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Sterilization and disinfection- Phrmaceutical Microbiology
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- 2. INTRODUCTION • Microorganisms arethe agents of contamination, infection, and decay. • Hence it becomes necessary to remove them from materials and areas. 2
- 3. STERILIZATION The process offreeing an article from microorganisms including their spores.
- 4. DISINFECTION: Reducing the numberof pathogenic microorganisms to the point where they no longer cause diseases. 4
- 5. Bacteriostatic Agent: An agentthat inhibits the growth of bacteria, but does not necessarily kill them. 5
- 6. Bactericide: An agent thatkills bacteria. Most do not kill Endospores. Sporicide: An agent that kills spores. 6
- 7. Methods of Sterilisation MicrobialControl Methods Physical Agents Chemical Agents Mechanical Remova Methods 7
- 8. Physical Agents Heat Radiation DryMoist Incineration Dry Oven Steam Under Pressure Boiling Water/Hot Water Pasteurization Sterilization Ionizing Non Ionizing X Ray, Cathode, Gamma Disinfection UV Sterilization Disinfection 8
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- 10. Mechanical Removal Methods Filtration Air Liquids DisinfectionSterilization 10
- 11. Physical Methods ofSterilisation 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, 11
- 12. Uses: • To steriliseForceps, Scissors, Scalpels, Swabs, glassware • Pharmaceuticals products like Liquid paraffin, dusting powder, fats and grease. 12
- 13. FLAMING • Inoculation loopor Wire, the tip of Forceps and spatulas, glass test tube and conical flask mouths are held in a bunsen flame till they are red hot. 13
- 14. INCINERATION This is anexcellent method of destroying materials such as contaminated cloth, animal carcasses and pathological materials. 14
- 15. MOIST HEAT STERILIZATION Killsmicroorganisms by coagulating their proteins. 15
- 16. MOIST HEAT STERILISATIONIS 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. 16
- 17. Pasteurisation •Process of killingof 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) 17
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- 19. 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 coaguable proteins can be sterilized by heating for 1 hr at 56oC in a water bath for several successive days. 19
- 20.
- 21. INSPISSATOR • Sterilizes byheating at 80-85oC for half an hour for 3 successive days • Used to sterilize media such as Lowenstein-Jensen & Loefller’s serum 21
- 22. TEMPERATURE AT 100OC Boiling: Killsvegetative 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 22
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- 24. STEAM AT ATMOSPHERICPRESSURE • 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 24
- 25. • For mediacontaining sugar and gelatin exposure of 100oC for 20 min for 3 successive days is used • The process is termed as Tyndallisation /Intermittent Sterilization 25
- 26. STEAM UNDER PRESSURE- AUTOCLAVE • Works on the principle of Steam under pressure •Invented by Charles Chamberland in 1879. 26
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- 28. • Autoclave consistsof 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 28
- 29. • Articles tobe sterilised are placed in the basket provided • Sterilisation is carried out under pressure at 121º for 15 mnts. 29
- 30. METHODS OF TESTINGTHE EFFECTIVENES OF AUTOCLAVES ARE AS FOLLOWS: 1. BOWIE DICK TEST PACK – It is a large pack with a chemical indicator both on outside and the inside to verify that steam has penetrated the pack. 2. MECHANICAL- Chart and gauges usually carried out by Biomed Engineer. 30
- 31. 3. CHEMICAL- bythe use of autoclave tapes, strips and card. A daily test in an empty chamber using a heat sensitive tape. This is for high vacuum/high pressure autoclaves. Ex. Routine use of Browne's TST strips or tube. 31
- 32. CHEMICAL AGENTS Chemical agentsact by • Protein coagulation • Disruption of the cell membrane • Removal of Sulphydryl groups • Substrate competition 32
- 33. ALCOHOLS • Ethanol /Isopropylalcohol are frequently used • No action on spores • Concentration recommended 60-90% in water Uses • Disinfection of clinical thermometer. • Disinfection of the skin – Venupuncture 33
- 34. 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 34
- 35. USES FORMALDEHYDE • To preserveanatomical specimens • Destroying Anthrax spores in hair and wool • 10% Formalin+0.5% Sodium tetra borate is used to sterilise metal instruments 35
- 36. USES GLUTARALDEHYDE • Used totreat corrugated rubber anesthetic tubes, Face masks, Plastic endotracheal tubes, Metal instruments and polythene tubing 36
- 37. DYES Two groups ofdyes are used Aniline dyes Acridine dyes 37
- 38. ANILINE DYES • AreBrilliant green, Malachite green & Crystal violet • Active against Gram positive bacteria • No activity against tubercle bacilli 38
