Sterilization
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Sterilization

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Sterilization Sterilization Presentation Transcript

  • DR.B.V.RAMANA, M.D.,
    • 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.
      • Relative Resistance of Microbial Forms
    Highest resistance Moderate resistance Least resistance bacterial endospore (Bacillus & Clostridium) protozoan cyst some fungal spores some naked virus vegetative bacteria that have higher resistance ( M. tuberculosis, S.aureus, Pseudomonas) most bacterial vegetative cells ordinary fungal spores & hypae enveloped virus Yeasts Trophozoites
  • 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.
    • 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.
  • a suffix indicating that the antimicrobial agent will kill or destroy a certain group of microorganism suffix “ cide ” – meaning to kill viricide – destroys virus fungicide – destroys fungi bactericide – destroys bacteria Suffix “ static/stasis ” – meaning to stand still a suffix indicating that the agent will prevent the growth or multiplication of the type of organism but are not killed outright bacteriostatic - prevents the growth of bacteria fungistatic – prevents the growth of fungi
    • Uses of sterilisation:
      • Sterilisation of materials, instruments used in surgical and diagnostic procedures.
      • Sterilisation of Media and reagents used in the microbiology laboratory.
      • Food and drug manufacturing to ensure safety from contaminating organisms.
  • Methods
    • Physical methods
    • Chemical methods
    • Physical methods:
        • Sunlight
        • Heat
            • Dry heat
            • Moist heat
        • Filtration
        • Radiation
    • Chemical methods:
          • Alcohols
          • Aldehydes
          • Phenols
          • Halogens
          • Oxidizing agents
          • Salts
          • Surface active agents
          • Dyes
          • Vapour phase disinfectants
  • Physical methods
    • Sun light:
      • Active germicidal effect due to its content of ultraviolet rays .
      • Natural method of sterilisation of water in tanks, rivers and lakes.
    • Heat :
      • Most reliable and commonly employed method .
      • Two types:
            • Dry heat
            • Moist heat
    • Heat :
    • Principle:
      • Dry heat kills the organism by
        • denaturation of the bacterial proteins,
        • oxidative damage
        • toxic effect of elevated levels of electrolytes .
    • Heat :
    • Principle:
      • Moist heat kills the organism by
        • denaturation of the bacterial proteins
        • coagulation of the bacterial proteins
    • Heat :
    • Factors influencing:
        • Nature of heat
        • Temperature and duration
        • Characteristic of organism and spores
        • Type of material
    • Heat :
    • Dry heat:
        • Red heat
        • Flaming
        • Incineration
        • Hot air oven
    • Dry heat:
        • Red heat: Materials are held in the flame of a bunsen burner till they become red hot.
            • Inoculating wires or loops
            • Tips of forceps
            • Needles
    • Dry heat:
        • 2. 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
    • Dry heat:
        • 3. Incineration:
        • Materials are reduced to ashes by burning.
        • Instrument used was incinerator.
            • Soiled dressings
            • Animal carcasses
            • Bedding
            • Pathological material
    • Dry heat:
        • 4. Hot air oven:
      • Hot air oven:
        • Most widely used method
        • Electrically heated and fitted with a fan to even distribution of air in the chamber.
        • Fitted with a thermostat that maintains the chamber air at a chosen temperature.
        • Temperature and time:
            • 160 C for 2 hours.
            • 170 C for 1 hour
            • 180 C for 30 minutes.
        • Uses:
          • Sterilisation of
            • Glassware like glass syringes, petridishes, pipettes and test tubes.
            • Surgical instruments like scalpels, scissors, forceps etc.
            • Chemicals like liquid paraffin, fats etc.
      • Precautions :
        • Should not be overloaded
        • Arranged in a manner which allows free circulation of air
        • Material to be sterilised 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 waremay crack by sudden cooling.
        • Sterilisation controls :
            • Spores of Bacillus subtilis subsp. niger
            • Thermocouples
            • Browne’s tube
    • Moist heat sterilisation:
        • A temperature below 100°C
        • A temperature of 100°C
        • A temperature above 100°C
        • A temperature below 100°C
            • Pasteurisation of milk
            • Inspissation
            • Vaccine bath
      • Pasteurisation of milk
        • Two types
            • Holder method ( 63°C for 30 minutes)
            • Flash method ( 72°C for 20 seconds followed by quickly cooling to 13°C)
      • Inspissation:
        • Heating at 80-85°C for half an hour daily on three consecutive days
        • Serum or egg media are sterilised
      • Vaccine bath:
        • Heating at 60°C for an hour daily in vaccine bath for several sucessive days.
        • Serum or body fluids can be sterilised by heating at 56°C for an hour daily for several sucessive days.
      • A temperature at 100°C
            • Boiling
            • Tyndallisation
            • Steam sterilisation
      • Boiling :
        • Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling.
      • Tyndallisation :
        • Steam at 100C for 20 minutes on three successive days
        • Used for egg , serum and sugar containing media.
