Sterilization and Disinfection

Detailed description of Sterilization for postgraduate microbiology and medical students

1. Sterilization & Disinfection Dr r Sathyajith 1 9:27 PM Dr R Sathyajith

2. Sterilization Latin sterilis means unable to produce offspring or barren Process by which an “article, surface or medium is freed of all living organisms either in vegetative or spore state ” When done using a chemical agent, it is referred to as ‘ Sterilant ’ Generally, it implies physical process rather than chemical agent. 2 9:27 PM Dr R Sathyajith

3. Disinfection & antiseptics Disinfection is the killing, inhibition or removal of all pathogenic organisms or organisms capable of causing infections. Antiseptics are chemical disinfectants that can be safely applied to the skin & mucous membrane. They are used to prevent infections by killing or inhibiting pathogenic growth. Antiseptics also reduce total microbial population and are less toxic as disinfectants. 3 9:27 PM Dr R Sathyajith

4. Common terms Bactericidal agents or germicides are those that can kill bacteria. Bacteriostatic agents – prevent multiplication of bacteria, which may remain alive. A chemical which is bactericidal at a particular concentration may only be bacteriostatic at higher dilutions Decontamination is the process of rendering an article or area free of danger from contaminants, including microbial, chemical, radioactive & other hazards. Cleaning - A process that removes foreign material (dirt, organic matter ). Must precede disinfection and sterilization. Usually done with soap and water or detergent. 4 9:27 PM Dr R Sathyajith

5. sterilization and disinfection Importance - Safety in the laboratory. The patient safety depends on using proper methods of sterilization to prepare instruments, needles, IV fluids. The accuracy and validity of microbiological tests. 9:27 PM 5 Dr R Sathyajith

6. Methods of sterilization Physical methods Sunlight Drying Heat Filtration Radiation Ultrasonic & sonic vibrations Dry heat Incineration Red heat Flaming Hot air sterilizers Microwave ovens Moist heat Pasteurization Boiling Steam under normal pressure Steam under pressure 6 9:27 PM Dr R Sathyajith

7. sunlight Role in spontaneous sterilization under natural conditions Has appreciable bactericidal properties Bacteria suspended in water are readily destroyed on exposure. Disinfectant action primarily due to UV rays & also heat rays. Under natural conditions sterilizing power varies according to circumstances. UV rays are screened out by glass or impurities/ ozone layer present in atmosphere. 7 9:27 PM Dr R Sathyajith

8. Drying Moisture is essential for growth of bacteria 80% of bacterial cell weight is water Drying hence has a deleterious effect Unreliable & of theoretical interest only Spores are not affected 8 9:27 PM Dr R Sathyajith

9. Refrigeration Refrigeration 5 o C Freezing -20 o C O rdinary refrigeration has a bacteriostatic effect, but does not kill, metabolic rate of some microbes is so reduced that they cannot reproduce or synthesize toxins Growth of pathogenic bacteria slows down at refrigerator temperature & can be used to preserve microorganisms 9 9:27 PM Dr R Sathyajith

10. filtration Filtration is commonly the mechanical operation which is used for the separation of microbes from fluids (liquids or gases) by interposing a medium through which only the fluid can pass . Different from Sieving/ Adsorption 10 9:27 PM Dr R Sathyajith

11. Filtration - uses Heat sensitive solutions – For sterilization of pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics etc. Separation of bacteriophages & bacterial toxins from bacteria Isolating organisms which are scanty in fluids Concentration of bacteria from liquids Isolation of viruses – pore size of 0.22mcm most virus & Mycoplasma pass through. 11 9:27 PM Dr R Sathyajith

12. filters - types Earthware filters Asbestos filters Sintered glass filters Membrane filters Syringe filters Vacuum & ‘In-line’ filters Pressure filters Air filters 12 9:27 PM Dr R Sathyajith

13. Earthware/candle filters Available in different grades of porosity Used for purification of water – I ndustrial & Drinking Fluid is forced by suction or pressure from inside to outside or vice versa After use sterilized by scrubbing with stiff brush - boiling – autoclaving Unglazed ceramic filters ( Chamberland & Doulton ) Compressed diatomaceous earth filters ( Berkefeld & Mandler ) 13 9:27 PM Dr R Sathyajith