- 39. ACRIDINE DYES • Acridinedyes in use are orange in colour • Effective against Gram positive than Gram negative • Important dyes are Proflavine, Acriflavine,Euflavine 39
- 40. HALOGENS • Iodine inaqueous 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 40
- 41. PHENOLS • Obtained bydistillation of coal tar • Phenols are powerful microbicidal substances • Phenolic derivatives have been widely used as disinfectants for various purposes in hospitals • Eg: Lysol, cresol 41
- 42. USES • Various combinationsare used in the control of pyogenic cocci in surgical & neonatal units in hospitals. • Aqueous solutions are used in treatment of wounds 42
- 43. GASES Ethylene Oxide •Colourless ,Highlypenetrating gas with a sweet ethereal smell. •Effective against all types of microorganisms including viruses and spores 43
- 44. USES • Specially usedfor 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. 44
- 45. FORMALDEHYDE GAS • Widelyemployed for fumigation of operation theatres and other rooms 45
- 46. BETA PROPIOLACTONE • Usedin fumigation • For sterilisation 0.2% BPL is used • Has a rapid biocidal activity • Very effective against viruses 46
- 47. SURFACE ACTIVE AGENTSAND METALLIC SALTS Substances which reduce the surface tension – Surface active agents 47
- 48. • Cations arewidely used in the form of quaternary ammonium compounds. • Markedly bactericidal, active against Gram positive organisms. • No action on spores, tubercle bacilli, viruses 48
- 49. METALLIC SALTS • Thesalts of silver, copper and mercury are used as disinfectants. • Act by coagulating proteins • Marked bacteriostatic, weak bactericidal and limited fungicidal activity 49
- 50. Sterilisation by filtration Filtrationhelps 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 50
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- 52. CANDLE FILTERS • Widelyused for purification of water Two types (a) Unglazed ceramic filter – Chamberland filter (b) Diatomaceous earth filters – Berkefeld filter 52
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- 54. ASBESTOS FILTER • Disposablesingle use discs • High adsorbing tendency • Carcinogenic Eg: Seitz filter 54
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- 56. SINTERED GLASS FILTER •Prepared by heat fusing powdered glass particles of graded size • Cleaned easily, brittle, expensive. 56
- 57.
- 58. MEMBRANE FILTERS • Madeof cellulose esters or other polymers Uses • Water purification & analysis • Sterilization & sterility testing • Preparation of solutions for parenteral use 58
- 59. RADIATION Two types ofradiations are used NON –IONISING IONISING 59
- 60. Non- Ionising radiation: •Electromagnetic rays with longer wavelength • Absorbed as heat • Can be considered as hot air sterilisation • Used in rapid mass sterilisation of prepacked Syringes and catheters Eg: UV rays 60
- 61. 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 61
- 62. ULTRASONIC AND SONICVIBRATION • Bactericidal • Microorganisms vary in their sensitivity, hence no practical value in sterilisation and disinfection 62
- 63. Sterilization methods Method Mechanismof action Apparatus to be sterilized Dry heat Oxidation of proteins Metals, Glassware, Ointment, Oils, Waxes, Powders i.e. items that are lacking water Moist heat Coagulation of proteins Aqueous solutions and suspensions, surgical dressing, metal instruments. Gaseous sterilization Ethylene oxide It is an alkylating agent. It acts by alkylating sulfydryl-, amino-, carboxyl- and hydroxyl- groups. heat labile articles such as bedding, textiles, rubber, plastics, syringes, disposable petri dishes, complex apparatus like heart-lung machine, respiratory and dental equipments. Formaldehyde Alkylating agent Fumigation of rooms and cabinets UV radiation dimer formation of pyrimidine, nucleotide damage Operating Rooms and T.B. laboratories Ionizing radiation (gamma radiations) Denaturation of essential cell constituents like enzymes, DNA sterilization of disposable plastic syringes, gloves, specimens containers and Petri Dishes. Filtration Retention Of Mo’s for heat labile substances e.g. sera, antibiotics. 63
- 64. Sterility test Media usedTest used incubation Test used Incubation Fluid thioglycolate media Membrane filtration 30-35˚C NLT 7days direct inoculation 30-35˚C NLT 14days Alternate Fluid thioglycolate media 30-35˚C NLT 7days 30-35˚C NLT 14 days Soyabean casein digest media 20-25˚C NLT 7days 20-25˚C NLT 14days S.No. media Test organism Fluid thioglycolate media Bacillus subtilis Staphylococcus aureus Pseudomonas aeruginosa Alternative Thioglycollate Bacteroides vulgatus Clostridium sporogenes Soyabean casein digest media 64
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