Sterilization physical methods

Sterilization physical methods

1. STERILIZATION BY PHYSICAL METHODS Dr.T.V.Rao MD 12/2/2012 Dr.T.V.Rao MD 1

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. 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. Classification There are two types of sterilization: physical and chemical. Physical sterilization includes: heat radiation filtration 2. Chemical sterilization includes: Alcohols Aldehydes Phenolics Oxidizing agents Quaternary ammonium compounds ethylene oxide gas Others 12/2/2012 Dr.T.V.Rao MD 4

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. 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. 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 composition 12/2/2012 Dr.T.V.Rao MD 7

8. 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. 12/2/2012 Dr.T.V.Rao MD 8

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. 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 12/2/2012 Dr.T.V.Rao MD 10

11. 12/2/2012 Dr.T.V.Rao MD 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 procedure

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 monitored 12/2/2012 Dr.T.V.Rao MD 12

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. Figure 9.1 A plot of microbial death rate 90% die 1 min 90% die 1 min Constant percentage of the extant population is killed each minute Time (min) Number of living microbes 12/2/2012 Dr.T.V.Rao MD 14

15. Methods Physical methods Chemical methods 12/2/2012 Dr.T.V.Rao MD 15

16. Physical methods: Physical methods: Sunlight Heat Dry heat Moist heat Filtration Radiation 12/2/2012 Dr.T.V.Rao MD 16

17. Chemical methods Chemical methods: Alcohols Aldehydes Phenols Halogens Oxidizing agents Salts Surface active agents Dyes Vapor phase disinfectants 12/2/2012 Dr.T.V.Rao MD 17

18. Physical Methods 12/2/2012 Dr.T.V.Rao MD 18

19. 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 How to Sterilize 12/2/2012 Dr.T.V.Rao MD 19

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 170 o C 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. 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 © 2012 Pearson Education Inc. 12/2/2012 Dr.T.V.Rao MD 21

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 65 o C for 30 minutes. High Temperature Short Time Pasteurization (HTST): Used today. Milk is exposed to 72 o C for 15 seconds. 12/2/2012 Dr.T.V.Rao MD 22

23. Inspissation: 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 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. 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. Heat : Factors influencing: Nature of heat Temperature and duration Characteristic of organism and spores Type of material 12/2/2012 Dr.T.V.Rao MD 25

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. Heat : Dry heat: Red heat Flaming Incineration Hot air oven 12/2/2012 Dr.T.V.Rao MD 27

28. 12/2/2012 Dr.T.V.Rao MD 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.

29. Dry heat: 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 12/2/2012 Dr.T.V.Rao MD 29

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 tubes 12/2/2012 Dr.T.V.Rao MD 30

31. Incineration: Materials are reduced to ashes by burning. Instrument used was incinerator. Soiled dressings Animal carcasses Bedding Pathological material 12/2/2012 Dr.T.V.Rao MD 31

32. 12/2/2012 Dr.T.V.Rao MD 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 heat

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. Uses of Hot Air Oven Sterilisation of Glassware like glass syringes, petri dishes, pipettes and test tubes. Surgical instruments like scalpels, scissors, forceps etc. Chemicals like liquid paraffin, fats etc. 12/2/2012 Dr.T.V.Rao MD 34

35. Precautions : Should not be overloaded Arranged in a manner which allows free circulation of air Material to be sterilized 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 ware may crack by sudden cooling. 12/2/2012 Dr.T.V.Rao MD 35

36. Sterilisation controls : Sterilisation controls Spores of Bacillus subtilis subsp. niger Thermocouples Browne’s tube 12/2/2012 Dr.T.V.Rao MD 36

37. Sterilizing below100°C temperature below 100° Pasteurization of milk Inspissation Vaccine bath 12/2/2012 Dr.T.V.Rao MD 37

38. Principle of Pasteurization 12/2/2012 Dr.T.V.Rao MD 38

39. A temperature at 100°C A temperature at 100°C Boiling Tyndallisation Steam sterilisation 12/2/2012 Dr.T.V.Rao MD 39

40. Boiling : 1 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 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. Temperatures above 100°C 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. 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. Figure 9.6 Autoclave-overview 12/2/2012 Dr.T.V.Rao MD 43

44. Autoclave: Closed Chamber with High Temperature and Pressure

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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 minutes 12/2/2012 Dr.T.V.Rao MD 46

47. 12/2/2012 Dr.T.V.Rao MD 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 closed

48. Uses of Autoclaves: Uses : Useful for materials which can not withstand high temp. To sterilize culture media, rubber material, gowns, dressings, gloves etc. 12/2/2012 Dr.T.V.Rao MD 48

49. Sterilisation controls: Sterilisation controls: Thermocouples Bacterial spores- Bacillus stearothermophilus Browne’s tube Autoclave tapes 12/2/2012 Dr.T.V.Rao MD 49

50. Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Nutrient medium containing pH color indicator Endospore strip Incubation After autoclaving, flexible vial is squeezed to break ampule and release medium onto spore strip. Yellow medium means spores are viable; autoclaved objects are not sterile. Red medium means spores were killed; autoclaved objects are sterile. Sterility Controls 12/2/2012 Dr.T.V.Rao MD 50

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. Purification of water. 12/2/2012 Dr.T.V.Rao MD 51

52. FILTRATION STERILIZATION This method is commonly used for sensitive pharmaceuticals and protein solutions in biological research. A filter with pore size 0.2 µm will effectively remove bacteria . If viruses must also be removed, a much smaller pore size around 20 nm is needed. Prions are not removed by filtration. The filtration equipment and the filters themselves may be purchased as presterilized disposable units in sealed packaging, or must be sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile filter membranes. To ensure sterility, the filtration system must be tested to ensure that the membranes have not been punctured prior to or during use. To ensure the best results, pharmaceutical sterile filtration is performed in a room with highly filtered air ( HEPA filtration) or in a laminar flow cabinet or "flowbox", a device which produces a laminar stream of HEPA filtered air. HEPA filters are critical in the prevention of the spread of airborne bacterial and viral organisms and, therefore, infection. Typically, medical-use HEPA filtration systems also incorporate high-energy ultra-violet light units to kill off the live bacteria and viruses trapped by the filter media.

53. Several Types of Filters Types of filters: Candle filters Asbestos disc filters Sintered glass filters Membrane filters Air filters Syringe filters 12/2/2012 Dr.T.V.Rao MD 53

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. The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 μ m (filtration membranes are most popular for this purpose). The filtering Depends on Pore Size 12/2/2012 Dr.T.V.Rao MD 55

56. Candle filters 12/2/2012 Dr.T.V.Rao MD 56

57. The roles of HEPA filters in biological flow safety cabinets Exhaust HEPA filter Blower Supply HEPA filter Light High-velocity air barrier Safety glass viewscreen 12/2/2012 Dr.T.V.Rao MD 57

58. Radiations : Radiations : Ionizing radiations Non - Ionizing radiations 12/2/2012 Dr.T.V.Rao MD 58

59. Ionising radiations: Ionizing radiations: X rays Gamma rays Cosmic rays Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation) 12/2/2012 Dr.T.V.Rao MD 59

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 © 2012 Pearson Education Inc. 12/2/2012 Dr.T.V.Rao MD 60

61. Forms of Radiation 12/2/2012 Dr.T.V.Rao MD 61

62. Physical Methods of Microbial Control: Radiation: Three types of radiation kill mic robes: 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. 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. 12/2/2012 Dr.T.V.Rao MD 63

64. Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students Email [email protected] 12/2/2012 Dr.T.V.Rao MD 64