      • Steam steriliser :
        • Steam at 100°C for 90 minutes.
        • Used for media which are decomposed at high temperature.
      • A temperature above 100°C
          • Autoclave :
          • -Steam above 100 ° C has a better killing power than dry heat.
          • -Bacteria are more susceptible to moist heat.
    • 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.
  •  
    • Sterilisation conditions:
      • Temperature – 121 °C
      • Chamber pressure -15 lb per square inch.
      • Holding time – 15 minutes
      • Others :
        • 126°C for 10 minutes
        • 133°C for 3 minutes
    • Uses :
        • Useful for materials which can not withstand high temp.
        • To sterilise culture media, rubber material, gowns, dressings, gloves etc.
    • Sterilisation controls:
        • Thermocouples
        • Bacterial spores- Bacillus stearothermophilus
        • Browne’s tube
        • Autoclave tapes
    • 3. Filtration:
        • Useful for substances which get damaged by heat.
        • To sterilise sera, sugars and antibiotic solutions.
        • To obtain bacteria free filtrates of clinical samples.
        • Purification of water.
    • Types of filters:
        • Candle filters
        • Asbestos disc filters
        • Sintered glass filters
        • Membrane filters
        • Air filters
        • Syringe filters
        • Candle filters
        • Membrane filters
    • Radiations :
        • Ionising radiations
        • Non - Ionising radiations
      • Ionising radiations:
          • X rays
          • Gamma rays
          • Cosmic rays
        • Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation)
    • Non-Ionising radiation:
          • Infra red rays
          • Ultraviolet (UV) rays
      • Infra red is used for rapid mass sterilisation of syringes and catheters.
      • Ultraviolet radiation is used for disinfecting enclosed areas such as bacterial laboratory, inoculation hood, laminar flow and operation theatres.
  • Chemical Methods
    • A variety of chemical agents are used as antiseptics and disinfectants.
    • Factors influencing the potency of a disinfectant:
        • Concentration
        • Time of action
        • pH
        • Temperature
        • Nature of organism
        • Presence of organic matter
      • High level disinfectants
      • Intermediate level disinfectants
      • low level disinfectants
    • High level disinfectants:
    • Glutaraldehyde, Hydrogen peroxide, peracitic acid and chlorine compounds.
    • Effectiveness may be equal to that of sterilisation.
    • Used for:
        • Endoscopes
        • Cystoscopes
        • Surgical instruments with plastic components
    • Intermediate level disinfectants
    • May not effective against spores
    • Includes alcohols, iodophores and phenols
    • Used for:
      • Laryngoscopes
      • Fiber optic endoscopes
    • Low level disinfectants:
      • Many organisms can survive on exposure to these disinfectants.
      • Used for items which come in contact with the patients but they do not penetrate into tissues.
      • Stethoscopes, ECG electrodes etc.
    • 1.Alcohols :
      • Ethyl alcohol and Isopropyl alcohol are commonly used.
      • Act by denaturing of bacterial proteins.
      • No sporicidal and virucidal activity.
      • Used as skin antiseptics.
      • Isopropyl alcohol is preferred to Ethyl alcohol as it is better fat solvent, more bactericidal and less volatile.
    • 2. Aldehydes:
      • Formaldehyde
      • Glutaraldehyde
      • Formaldehyde:
      • Bactericidal, sporicidal and virucidal
      • Used in both aqueous solution and gaseous forms
      • A 10% aqueous solution is routinely used.
      • Uses:
          • preservation of tissues for pathological examination
          • To sterilise bacterial vaccines
          • To prepare toxoid from toxin
          • For killing of bacterial cultures and suspensions
          • For destroying anthrax spores in hair and wool.
    • Glutaraldehyde :
    • Effective against bacteria , fungi and viruses.
    • Less toxic and irritant to eyes and skin than formaldehyde
    • Used as 2% buffered solution
    • Available commercially as CIDEX
    • Uses:
      • For sterilisation of cystoscopes, endoscopes and bronchoscopes
      • For sterilisation of plastic endotracheal tubes, face masks, rubber anesthetic tubes
    • 3.Phenols:
    • Lister,the father of antiseptic surgery, first introduced the use of phenol ( Carbolic acid) in surgery (1867).
    • Produced by distillation of coal tar between temperatures of 170°C and 270°C.
    • Bactericidal action due to cell membrane damage.
    • Commonly used Phenol derivatives are
          • cresol
          • chlorhexidine
          • chloroxylenol
          • hexachlorophanes
    • Cresols :
    • Lysol is a solution of cresols in soap.
    • Active against a wide range of organisms.
    • uses:
      • For sterilisation of infected glass ware
      • Cleaning of floors
      • Disinfection of excreta
    • Chlorhexidine:
      • Eg.Salvlon – (Chlorhexidine and cetrimide)
      • More active against Gram positive than Gram negative
      • Good fungicidal activity.
      • No action on spores and little activity against viruses
    • Chloroxylenol :
      • Eg.dettol
      • Less toxic and less irritant.