14. Earthware/candle filters 14 9:27 PM Dr R Sathyajith

15. Earthware/candle filters 15 9:27 PM Dr R Sathyajith

16. Asbestos filter Disposable, single use discs Filter disk is made up of asbestos (Mg trisilicate) High adsorbing capacity Alkalinize filtered fluid. Carcinogenic potential of asbestos has discouraged its use E.g. Seitz filter, Carlson & Sterimat 16 9:27 PM Dr R Sathyajith

17. Seitz filter 17 9:27 PM Dr R Sathyajith

18. Sintered glass filter Sintered glass is a glass mesh used for filtration Prepared by heat fusing finely powdered glass particles Pore size can be controlled by particle size of the glass powder Easy to clean L ow absorption properties Cons - Fragile & expensive 18 9:27 PM Dr R Sathyajith

19. Sintered glass filter 19 9:27 PM Dr R Sathyajith

20. Membrane filter Consists of polymeric material like cellulose nitrate, cellulose diacetate , polycarbonate & polyester. Made as disks from 13-293mm diameter & 0.015-12mcm porosities Average pore diameter (APD) 0.22mcm MC used in sterilization 20 9:27 PM Dr R Sathyajith

21. Membrane filter 21 9:27 PM Dr R Sathyajith

22. Membrane filter 22 9:27 PM Dr R Sathyajith

23. Membrane filter Uses Water purification & analysis Sterilization & sterility testing Preparation of sterile solutions for parenteral use Bacterial count of water – Known amount of water is filtered & the membrane disk is placed upside down on a culture medium & incubated & colonies are counted to know the viable count 23 9:27 PM Dr R Sathyajith

24. Syringe filters Membrane of 13-25mm diameter is fitted into syringe like holders of stainless steel or polycarbonate For sterilization, the fluid is forced through the disk membrane by pressing the piston of the syringe. 24 9:27 PM Dr R Sathyajith

25. Syringe filters 25 9:27 PM Dr R Sathyajith

26. Vacuum & in-line filters Membrane diameter of 25 & 45mm are used In-line filter holder of Teflon or stainless steel & aluminium Vacuum holders of borosilicate glass, polycarbonate or stainless steel Used for sterilizing large volumes of liquids 26 9:27 PM Dr R Sathyajith

27. Vacuum filters 27 9:27 PM Dr R Sathyajith

28. In-line filters 28 9:27 PM Dr R Sathyajith

29. Pressure filters Large membranes 100-293mm in diameter or filter cartridges are used Housed in pressure filter holders Used for very pure water for lab use If fitted with Teflon filter, then autoclavable with filter in situ 29 9:27 PM Dr R Sathyajith

30. Pressure filters 30 9:27 PM Dr R Sathyajith

31. Air filters HEPA – High efficiency particulate filters Large volumes of air can be rapidly freed from infection by passage through them Remove fine particles of size 0.3mcm or larger Widely used in laminar air flow (LAF) cabinets/chambers Available in different efficiencies from 95.0% to 99.997% depending on the need Do not filter out gasses, chemical vapors and odor molecules 31 9:27 PM Dr R Sathyajith

32. Hepa in laf safety cabinet 9:27 PM 32 Supply HEPA filter Exhaust HEPA filter High-velocity air barrier Safety glass viewscreen Dr R Sathyajith

33. HEPA First used to remove radioactive contaminants from the air in the development of the atomic bomb Working with hazardous items such as the HIV virus, TB bacilli & other lethal bacteria and viruses for the sake of research requires HEPA filtration 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 33 9:27 PM Dr R Sathyajith

34. HEPA 34 9:27 PM Dr R Sathyajith

35. hepa 35 9:27 PM Dr R Sathyajith

36. Radiation - nonionizing 2 types – IR & UV Effectiveness of UV related to its wavelength Most effective is 240-280nm range Produced artificially by mercury vapor lamp Unlike ionizing radiation, energy of UV is lower & penetration power is poor Does not penetrate glass, dirt films, water etc. 36 9:27 PM Dr R Sathyajith

