Sterilization physical methods

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Sterilization physical methods

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Sterilization physical methods

  1. 1. STERILIZATION BY PHYSICAL METHODS Dr.T.V.Rao MD12/2/2012 Dr.T.V.Rao MD 1
  2. 2. Why we need Sterilization• 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.12/2/2012 Dr.T.V.Rao MD 2
  3. 3. How can microorganisms be killed? Denaturation of proteins (e.g. wet heat, ethylene oxide) Oxidation (e.g. dry heat, hydrogen peroxide) Filtration Interruption of DNA synthesis/repair (e.g. radiation) Interference with protein synthesis (e.g. bleach) Disruption of cell membranes (e.g. phenols)12/2/2012 Dr.T.V.Rao MD 3
  4. 4. Classification There are two types of 2. Chemical sterilization sterilization: physical and includes: chemical.  Alcohols1. Physical sterilization  Aldehydes includes:  Phenolics heat  Oxidizing agents radiation  Quaternary ammonium filtration compounds  ethylene oxide gas  Others12/2/2012 Dr.T.V.Rao MD 4
  5. 5. 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.12/2/2012 Dr.T.V.Rao MD 5
  6. 6. Antiseptics :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.12/2/2012 Dr.T.V.Rao MD 6
  7. 7. Factors that influence efficacy of disinfection/sterilization Contact time Physico-chemical environment (e.g. pH) 3 Presence of organic material 4 Temperature 5 Type of microorganism 6 Number of microorganisms 7 Material composition12/2/2012 Dr.T.V.Rao MD 7
  8. 8. Uses of sterilisation: 1. Sterilisation of materials, instruments used in surgical and diagnostic procedures. 2. Sterilisation of Media and reagents used in the microbiology laboratory. 3. Food and drug manufacturing to ensure safety from contaminating organisms.12/2/2012 Dr.T.V.Rao MD 8
  9. 9. Understanding the Terminology• 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• fungi static – prevents the growth of fungi 12/2/2012 Dr.T.V.Rao MD 9
  10. 10. Relative Resistance of Microbial FormsHighest resistance Moderate resistance Least resistancebacterial endospore protozoan cyst most bacterial vegetative cells(Bacillus & Clostridium) some fungal spores ordinary fungal spores & hypae some naked virus enveloped virus vegetative bacteria that Yeasts have higher resistance Trophozoites ( M. tuberculosis, S.aureus, Pseudomonas) 12/2/2012 Dr.T.V.Rao MD 10
  11. 11. What to sterilize?• It is mandatory to sterilize : – all instruments that penetrate soft tissues and bone. – Instruments that are not intended to penetrate the tissues, but that may come into contact with oral tissues.• If the sterilization procedure may damage the instruments, then, sterilization can be replaced by Disinfection procedure12/2/2012 Dr.T.V.Rao MD
  12. 12. Ideal sterilization/disinfection process• Highly efficacious• Fast• Good penetrability• Compatible with all materials• Non-toxic• Effective despite presence of organic material• Difficult to make significant mistakes in process• Easily monitored12/2/2012 Dr.T.V.Rao MD 12
  13. 13. Control of Microbial Growth: Rate of Microbial Death When bacterial populations are heated or treated antimicrobial chemicals, they usually die at a constant rate.12/2/2012 Dr.T.V.Rao MD 13
  14. 14. Figure 9.1 A plot of microbial death rateNumber of living microbes 90% die Constant percentage of the extant population 1 min is killed each minute 90% die 1 min Time (min) 12/2/2012 Dr.T.V.Rao MD 14
  15. 15. Methods1.Physical methods2.Chemical methods12/2/2012 Dr.T.V.Rao MD 15
  16. 16. Physical methods:• Physical methods: 1.Sunlight 2.Heat 1.Dry heat 2.Moist heat 3.Filtration 4.Radiation12/2/2012 Dr.T.V.Rao MD 16
  17. 17. Chemical methods• Chemical methods: 1. Alcohols 2. Aldehydes 3. Phenols 4. Halogens 5. Oxidizing agents 6. Salts 7. Surface active agents 8. Dyes 9. Vapor phase disinfectants12/2/2012 Dr.T.V.Rao MD 17
