Microorganisms can cause infection and are present everywhere. Sterilization aims to remove or destroy microorganisms from surfaces and materials. Various physical and chemical methods are used for sterilization with different effectiveness against bacterial vegetative cells, spores, viruses, and other microbes. Common physical sterilization methods include heat, radiation, and filtration, while chemical methods include alcohols, aldehydes, phenols, halogens, and other disinfecting agents. The choice of sterilization method depends on the type of material or substance being sterilized.

1. DR.B.V.RAMANA, M.D.,

2. 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.

3. 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

4. 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.

5. 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.

6. 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 fungistatic – prevents the growth of fungi

7. 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.

8. Methods Physical methods Chemical methods

9. Physical methods: Sunlight Heat Dry heat Moist heat Filtration Radiation

10. Chemical methods: Alcohols Aldehydes Phenols Halogens Oxidizing agents Salts Surface active agents Dyes Vapour phase disinfectants

11. Physical methods

12. Sun light: Active germicidal effect due to its content of ultraviolet rays . Natural method of sterilisation of water in tanks, rivers and lakes.

13. Heat : Most reliable and commonly employed method . Two types: Dry heat Moist heat

14. Heat : Principle: Dry heat kills the organism by denaturation of the bacterial proteins, oxidative damage toxic effect of elevated levels of electrolytes .

15. Heat : Principle: Moist heat kills the organism by denaturation of the bacterial proteins coagulation of the bacterial proteins

16. Heat : Factors influencing: Nature of heat Temperature and duration Characteristic of organism and spores Type of material

17. Heat : Dry heat: Red heat Flaming Incineration Hot air oven

18. 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

19. 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

20. Dry heat: 3. Incineration: Materials are reduced to ashes by burning. Instrument used was incinerator. Soiled dressings Animal carcasses Bedding Pathological material

21. Dry heat: 4. Hot air oven:

22. 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.

23. Uses: Sterilisation of Glassware like glass syringes, petridishes, pipettes and test tubes. Surgical instruments like scalpels, scissors, forceps etc. Chemicals like liquid paraffin, fats etc.

24. Precautions : Should not be overloaded Arranged in a manner which allows free circulation of air Material to be sterilised 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 waremay crack by sudden cooling.

25. Sterilisation controls : Spores of Bacillus subtilis subsp. niger Thermocouples Browne’s tube

26. Moist heat sterilisation: A temperature below 100°C A temperature of 100°C A temperature above 100°C

27. A temperature below 100°C Pasteurisation of milk Inspissation Vaccine bath

28. Pasteurisation of milk Two types Holder method ( 63°C for 30 minutes) Flash method ( 72°C for 20 seconds followed by quickly cooling to 13°C) 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 sucessive days. Serum or body fluids can be sterilised by heating at 56°C for an hour daily for several sucessive days.

29. A temperature at 100°C Boiling Tyndallisation Steam sterilisation

30. Boiling : 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 steriliser : Steam at 100°C for 90 minutes. Used for media which are decomposed at high temperature.

31. 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.

32. 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.

34. 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

35. Uses : Useful for materials which can not withstand high temp. To sterilise culture media, rubber material, gowns, dressings, gloves etc.

36. Sterilisation controls: Thermocouples Bacterial spores- Bacillus stearothermophilus Browne’s tube Autoclave tapes

37. 3. Filtration: Useful for substances which get damaged by heat. To sterilise sera, sugars and antibiotic solutions. To obtain bacteria free filtrates of clinical samples. Purification of water.

38. Types of filters: Candle filters Asbestos disc filters Sintered glass filters Membrane filters Air filters Syringe filters

39. Candle filters

40. Membrane filters

41. Radiations : Ionising radiations Non - Ionising radiations

42. Ionising radiations: X rays Gamma rays Cosmic rays Gamma radiation are commercially used for sterilisation of disposable items. (cold sterilisation)

43. 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.

44. Chemical Methods

45. A variety of chemical agents are used as antiseptics and disinfectants. Factors influencing the potency of a disinfectant: Concentration Time of action pH Temperature Nature of organism Presence of organic matter

46. High level disinfectants Intermediate level disinfectants low level disinfectants

47. High level disinfectants: Glutaraldehyde, Hydrogen peroxide, peracitic acid and chlorine compounds. Effectiveness may be equal to that of sterilisation. Used for: Endoscopes Cystoscopes Surgical instruments with plastic components

48. Intermediate level disinfectants May not effective against spores Includes alcohols, iodophores and phenols Used for: Laryngoscopes Fiber optic endoscopes

49. Low level disinfectants: Many organisms can survive on exposure to these disinfectants. Used for items which come in contact with the patients but they do not penetrate into tissues. Stethoscopes, ECG electrodes etc.

50. 1.Alcohols : Ethyl alcohol and Isopropyl alcohol are commonly used. Act by denaturing of bacterial proteins. No sporicidal and virucidal activity. Used as skin antiseptics. Isopropyl alcohol is preferred to Ethyl alcohol as it is better fat solvent, more bactericidal and less volatile.