37. Radiation – nonionizing UV 37 9:27 PM Dr R Sathyajith

38. Radiation – nonionizing UV Microbial sensitivity to UV radiation Bacterial spores are more resistant than vegetative cells Viruses are inactivated & are more sensitive than spores HIV not inactivated Practical uses Disinfect water Disinfect closed areas like OT, wards, labs & ventilated safety cabinets . 38 9:27 PM Dr R Sathyajith

39. Radiation – nonionizing UV 39 9:27 PM Dr R Sathyajith

40. Radiation – nonionizing UV UV light sterilization offers great potential for tackling HAI’s or Healthcare Associated Infections transmitted through the surrounding environments of the healthcare institutions . Effective against – MRSA, VRE, C. difficile , A. baumannii , ESBL, Influenza virus A. Disadvantages – Uninterrupted line of sight required – Production of ozone 40 9:27 PM Dr R Sathyajith

41. Radiation – nonionizing UV 41 9:27 PM Dr R Sathyajith

42. Radiation - nonionizing Hazards of UV – Burns skin & damages eyes – Human exposure should be avoided Infrared IR can be considered a form of hot air sterilization Used for rapid mass sterilization of prepacked items like syringes & catheters 42 9:27 PM Dr R Sathyajith

43. Radiation – nonionizing IR Bacti – Cinerator (IR Sterilizer) Sterilizes needles, loops, and mouth of culture tube, small bottle in 5-7 seconds at optimum sterilizing temperature above 800° C Maximum temperature towards 930º C . 43 9:27 PM Dr R Sathyajith

44. Radiation – nonionizing IR 44 9:27 PM Dr R Sathyajith

45. Radiation – Ionising Very high penetrating power Highly lethal to all cells – Humans/ Bacteria Causes structural defects in microbial DNA synthesis leading to cell death Bacterial spores are resistant E.g. X-rays, Gamma rays & cosmic rays. 45 9:27 PM Dr R Sathyajith

46. Radiation – Ionising gamma 46 9:27 PM Dr R Sathyajith

47. Radiation – Ionising radiation Cold sterilization No appreciable increase in temperature Commercial plants use gamma rays emitted from radioactive elements like Cobalt 60 Uses – Sterilizing in pharmacy & medicine ( Abx , hormones, sutures, vaccines) / packaged disposable articles ( IV lines, catheter, syringes, Gloves) 47 9:27 PM Dr R Sathyajith

48. Ultrasonic & sonic vibrations Have bactericidal properties Effects variable on different bacteria Unreliable & of no practical value in medicine Sometimes used for fine cleaning to remove soil from joints, crevices, lumens and other difficult to clean areas 48 9:27 PM Dr R Sathyajith

49. Ultrasonic sterilizers 49 9:27 PM Dr R Sathyajith

50. heat Most reliable Universally applicable Method of choice – whenever applicable 2 types – Dry & Moist Materials damaged by heat – Sterilized at low temperature or longer periods or repeated cycles Recommended minimum holding time does not include the time taken to reach the specified temperature 50 9:27 PM Dr R Sathyajith

51. Decimal reduction time (DRT) DRT also called D-value refers to the time required at a certain temperature to kill 90% of the organisms being studied Thus after a colony is reduced by 1 D, only 10% of the original organisms remain. E.g. if a organism is reduced by 90% after exposure to temperatures of 300° F for 20 minutes, the D-value would be written as D 300F = 20 minutes Sterilization-resistant organism are given a unique D-value D-value determination is often carried out to measure efficiency 51 9:27 PM Dr R Sathyajith

52. DRT 9:27 PM 52 Dr R Sathyajith

53. Z value U sed in thermal death time calculations The z-value of an organism is the temperature, in degrees (Fahrenheit or Celsius), that is required for the thermal destruction curve to move one log cycle Simply, temperature required for one log reduction in the D-value 53 9:27 PM Dr R Sathyajith

