destroying instruments. Higher temperature are corrosive, resulting in loss of hardness.

Incinerator : 

Incinerator Widely used method for waste treatment. Used when culture containers are disposable. Waste material is converted to ash.

Moist Heat : 

Moist Heat Spore are killed by moist heat. Culture media & other liquids required to retain their content of water. Not applicable to waterproof materials such as oils and greases or dry materials.

Pasteurization : 

Pasteurization Moist heat at temperature below 1000 C. Heat labile fluids may be disinfected not sterilized by heating at 560 C for 30 min. Sufficient to kill mesophilic bacteria but not spores. For serum, or other body fluids containing proteins, temp to rise above 59o C. UHT; 140o C less than 1 sec. Cold Pasteurization. High pressure pasteurization.

Washer Disinfectors : 

Washer Disinfectors Washing machines using hot water, steam and detergents, may be used. Washing action at 710 C for 3 min or 800 C for 1 minute sufficient to kill vegetative organism. Recommended for instruments contaminated with HBV and HIV. Accordingly it is set at 93o C for 10 min.

Boiling at 100o C : 

Boiling at 100o C Heating in boiling water at 100o C for 5 minutes sufficient to kill all vegetative bacteria, HBV and some bacterial spore. Used only in case of emergencies to sterilize medical and surgical equipment. Heat labile articles and hollow or porous items where water will not penetrate lumen cannot be disinfected this way.

Steaming at 100o C : 

Steaming at 100o C To prevent glass from cracking which happens when it is heated directly. Also in case of heat labile culture media. Pure steam in equilibrium with boiling water at normal atmospheric pressure Exposure to this temperature for 5 minutes will kill microorganism.

Free Steaming : 

Free Steaming Koch and Arnold steamers are used. Useful for selective heat labile media like DCA, XLD, TCBS and Slenite F broth. These media do not support the growth of heat resistant bacteria. Tyndallization- 20 min for 3 succesive days.

Steam sterilization : 

Steam sterilization Irreversibly coagulates and denatures microbial enzymes and proteins. Important parameters for effectiveness Exposure time Temperature of process Level of moisture. Pressure

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1) Advantages Non-toxic Cycle easy to control and monitor Inexpensive Rapidly microbiocidal Least affected by organic/inorganic soils Rapid cycle time Penetrates medical packing, device lumens 2) Disadvantages Deleterious for heat labile instruments Potential for burns

Autoclaves : 

Autoclaves Invented by Charles Chamberland in 1879. Precursor was the Steam digester invented by Denis Papin in 1679. At correct temp lethal to all bacteria, viruses, fungi & protozoa.

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Example of usage of autoclaves are: Hospitals & OPD- Porous load autoclaves. Mortuary- Bench top autoclaves. Microbiology Lab: Media preparators or fluid cycle steam sterilizers. Pharmaceutical- Fluid Cycle Sterilizer :

Types of autoclaves : 

Types of autoclaves Simple laboratory autoclave Transportable bench top autoclaves Large simple autoclave Downward displacement autoclave Multipurpose laboratory autoclave Pre vacuum High security autoclave. Porous load sterilizer. Low temperature steam. Steam flash pressure pulsing steam sterilization autoclaves

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Sterilization Hold Time Heat Penetration Time Condensation of steam: 3 effects Wets microorganisms. Liberates latent heat. Significant contraction of steam.

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Time and temperature with pressure required for sterilization by steam under pressure in autoclave. 121-124o c at 1.1 bar for 15 min. 134-138o c at 2.2 bar for 3 min. 1 bar=1 atm pressure=14.7 pounds per square inch

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Steam which is present must be Saturated: Dry. Pure. Steam supply: Superheated steam ( Dry gas) Wet steam

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Removal of air: Simple transportable (pressure-cooker) autoclaves. Downward displacement autoclaves. Porous load autoclaves. Items to be put in autoclave Unwrapped non porous items: Porous loads Discard loads Fluids in sealed containers. Nutrient media.

Simple laboratory autoclave : 

Simple laboratory autoclave Considered unsatisfactory. Not monitor temperature of load and therefore cannot prevent spoiling nutritive value. No safety interlock.

Transportable bench top autoclaves. : 

Transportable bench top autoclaves. Sophisticated version of air displacement autoclave. Possess automatic cycle control with indicator for cycle failure and thermal safety. Disadvantages of this are: Not fitted with vacuum assisted air removal. No assisted drying. Cannot be used for porous loads, packaged items or discard loads. Cant handle liquid loads

Large simple autoclave : 

Large simple autoclave Larger version of simple pressure cooker. No means of assisted air removal. Size too large to permit removal of air. Unsuitable for wrapped articles and make safe loads.

Downward displacement laboratory autoclaves : 

Downward displacement laboratory autoclaves Provision for removal of air from chamber{ balanced pressure steam trap} Other devices assist the drying of the load. No vacuum assistance for air removal. Air removal was found to be inadequate. Only value is sterilization of unwrapped non-porous metallic items. Cooling may take many hours to be below 80o C

Multipurpose laboratory autoclaves : 

Multipurpose laboratory autoclaves Different types of load require different cycles required for these different loads. Special feature Presence of efficient means of assisted air removal and drying, assisted cooling and a temperature sensitive probe reading directly from the load.