  18. 18. Physical Methods12/2/2012 Dr.T.V.Rao MD 18
  19. 19. How to Sterilize Materials Method1 Inoculating wires and loops Red heat2 Glass ware- syringes, petridishes, testtubes, flasks Hot –air oven etc.3 Disposable syringes, and other disposable items Gamma radiation4 Culture media Autoclaving5 Culture media containing serum and egg Tyndallisation6 Toxin , serum, sugar, and antibiotic solutions Filtration7 Cystoscope and endoscope Glutaraldehyde8 Infected soiled dressings Incineration9 Skin Iodine, alcohol 12/2/2012 Dr.T.V.Rao MD 1910 Milk Pasteurisation
  20. 20. Physical Methods of Microbial Control Dry Heat:Direct Flaming: Used to sterilize inoculating loops and needles. Heat metal until it has a red glow.Incineration: Effective way to sterilize disposable items (paper cups, dressings) and biological waste.Hot Air Sterilization: Place objects in an oven. Require 2 hours at 170oC for sterilization. Dry heat is transfers heat less effectively to a cool body, than moist heat.12/2/2012 Dr.T.V.Rao MD 20
  21. 21. Physical Methods of Microbial Control• Heat-Related Methods – Moist heat • Pasteurization – Used for milk, ice cream, yogurt, and fruit juices – Not sterilization » Heat-tolerant microbes survive – Pasteurization of milk » Batch method » Flash pasteurization » Ultrahigh-temperature pasteurization 12/2/2012 Dr.T.V.Rao MD 21© 2012 Pearson Education Inc.
  22. 22. Physical Methods of Microbial Control Moist Heat (Continued):Pasteurization: Developed by Louis Pasteur to prevent the spoilage of beverages. Used to reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.  Classic Method of Pasteurization: Milk was exposed to 65oC for 30 minutes.  High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72oC for 15 seconds.12/2/2012 Dr.T.V.Rao MD 22
  23. 23. Inspissation: 1. Inspissation:  Heating at 80-85°C for half an hour daily on three consecutive days  Serum or egg media are sterilised 2. Vaccine bath:  Heating at 60°C for an hour daily in vaccine bath for several successive days.  Serum or body fluids can be sterilised by heating at 56°C for an hour daily for several successive days.12/2/2012 Dr.T.V.Rao MD 23
  24. 24. Sun light:• Sun light: – Active germicidal effect due to its content of ultraviolet rays . – Natural method of sterilisation of water in tanks, rivers and lakes.12/2/2012 Dr.T.V.Rao MD 24
  25. 25. Heat : • Factors influencing: • Nature of heat • Temperature and duration • Characteristic of organism and spores • Type of material12/2/2012 Dr.T.V.Rao MD 25
  26. 26. Heat effectively kills Majority of Microbes Heat :• Principle: – Dry heat kills the organism by • denaturation of the bacterial proteins, • oxidative damage • toxic effect of elevated levels of electrolytes. 12/2/2012 Dr.T.V.Rao MD 26
  27. 27. Heat :• Dry heat: 1.Red heat 2.Flaming 3.Incineration 4.Hot air oven12/2/2012 Dr.T.V.Rao MD 27
  28. 28. Dry-Heat Sterilization• Involves heating at atmospheric pressure and often use a fan to obtain uniform temperature by circulation.• Heat at 180º for half hour , 170º for 1 hr., or 160º C for 2 hrs.• Times are the periods during which object is maintained at the respective temp.12/2/2012 Dr.T.V.Rao MD
  29. 29. Dry heat:• Dry heat: 1. Red heat: Materials are held in the flame of a bunsen burner till they become red hot. » Inoculating wires or loops » Tips of forceps » Needles12/2/2012 Dr.T.V.Rao MD 29
  30. 30. Dry heat: • 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 tubes12/2/2012 Dr.T.V.Rao MD 30
  31. 31. Incineration: • Materials are reduced to ashes by burning. • Instrument used was incinerator. • Soiled dressings • Animal carcasses • Bedding12/2/2012 • Pathological material Dr.T.V.Rao MD 31
