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sterilization and disinfection


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This is a series of lectures on microbiology useful for undergraduate medical and paramedical students

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sterilization and disinfection

  1. 1. STERILIZATION AND DISINFECTION Dr. Ashish Jawarkar M.D. Parul Sevashram Hospital
  2. 2. INTRODUCTION Microorganisms are the agents of contamination, infection, and decay.  Hence it becomes necessary to remove them from materials and areas.  Early civilization practiced salting, smoking, pickling and exposure to sunlight . 
  3. 3. In mid 1800s Lister developed Aseptic techniques to prevent contamination of surgical wounds. Prior to this development: • Nosocomial infections caused death in 10% of surgeries. • Up to 25% mothers delivering in hospitals died due to infection.
  4. 4. DEFINITION STERILIZATION The process of freeing an article from microorganisms including their spores.
  5. 5. DISINFECTION: Reducing the number of pathogenic microorganisms to the point where they no longer cause diseases.
  6. 6. Sepsis: Comes from Greek for decay or putrid. Indicates bacterial contamination. Asepsis: Absence of significant contamination.  Aseptic techniques are used to prevent contamination of surgical instruments, medical personnel, and the patient during surgery.  Aseptic techniques are also used to prevent bacterial contamination in food industry.
  7. 7. Bacteriostatic Agent: An agent that inhibits the growth of bacteria, but does not necessarily kill them.
  8. 8. Bactericide: An agent that kills bacteria. Most do not kill Endospores. Sporicide: An agent that kills spores.
  9. 9. Methods of Sterilisation Microbial Control Methods Physical Agents Chemical Agents Mechanical Remova Methods
  10. 10. Physical Agents Heat Dry Radiation Moist Incineration Steam Under Pressure Dry Oven Ionizing Non Ionizing X Ray, Cathode, Gamma UV Sterilization Boiling Water/Hot Water Pasteurization Disinfection Sterilization Disinfection
  11. 11. Chemical Agent Gas Disinfection erilization Animate Liquids Inanimate Chemotherapy Antiseptics Sterilization Disinfectio
  12. 12. Mechanical Removal Methods Filtration Air Liquids Disinfection Sterilization
  13. 13. Physical Methods of Sterilisation Sterilisation By Dry Heat: • 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, Hot Air Oven
  14. 14. Uses:  To sterilise Forceps, Scissors, Scalpels, Swabs.  Pharmaceuticals products like Liquid paraffin, dusting powder, fats and grease.
  15. 15. FLAMING  Inoculation loop or Wire, the tip of Forceps and spatulas are held in a bunsen flame till they are red hot.
  16. 16. Flaming
  17. 17. INCINERATION This is an excellent method of destroying materials such as contaminated cloth, animal carcasses and pathological materials.
  18. 18. Incineration
  19. 19. Dry heat - HOT AIR OVEN  Holding temp & time: 1600C for 1 hr  Used for glassware, forceps, swabs, water impermeable oils, waxes & powders  Before placing in hot air oven  Dry glassware completely  Plug test tubes with cotton wool  Wrap glassware in Kraft papers  Don’t over load the oven  Allow free circulation of air between the material
  20. 20. Dry heat - HOT AIR OVEN  Sterilization controls: to check whether the equipment is working properly  Chemical  Color controls: Browne’s tubes change from red to green  Thermocouples  Biological controls: paper strips containing106 spores of Clostridium tetani  Place strips in oven along with other material for the sterilization  Later culture the strips in thioglycollate broth or RCM at 37 0C for 5 days  Growth in medium indicates failure of sterilization
  21. 21. Dry heat – Hot air oven
  22. 22. MOIST HEAT STERILIZATION Kills microorganisms by coagulating their proteins.
  23. 23. MOIST HEAT STERILISATION IS CARRIED OUT WITH FOLLOWING METHODS  Temp below 100oC: “Pasteurisation”, Inspissator.  Temperature at 100oC: Boiling.  Steam at atmospheric pressure: Koch/Arnold’s steamer.  Steam under pressure: Autoclave.
  24. 24. Pasteurisation  Process of killing of pathogens in the milk but does not sterilize it .  Milk is heated at 63oC for 30 mins. (HOLDER METHOD)  At 72oC for 15-20 Sec. Rapid cooling to 13oC (FLASH PROCESS)
  25. 25. Water Bath
  26. 26. HOT WATER BATH  To inactivate non sporing bacteria for the preparation of vaccines - Special vaccine bath at 60oC for one hour is used  Serum or body fluids containing coagulable proteins can be sterilized by heating for 1 hr at 56oC in a water bath for several successive days.
  27. 27. Inspissator
  28. 28. INSPISSATOR  Sterilizes by heating at 80-85oC for half an hour for 3 successive days  Used to sterilize media such as Lowenstein-Jensen & Loefller’s serum
  29. 29. TEMPERATURE AT 100OC Boiling: Kills vegetative forms of bacterial pathogens.  Hepatitis virus: Can survive up to 30 minutes of boiling. Endospores: Can survive up to 20 hours or more of boiling
  30. 30. Steam Sterilizer
  31. 31. STEAM AT ATMOSPHERIC PRESSURE  Steam is generated using a steamer (Koch/ Arnold)  Consists of a Tin cabinet  Has a conical lid to enable the drainage of condensed steam  Perforated tray above ensures materials are surrounded by steam.  For routine sterilization exposure of 90 mins is used