54. Z value 54 9:27 PM Dr R Sathyajith

55. D-value & z-value The Z-value is 10, D-value is 4.5 minutes at 150°F, what is the D-value for 160°F ? The new D-value is 0.45 minutes This means that each 10°F increase in temperature will reduce our D-value by 1 log. Conversely , a 10°F decrease in temperature will increase our D-value by 1 log. So, the D-value for a temperature of 140°F would be 45 minutes. 55 9:27 PM Dr R Sathyajith

56. Heat – factors influencing Nature of heat Dry or Moist Temperature & Time Time required is inversely proportional to temperature Thermal death time (TDT) – Minimum time required to kill a suspension of organisms at a predetermined temperature in a specified environment 56 9:27 PM Dr R Sathyajith

57. Heat – factors influencing Number of microbes More number of microbes there are to begin with, longer it takes to eliminate the entire population Characteristics of organisms Vegetative bacteria & virus are more susceptible & spores are more resistant. Nature of contaminated material Presence of organic substances, proteins, nucleic acid, starch, fats & oils, increase the TDT High acid/ alkaline pH increases bacterial killing 57 9:27 PM Dr R Sathyajith

58. Heat - moa Dry heat Protein denaturation Oxidative damage High electrolyte level toxicity Moist heat Coagulation & Denaturation of enzymes/ structural proteins 58 9:27 PM Dr R Sathyajith

59. red heat - flaming Red heat - Item to sterilized are held almost vertically in a Bunsen flame until red hot along their whole length, almost up to the tip of their metal holder. Items sterilized by red heat – Inoculating wire loops Points of forceps Flaming – Only exposed to the flame for a few seconds & not heating to red hot. E.g Scalpel blades, glass slides, mouth of culture tubes 59 9:27 PM Dr R Sathyajith

60. red heat 60 9:27 PM Dr R Sathyajith

61. flaming 61 9:27 PM Dr R Sathyajith

62. Dry heat - incineration Excellent & safe method Material destroyed completely – reduced to ashes Very high temperatures reached Used for – Pathological wastes, surgical dressing, animal carcasses, plastics like PVC/ polythene Avoid – Materials made of polystyrene as they emit black dense smoke 62 9:27 PM Dr R Sathyajith

63. incinerator 63 9:27 PM Dr R Sathyajith

64. Dry heat – hot air oven 64 9:27 PM Dr R Sathyajith

65. Dry heat – hot air oven Most widely used method by dry heat Used - When can withstand high temperatures & when affected by contact with steam Parts – E lectric heater, Fan to provide forced air circulation, Temperature indicator, Control thermostat & timer, Open mesh shelving, Heating coils on wall/floor & adequate wall insulation – Inner layer poor conductor & outer metallic 65 9:27 PM Dr R Sathyajith

66. Dry heat – hot air oven 66 9:27 PM Dr R Sathyajith

67. Dry heat – hot air oven Preparation of load No overloading – to allow free circulation of air Articles should be thoroughly clean & perfectly dry Test tubes & flasks – Should be wrapped in paper Avoid – Rubber except silicon, Cotton plugs, Plastics 67 9:27 PM Dr R Sathyajith

68. Dry heat – hot air oven Sterilizing cycle Sterilization hold time – It is set to 160 o C for 2hours or 170 o C for 1hour or 180 o C for 30min Cutting instruments like used in ophthalmic surgery should be sterilized at 150 o C for 2hours Oils, glycerols & dusting powder holding time recommended is 150 o C for 1hour. 68 9:27 PM Dr R Sathyajith

69. hot air oven - Uses Cooling takes several hours – Open the door after chamber & load have cooled below 80 o C or else glassware will crack Glassware such as tubes, flasks, all glass syringes, petri dishes, pipettes. Metal instruments like forceps, scissors & scalpels Nonaqueous materials & powders, oils & greases in sealed containers, swab sticks placed in test tubes 69 9:27 PM Dr R Sathyajith