  32. 32. Dry-Heat Sterilization Disadvantages • Disadvantages: –Less reliable than autoclaving –Large temp difference may arise within device. –sharp instruments get dulled –Many materials do not tolerate dry heat12/2/2012 Dr.T.V.Rao MD
  33. 33. 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.12/2/2012 Dr.T.V.Rao MD 33
  34. 34. Uses of Hot Air Oven – Sterilisation of 1.Glassware like glass syringes, petri dishes, pipettes and test tubes. 2.Surgical instruments like scalpels, scissors, forceps etc. 3.Chemicals like liquid paraffin, fats etc.12/2/2012 Dr.T.V.Rao MD 34
  35. 35. – Precautions : 1. Should not be overloaded 2. Arranged in a manner which allows free circulation of air 3. Material to be sterilized should be perfectly dry. 4. Test tubes, flasks etc. should be fitted with cotton plugs. 5. petridishes and pipetts should be wrapped in paper. 6. Rubber materials and inflammable materials should not be kept inside. 7. The oven must be allowed to cool for two hours before opening, since glass ware may crack by12/2/2012 sudden cooling. Dr.T.V.Rao MD 35
  36. 36. Sterilisation controls : • Sterilisation controls 1.Spores of Bacillus subtilis subsp. niger 2. Thermocouples 3.Browne’s tube12/2/2012 Dr.T.V.Rao MD 36
  37. 37. Sterilizing below100°C 1. temperature below 100° Pasteurization of milk 1.Inspissation 2.Vaccine bath12/2/2012 Dr.T.V.Rao MD 37
  38. 38. Principle of Pasteurization12/2/2012 Dr.T.V.Rao MD 38
  39. 39. A temperature at 100°C II. A temperature at 100°C 1. Boiling 2. Tyndallisation 3. Steam sterilisation12/2/2012 Dr.T.V.Rao MD 39
  40. 40. Boiling : 1 Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling. 2. Tyndallisation :  Steam at 100C for 20 minutes on three successive days  Used for egg , serum and sugar containing media. 3. Steam sterilizer :  Steam at 100°C for 90 minutes.  Used for media which are decomposed at high temperature.12/2/2012 Dr.T.V.Rao MD 40
  41. 41. Temperatures above 100°C III. 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.12/2/2012 Dr.T.V.Rao MD 41
  42. 42. Components of autoclave:• 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.12/2/2012 Dr.T.V.Rao MD 42
  43. 43. Figure 9.6 Autoclave-overview12/2/2012 Dr.T.V.Rao MD 43
  44. 44. Autoclave: Closed Chamber withHigh Temperature and Pressure
  45. 45. 12/2/2012 Dr.T.V.Rao MD 45
  46. 46. Sterilisation conditions• 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 minutes12/2/2012 Dr.T.V.Rao MD 46
  47. 47. Sterilization – instrument Packing• Often instruments are packed for sterilization to be stored and handled without being contaminated.• Packing depend on the intended shelf life after sterilization.• The available packing options are: – Textile has shelf life of 1 month – Paper has shelf life of 1 – 6 months – Nylon, glass, and metal have shelf life of 1 year if tightly closed12/2/2012 Dr.T.V.Rao MD
  48. 48. Uses of Autoclaves:• Uses : 1. Useful for materials which can not withstand high temp. 2. To sterilize culture media, rubber material, gowns, dressings, gloves etc.12/2/2012 Dr.T.V.Rao MD 48
  49. 49. Sterilisation controls:• Sterilisation controls: 1. Thermocouples 2. Bacterial spores- Bacillus stearothermophilus 3. Browne’s tube 4. Autoclave tapes12/2/2012 Dr.T.V.Rao MD 49
  50. 50. Sterility Controls Yellow medium means spores areCap that allows viable; autoclavedsteam to penetrate objects are not sterile.Flexible plasticvialCrushable glass IncubationampuleNutrient mediumcontaining pHcolor indicator Red medium means spores were After autoclaving, flexible killed; autoclavedEndospore strip vial is squeezed to break objects are ampule and release sterile. medium onto spore strip. 12/2/2012 Dr.T.V.Rao MD 50