  32. 32.  For media containing sugar and gelatin exposure of 100oC for 20 min for 3 successive days is used  The process is termed as Tyndallisation /Intermittent Sterilization
  33. 33. STEAM UNDER PRESSURE - AUTOCLAVE  Works on the principle of Steam under pressure  Invented by Charles Chamberland in 1879.
  34. 34. AUTOCLAVE
  35. 35.  Autoclave consists of a vertical or a horizontal cylinder.  One end has an opening which is meant for keeping materials to be sterilised.  The lid is provided with a Pressure gauge, to measure the pressure  A safety valve is present to permit the escape of steam from the chamber
  36. 36.  Articles to be sterilised are placed in the basket provided  Sterilisation is carried out under pressure at 121º for 15 mnts.
  37. 37. CHEMICAL AGENTS Chemical agents act by  Protein coagulation  Disruption of the cell membrane  Removal of Sulphydryl groups  Substrate competition
  38. 38. ALCOHOLS  Ethanol /Isopropyl alcohol are frequently used  No action on spores  Concentration recommended 60-90% in water Uses  Disinfection of clinical thermometer.  Disinfection of the skin – Venupuncture
  39. 39. ALDEHYDES  Formaldehyde & Glutaraldehyde are frequently used  Formaldehyde is bactericidal, sporicidal & has a lethal effect on viruses.  Glutaraldehyde is effective against Tubercle bacilli, fungi and viruses
  40. 40. USES FORMALDEHYDE  To preserve anatomical specimens  Destroying Anthrax spores in hair and wool  10% Formalin+0.5% Sodium tetra borate is used to sterilise metal instruments
  41. 41. USES GLUTARALDEHYDE  Used to treat corrugated rubber anesthetic tubes, Face masks, Plastic endotracheal tubes, Metal instruments and polythene tubing
  42. 42. DYES Two groups of dyes are used Aniline dyes Acridine dyes
  43. 43. ANILINE DYES  Are Brilliant green, Malachite green & Crystal violet  Active  No against Gram positive bacteria activity against tubercle bacilli
  44. 44. ACRIDINE DYES  Acridine dyes in use are orange in colour  Effective against Gram positive than Gram negative  Important dyes are Proflavine, Acriflavine,Euflavine
  45. 45. HALOGENS  Iodine in aqueous and alcoholic solution has been used widely as a skin disinfectant  Actively bactericidal with moderate against spores  Chlorine and its compounds have been used as disinfectants in water supplies & swimming pools
  46. 46. PHENOLS  Obtained by distillation of coal tar  Phenols are powerful microbicidal substances  Phenolic derivatives have been widely used as disinfectants for various purposes in hospitals  Eg: Lysol, cresol
  47. 47. USES  Various combinations are used in the control of pyogenic cocci in surgical & neonatal units in hospitals.  Aqueous solutions are used in treatment of wounds
  48. 48. GASES Ethylene Oxide Colourless ,Highly penetrating gas with a sweet ethereal smell. Effective against all types of microorganisms including viruses and spores
  49. 49. USES  Specially used for sterilising heart-lung machines,respirators,sutures,dental equipments, books and clothing.  Also used to sterilise Glass, metal and paper surfaces ,plastics, oil,some foods and tobacco.
  50. 50. FORMALDEHYDE GAS  Widely employed for fumigation of operation theatres and other rooms
  51. 51. BETA PROPIOLACTONE  Used  For in fumigation sterilisation 0.2% BPL is used  Has a rapid biocidal activity  Very effective against viruses
  52. 52. SURFACE ACTIVE AGENTS AND METALLIC SALTS Substances which reduce the surface tension – Surface active agents
  53. 53.  Cations are widely used in the form of quaternary ammonium compounds.  Markedly bactericidal, active against Gram positive organisms.  No action on spores, tubercle bacilli, viruses
  54. 54. METALLIC SALTS  The salts of silver, copper and mercury are used as disinfectants.  Act by coagulating proteins  Marked bacteriostatic, weak bactericidal and limited fungicidal activity
  55. 55. Sterilisation by filtration Filtration helps to remove bacteria from heat labile liquids such as sera and solutions of sugar, Antibiotics. The following filters are used Candle filters Asbestos filters Sintered glass filter Membrane filters
  57. 57. CANDLE FILTERS  Widely used for purification of water Two types (a) Unglazed ceramic filter – Chamberland filter (b) Diatomaceous earth filters – Berkefeld filter
  59. 59. ASBESTOS FILTER  Disposable  High single use discs adsorbing tendency  Carcinogenic Eg: Seitz filter
  61. 61. SINTERED GLASS FILTER  Prepared by heat fusing powdered glass particles of graded size  Cleaned easily, brittle, expensive.
  63. 63. MEMBRANE FILTERS  Made of cellulose esters or other polymers Uses  Water purification & analysis  Sterilization & sterility testing  Preparation of solutions for parenteral use
  64. 64. RADIATION Two types of radiations are used NON –IONISING IONISING
  65. 65. Non- Ionising radiation:  Electromagnetic  Absorbed  Can rays with longer wavelength as heat be considered as hot air sterilisation  Used in rapid mass sterilisation of prepacked Syringes and catheters Eg: UV rays
  66. 66. IONISING RADIATIONS  X- rays, gamma rays & cosmic rays.  High penetrative power  No appreciable increase in the temperature – COLD STERILISATION  Sterilise plastics Syringes, catheters, grease fabrics metal foils
  67. 67. ULTRASONIC AND SONIC VIBRATION  Bactericidal  Microorganisms vary in their sensitivity, hence no practical value in sterilisation and disinfection
  68. 68. THANK YOU