70. hot air oven - Sterilization controls Biological control An envelope containing filter paper strip impregnated with 10 6 spores of Bacillus atrophaeus ( formerly Bacillus subtilis subsp niger ) is inserted Non toxigenic strains of Clostridium tetani can be also used After sterilization , strips are removed & inoculated into tryptone soy broth/Cooked Meat media Incubated anaerobically at 37 o C for 5days No growth indicates proper sterilization 70 9:27 PM Dr R Sathyajith

71. Bacillus atrophaeus 9:27 PM 71 Dr R Sathyajith

72. hot air oven - Sterilization controls Chemical indicators Browne’s tubes No. 3 containing red solution is inserted Color change from red to green indicates proper sterilization Thermocouples May be used periodically 72 9:27 PM Dr R Sathyajith

73. hot air oven - Sterilization controls 73 9:27 PM Dr R Sathyajith

74. hot air oven vs autoclave Safe – No water used & no pressure build up inside Smaller – But as effective as autoclave Cheaper – Suitable for small laboratory Rapid – also higher temperatures reached Dry heat penetrates more slowly 74 9:27 PM Dr R Sathyajith

75. Dry heat – microwave oven H eating effect if not uniform Not reliable method for strerilization 75 9:27 PM Dr R Sathyajith

76. Moist heat Temp below 100 o C Pasteurization Inspissation Water bath Vaccine preparation Temp at 100 o C Boiling Koch & Arnold steamer Tyndallization Temp above 100 o C Autoclave 76 9:27 PM Dr R Sathyajith

77. pasteurization Used in dairy industry for milk All non sporing pathogens like Mycobacteria, Brucella & Salmonella are destroyed Coxiella burnetii is relatively heat resistant & may survive holder method All f/b rapid cooling to <13 o C 77 9:27 PM Dr R Sathyajith

78. pasteurization 9:27 PM 78 Dr R Sathyajith

79. pasteurization Holder method – 60 o C for 30min Flash method/ High temperature short term (HTST) – 72 o C for 15-20sec Higher-Heat Shorter Time (HHST) – 90 o C for 0.5sec Ultra high temperature (UHT) sterilization – 140-150 o C for 1-3sec, Shelf stable Source - International dairy food association (IDFA) guidelines 79 9:27 PM Dr R Sathyajith

80. inspissation Literally means the process of thickening by dehydration Also called fractional sterilization Used when heating high-protein (Egg) containing culture media like Lowenstein-Jenson & Loeffler’s serum slope. Heated at 80 o C for 30min (2hrs*) on 3 successive days Arnold’s inspissator used * Handbook of Media for Clinical Microbiology By James W. Snyder, Ronald M. Atlas 80 9:27 PM Dr R Sathyajith

81. inspissator 81 9:27 PM Dr R Sathyajith

82. Water bath Washing or rinsing laundry or eating utensils at 70-80 o C for few minutes will kill most non sporing microorganisms 82 9:27 PM Dr R Sathyajith

83. boiling Boiling at 100 o C for 10-30min kill all vegetative forms Sporing bacteria require prolonged periods of boiling Hard water should not be used Adding 2% NaHCo3 increases efficacy Not recommended for sterilization, only means of disinfection Disinfection of surgical instruments before sterilizing Disinfection in field conditions & when sterility not essential 83 9:27 PM Dr R Sathyajith

84. boiling 84 9:27 PM Dr R Sathyajith

85. Tyndallization Fractional/ Intermittent sterilization Boiling & exposure to steam at 100 o C for 20min for 3 consecutive days Used for heat sensitive culture media 1 st day – Vegetative cells, 2 nd day – Early germinating spores, 3 rd day – Late germinating spores Failure to kill some anaerobic spores & thermophiles. Works for broth media that supports the growth of spores & not water 85 9:27 PM Dr R Sathyajith

86. Koch & Arnold steamer 86 9:27 PM Dr R Sathyajith

87. Moist heat - autoclave Process of sterilization using saturated steam under high pressure above 100 o C Principle – water boils when its vapor pressure equals that of surrounding atmosphere. When pressure inside a closed vessel increases, the temperature at which water boils also increases Steam condenses to water & gives up its latent heat to the cooler surface it comes in contact with. Energy available from this latent heat is considerable 87 9:27 PM Dr R Sathyajith