Multipurpose laboratory autoclaves ( Contd) : 

Multipurpose laboratory autoclaves ( Contd) Aqueous media Accelerated cooling required to avoid damage to nutrient properties of culture medium. Cooling to below 80o C before autoclave is opened minimize risk of breakage and explosions. Volume of DIN bottle which is used should be not over 80%. Duration of heating up period should be controlled automatically with a thermocouple placed in one of the largest bottle or simulators, which reproduces thermal characteristic of bottle.

Multipurpose laboratory autoclaves ( Contd) : 

Multipurpose laboratory autoclaves ( Contd) 121o c for 15 min. Cooling of load should be assisted so that media are cooled to under 80o c in 30 min. Methods of cooling. Cooling time duration set by a thermocouple placed in simulator Air blasting. Glasswares are satisfactorily processed as part of a fluid cycle.

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High security autoclave Porous load sterilizer Low temperature steam

Gas sterilization : 

Gas sterilization For delicate heat labile equipments. Monitoring their efficacy by biological test.

1. Low temperature steam formaldehyde : 

1. Low temperature steam formaldehyde Steam at sub atmospheric pressure (temp below 100o C) kills the spores of thermophilic bacteria very slowly. Sporicidal at high concentration in presence of moisture. Synergism between formaldehyde and steam. Difficulty in combining the two.

2. Ethylene oxide sterilizer : 

2. Ethylene oxide sterilizer Useful also for small proportion of medical and surgical devices which cannot withstand autoclaving. Kills micro organisms by altering their DNA by alkylation Lots of disadvantages of using this method Therefore only materials with documented ethylene oxide penetration and dissipation properties should be used as wrappers. No current technology can replace sterilization with ethylene oxide completely. Sterilization with this does not fail as frequently as sterilization with plasma.

Low temperature sterilization by ozone : 

Low temperature sterilization by ozone The 125 l ozone sterilizer uses medical grade oxygen water and electricity to generate ozone within the sterilizer to provide efficient sterilants without producing toxic chemicals or using high temperatue. End of cycle O2 and H2O are formed.

Liquid Sterilization : 

Liquid Sterilization To sterilize immersible devices like endoscopes etc with 35% liquid Peracetic acid. Done using STERIS system 1 Acid is diluted with sterile filtered water. Commercially available spores can be used for monitoring sterilization. Disadvantage is high cost.

Sterilizing Filter : 

Sterilizing Filter Aqueous liquids sterilized by forced passage through filter of porosity small enough to retain any microorganism. 1) Membrane filters: Manufactured from variety of polymeric material such as cellulose diacetate, polycarbonate and polyester, as disc.

Membrane Filter (Contd) : 

Membrane Filter (Contd) Membrane made in 2 ways Capillary pore membranes: For viruses Labyrinthine pore membranes: Bacteria & yeast Exact procedure for use varies with form in which filter is supplied. Filters may be supplied with plastic holders pre sterilized for single use, or mounted in re-usable holders and fitted to filtration vessels.

Sterilizing Filter : 

Sterilizing Filter 2)Syringe filters: Membrane 13-25mm diameter. Fitted in syringe like holders of stainless steel or polycarbonate. Used for sterilization of small volumes of fluid. 3)Vacuum and in- line filters: Membranes of 25-45 mm diameter are used either with in line filter holders of Teflon or stainless steel and aluminum. Used for sterilization of large volumes of air and liquid.

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4) Pressure filtration: Large membranes, 100-293 mm in diameter, housed in pressure filter holders. Production of pure water. 5) Air Filters: Large volume of air rapidly freed from infection by passage through HEPA (High efficiency particle arrester)

Air Filters (Contd) : 

Air Filters (Contd) Principal use is to render safe the air withdrawn from an exhaust ventilated safety cabinets used for work with pathogens. To decontaminate air input into laminar flow cabinet. Fitted with disposable pre filter which reduces load collected by main filter. Main HEPA should have 99.99% efficiency.


STERILIZATION BY RADIATION IONIZING radiation - γ radiation from radioactive elements ,usually Co60., E.g.. Sterilization of Disposable Syringes. Bacillus pumilis used for testing. ULTRAVIOLET RAYS: Mercury vapor lamps emitting radiation in the range of 250-260nm are bactericidal & to a lesser extent sporicidal.

OT Sterilization : 

OT Sterilization Brief stay of patient. Need to prevent surgical site infection Microbiological monitoring required.

OT Sterilization : 

OT Sterilization Formalin fumigation of theatre, corridor Cleaning daily. UV radiation for 12-16 hours. Ultrasonic vibrations for microsurgical instruments with hinged area and serrated edges.

Sterility Monitoring of OT : 

Sterility Monitoring of OT Swabbing and culturing Settle plate method Slit sampler Rodac system.

References : 

References Mackie & McCartney Practical Medical Microbiology 14 th edition Murray, Scot et al Text book of medical microbiology Bailey & Scot, Diagnostic Medical Microbiology Twelfth Edition Ananthnarayan and Paniker, Textbook of microbiology, 8 th edition.

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