51. 2. Aldehydes: Formaldehyde Glutaraldehyde

52. Formaldehyde: Bactericidal, sporicidal and virucidal Used in both aqueous solution and gaseous forms A 10% aqueous solution is routinely used. Uses: preservation of tissues for pathological examination To sterilise bacterial vaccines To prepare toxoid from toxin For killing of bacterial cultures and suspensions For destroying anthrax spores in hair and wool.

53. Glutaraldehyde : Effective against bacteria , fungi and viruses. Less toxic and irritant to eyes and skin than formaldehyde Used as 2% buffered solution Available commercially as CIDEX Uses: For sterilisation of cystoscopes, endoscopes and bronchoscopes For sterilisation of plastic endotracheal tubes, face masks, rubber anesthetic tubes

54. 3.Phenols: Lister,the father of antiseptic surgery, first introduced the use of phenol ( Carbolic acid) in surgery (1867). Produced by distillation of coal tar between temperatures of 170°C and 270°C. Bactericidal action due to cell membrane damage. Commonly used Phenol derivatives are cresol chlorhexidine chloroxylenol hexachlorophanes

55. Cresols : Lysol is a solution of cresols in soap. Active against a wide range of organisms. uses: For sterilisation of infected glass ware Cleaning of floors Disinfection of excreta

56. Chlorhexidine: Eg.Salvlon – (Chlorhexidine and cetrimide) More active against Gram positive than Gram negative Good fungicidal activity. No action on spores and little activity against viruses

57. Chloroxylenol : Eg.dettol Less toxic and less irritant. Readily inactivated by organic matter Inactive against pseudomonas.

58. Hexachlorophane: More active against Gram positive than Gram negative bacteria. Bacteriostatic at high dilutions. Applied on skin as prophylaxis against staphylococcal infections. Potentially toxic and should be used with care.

59. 4.Halogens: Chlorine Iodine Commonly used disinfectants Bactericidal, sporicidal and virucidal

60. Chlorine : In the form of Bleaching powder, Sodium hypochlorite Chloramine Disinfection is due to release of free chlorine. Reacts with water to form hypochlorus acid . Bactericidal, virucidal, fungicidal and sporicidal. Used in water supplies, swimming pools, food and dairy industries .

61. Iodine : Used as skin disinfectant. Bactericidal and moderate action on spores. Betadine is one example.

62. 5.Oxidising agents: Hydrogen peroxide Effective at concentration of 3-6% Kills spores at 10- 25% Used to disinfect Contact lenses Surgical prostheses Plastic implants

63. 6.Salts: Salts of heavy metals have toxic effect on bacteria. Salts of copper, silver and mercury are used as disinfectants. Act by coagulation of bacterial proteins. Mercuric chloride, once used as disinfectant is highly toxic. Thiomersol and mercurochrome are less toxic Copper salts are used as fungicides.

64. 7.Surface active agents: Substances which alter energy relationships at interfaces , producing a reduction of surface tension, are known as surface active agents or surfactants. Anionic cationic nonionic amphoteric compounds.

65. The anionic compounds: e.g.:- common soaps, Have strong detergent but weak antimicrobial properties . These agents are most active at acidic pH. Effective against Gram positive organisms but are relatively ineffective against Gram negative species.

66. Cationic surfactants: Quaternary ammonium compounds are the most important cationic surfactants. These compounds are bactericidal for a wide range of organisms, gram positive species are more susceptible. The common cationic compounds are acetyl trimethyl ammonium bromide (cetavalon or cetrimide) and benzalkonium chloride.

67. Amphoteric compounds: known as ‘Tego’ compounds possess detergent properties of anionic and antimicrobial activity of cationic compounds. They are active against a wide range of Gram positive and Gram negative organisms and some viruses.

68. 8.Dyes : Two groups of dyes have been used extensively as skin and wound antiseptics aniline dyes acridine dyes Both are bacteriostatic in high dilution but have low bactericidal action. Aniline dyes include crystal violet, brilliant green and malachite green. They are more active against Gram positive bacteria than Gram negative bacteria.

69. Acridine dyes include acriflavine , euflavine, proflavine and aminacrine. They are more active against Gram positive bacteria than Gram negative bacteria. Gentian violet and acriflavine are two widely used dyes for skin disinfection especially in Gram positive bacterial infections.

70. 9.Vapour phase disinfectants: Formaldehyde gas Ethylene oxide Betapropiolactone

71. Formaldehyde gas: Used for fumigation of operation theatres, wards and laboratories etc. It is generated by adding 150 gm of KMnO4 to 280 ml of formalin for 1000 cu. Feet of room volume. The doors should be sealed and left unopened for 48 hours. The gas is toxic and irritant when inhaled. After completion of sterilisation the irritant vapours are nullified by exposure to ammonia vapour.

72. Ethylene oxide: It is a colourless liquid with a boiling point of 10.7°C. Effective against all types of organisms including viruses and spores. It has a potential toxicity to human beings, including mutagenicity and carcinogenicity . It is highly inflamable. Used for sterilising plastic and rubber articles, respirators, heart lung machines, sutures, dental equipments etc.

73. Betapropiolactone : Condensation product of ketane and formaldehyde. It has a boiling point of 163°C. Used in 0.2%. Effective against all types of organisms including viruses. More efficient for fumigation than formaldehyde. Used for inactivation of vaccines.

74. 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