88. Autoclave - parts Vertical/ Horizontal Stainless steel/ Gun metal Screw clamps to fasten lid Airtight by washer Heated by gas/ electricity 88 9:27 PM Dr R Sathyajith

89. Autoclaves - types Steam jacketed autoclave : Double walled chamber in which steam at 121 degree is introduced from an external source. Steam enters the jacket from which it enters the chamber migrating downwards replacing the air which is expelled through a discharge tap at the bottom. 2. Flash autoclaves : It sterilizes at 134 degree for 3 – 5 minutes. Used in operation rooms in emergency situations. 9:27 PM 89 Dr R Sathyajith

90. Autoclave procedure 90 9:27 PM Dr R Sathyajith

91. autoclave 9:27 PM 91 Dr R Sathyajith

92. Autoclave procedure Sufficient water is put in the cylinder. Articles are kept above the perforated shelf Lid is screwed tight Autoclave is heated Discharge tap is kept open Safety valve is adjusted to required pressure Steam-air mixture is allowed to escape freely till all air is displaced 92 9:27 PM Dr R Sathyajith

93. Autoclave procedure 8. Discharge tap closed 9. Steam pressure rises inside 10. Safety valve opens when it reaches the desired s et level (15psi) 11. Excess steam escapes 12. Holding period calculated from now (15min) 13. Heater turned off when holding period over. 14. Cool the autoclave till pressure inside equals atmospheric pressure 15. Discharge tap is open & later lid is opened & items removed 93 9:27 PM Dr R Sathyajith

94. Autoclave - precautions Air must be allowed to escape from the chamber because the temperature of air-steam mixture is lower than that of pure steam If the discharge tap is opened when pressure inside is high, liquid media will boil violently & spill from the container & can also explode If discharge tap is opened after pressure inside has fallen below atmospheric pressure, excessive water would have evaporated & lost from the media Material to be arranged to ensure free circulation of steam inside the chamber 94 9:27 PM Dr R Sathyajith

95. Autoclave - uses MOA – Bacterial proteins coagulates & condensed water ensures moist condition fro killing Sterilizing culture media, aqueous solutions, rubber materials, dressing materials, gowns, dressing, linen, gloves, instruments & pharmaceutical products For all materials that are water containing, permeable or wettable Useful for materials u nable to withstand higher temperature of hot air. 95 9:27 PM Dr R Sathyajith

96. Autoclaves - controls Filter paper strip impregnated in 10 4 spores of Geobacillus stearothermophilus (ATCC 7953) used Placed in least accessible & coolest part Inoculated in TSB & incubated at 56 o C for 5days It withstands 121 o C for upto 12min 96 9:27 PM Dr R Sathyajith

97. Autoclaves - controls 9:27 PM 97 Cap that allows steam to penetrate Flexible plastic vial Crushable glass ampule Nutrient medium containing pH color indicator Endospore strip 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. Dr R Sathyajith

98. Autoclaves - controls Self adhesive sterilisation indicator tape with bands that turn dark brown. Supplied in 19 or 25mm widths . Paper sterilizer control strips with a heat sensitive colored dot that changes from yellow to purple. 98 9:27 PM Dr R Sathyajith

99. Autoclave vs hot air oven More lethal – action of moist heat Quicker in heating up articles Greater penetration of porous material like cotton plugs, paper & cloth wrappers, surgical linen, hollow apparatus. 99 9:27 PM Dr R Sathyajith

100. References Seymour S. Block: Disinfection, sterilization and preservation, Williams &Wilkins (ISBN 0812113640 ) CDC guideline for disinfection and sterilization in healthcare facilities http://www.cdc.gov/hicpac/pdf/guidelines/Disinfection_Nov_2008.pdf Textbook of microbiology, Surinder kumar , Jaypee Textbook of microbiology, Ananthanarayan & Panicker , 9 th Ed 9:27 PM 100 Dr R Sathyajith

101. Thank you 101 9:27 PM Dr R Sathyajith