  51. 51. Filtration:• . Filtration: • Useful for substances which get damaged by heat. • To sterilize sera, sugars and antibiotic solutions. • To obtain bacteria free filtrates of clinical samples.12/2/2012 • Purification of water. Dr.T.V.Rao MD 51
  52. 52. FILTRATION STERILIZATION This method is commonly used for  To ensure sterility, the filtration sensitive pharmaceuticals and protein system must be tested to ensure that solutions in biological research. the membranes have not been A filter with pore size 0.2 µm will punctured prior to or during use. effectively remove bacteria.  To ensure the best results, If viruses must also be removed, a pharmaceutical sterile filtration is much smaller pore size around 20 nm performed in a room with highly is needed. filtered air (HEPA filtration) or in a Prions are not removed by filtration. laminar flow cabinet or "flowbox", a device which produces a laminar The filtration equipment and the stream of HEPA filtered air. filters themselves may be purchased as presterilized disposable units in  HEPA filters are critical in the sealed packaging, prevention of the spread of airborne bacterial and viral organisms and, or must be sterilized by the user, therefore, infection. Typically, generally by autoclaving at a medical-use HEPA filtration systems temperature that does not damage also incorporate high-energy ultra- the fragile filter membranes. violet light units to kill off the live bacteria and viruses trapped by the filter media.
  53. 53. Several Types of Filters• Types of filters: 1. Candle filters 2. Asbestos disc filters 3. Sintered glass filters 4. Membrane filters 5. Air filters 6. Syringe filters12/2/2012 Dr.T.V.Rao MD 53
  54. 54. Filtration Sterilize solutions that may be damaged or denatured by high temperatures or chemical agents.12/2/2012 Dr.T.V.Rao MD 54
  55. 55. The filtering Depends on Pore Size The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 μm (filtration membranes are most popular for this purpose).12/2/2012 Dr.T.V.Rao MD 55
  56. 56. Candle filters12/2/2012 Dr.T.V.Rao MD 56
  57. 57. The roles of HEPA filters in biological flow Safety glass Exhaust HEPA viewscreen filter safety cabinets Blower Supply HEPA filter Light High-velocity air barrier12/2/2012 Dr.T.V.Rao MD 57
  58. 58. Radiations : • Radiations : •Ionizing radiations •Non - Ionizing radiations12/2/2012 Dr.T.V.Rao MD 58
  59. 59. Ionising radiations: – Ionizing radiations: 1. X rays 2. Gamma rays 3. Cosmic rays • Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation)12/2/2012 Dr.T.V.Rao MD 59
  60. 60. Physical Methods of Microbial Control • Radiation – Nonionizing radiation • Wavelengths greater than 1 nm • Excites electrons, causing them to make new covalent bonds – Affects 3-D structure of proteins and nucleic acids • UV light causes pyrimidine dimers in DNA • UV light does not penetrate well • Suitable for disinfecting air, transparent fluids, and surfaces of objects 12/2/2012 Dr.T.V.Rao MD 60© 2012 Pearson Education Inc.
  61. 61. Forms of Radiation12/2/2012 Dr.T.V.Rao MD 61
  62. 62. Physical Methods of Microbial Control: Radiation: Three types of radiation kill microbes: Ultraviolet light (Nonionizing Radiation): Wavelength is longer than 1 nanometer. Damages DNA by producing thymine dimers, which cause mutations. Used to disinfect operating rooms, nurseries, cafeterias. Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and cloth.12/2/2012 Dr.T.V.Rao MD 62
  63. 63. Non-Ionising radiation: 1. Infra red rays 2. 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.12/2/2012 Dr.T.V.Rao MD 63
  64. 64. Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students Email doctortvrao@gmail.com12/2/2012 Dr.T.V.Rao MD 64

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