      • Readily inactivated by organic matter
      • Inactive against pseudomonas.
    • Hexachlorophane:
    • More active against Gram positive than Gram negative bacteria.
    • Bacteriostatic at high dilutions.
    • Applied on skin as prophylaxis against staphylococcal infections.
    • Potentially toxic and should be used with care.
    • 4.Halogens:
        • Chlorine
        • Iodine
      • Commonly used disinfectants
      • Bactericidal, sporicidal and virucidal
    • Chlorine :
      • In the form of
          • Bleaching powder,
          • Sodium hypochlorite
          • Chloramine
      • Disinfection is due to release of free chlorine.
      • Reacts with water to form hypochlorus acid .
      • Bactericidal, virucidal, fungicidal and sporicidal.
      • Used in water supplies, swimming pools, food and dairy industries .
    • Iodine :
        • Used as skin disinfectant.
        • Bactericidal and moderate action on spores.
        • Betadine is one example.
    • 5.Oxidising agents:
      • Hydrogen peroxide
        • Effective at concentration of 3-6%
        • Kills spores at 10- 25%
        • Used to disinfect
            • Contact lenses
            • Surgical prostheses
            • Plastic implants
    • 6.Salts:
        • Salts of heavy metals have toxic effect on bacteria.
        • Salts of copper, silver and mercury are used as disinfectants.
        • Act by coagulation of bacterial proteins.
        • Mercuric chloride, once used as disinfectant is highly toxic.
        • Thiomersol and mercurochrome are less toxic
        • Copper salts are used as fungicides.
    • 7.Surface active agents:
    • Substances which alter energy relationships at interfaces , producing a reduction of surface tension, are known as surface active agents or surfactants.
      • Anionic
      • cationic
      • nonionic
      • amphoteric compounds.
    • The anionic compounds:
    • e.g.:- common soaps,
    • Have strong detergent but weak antimicrobial properties .
    • These agents are most active at acidic pH.
    • Effective against Gram positive organisms but are relatively ineffective against Gram negative species.
    • Cationic surfactants:
    • Quaternary ammonium compounds are the most important cationic surfactants.
    • These compounds are bactericidal for a wide range of organisms, gram positive species are more susceptible.
    • The common cationic compounds are acetyl trimethyl ammonium bromide (cetavalon or cetrimide) and benzalkonium chloride.
    • Amphoteric compounds:
    • known as ‘Tego’ compounds possess detergent properties of anionic and antimicrobial activity of cationic compounds.
    • They are active against a wide range of Gram positive and Gram negative organisms and some viruses.
    • 8.Dyes :
    • Two groups of dyes have been used extensively as skin and wound antiseptics
      • aniline dyes
      • acridine dyes
    • Both are bacteriostatic in high dilution but have low bactericidal action.
    • Aniline dyes include crystal violet, brilliant green and malachite green.
    • They are more active against Gram positive bacteria than Gram negative bacteria.
    • Acridine dyes include acriflavine , euflavine, proflavine and aminacrine.
    • They are more active against Gram positive bacteria than Gram negative bacteria.
    • Gentian violet and acriflavine are two widely used dyes for skin disinfection especially in Gram positive bacterial infections.
    • 9.Vapour phase disinfectants:
        • Formaldehyde gas
        • Ethylene oxide
        • Betapropiolactone
    • Formaldehyde gas:
    • Used for fumigation of operation theatres, wards and laboratories etc.
    • It is generated by adding 150 gm of KMnO4 to 280 ml of formalin for 1000 cu. Feet of room volume.
    • The doors should be sealed and left unopened for 48 hours.
    • The gas is toxic and irritant when inhaled.
    • After completion of sterilisation the irritant vapours are nullified by exposure to ammonia vapour.
    • Ethylene oxide:
    • It is a colourless liquid with a boiling point of 10.7°C.
    • Effective against all types of organisms including viruses and spores.
    • It has a potential toxicity to human beings, including mutagenicity and carcinogenicity .
    • It is highly inflamable.
    • Used for sterilising plastic and rubber articles, respirators, heart lung machines, sutures, dental equipments etc.
    • Betapropiolactone :
    • Condensation product of ketane and formaldehyde.
    • It has a boiling point of 163°C.
    • Used in 0.2%.
    • Effective against all types of organisms including viruses.
    • More efficient for fumigation than formaldehyde.
    • Used for inactivation of vaccines.
  • Materials Method 1 Inoculating wires and loops Red heat 2 Glass ware- syringes, petridishes, testtubes, flasks etc. Hot –air oven 3 Disposable syringes, and other disposable items Gamma radiation 4 Culture media Autoclaving 5 Culture media containing serum and egg Tyndallisation 6 Toxin , serum, sugar, and antibiotic solutions Filtration 7 Cystoscope and endoscope Glutaraldehyde 8 Infected soiled dressings Incineration 9 Skin Iodine, alcohol 10 Milk Pasteurisation
  •