Sterilization

1. Sterilization Why Sterilization ? Contaminated of surgical instruments will leads to infections. 2. Contamination in Microbiology lab, leads to difficulty in diagnosis and misdiagnosis. 3. Contamination in drug and food industries.

2. Sterilization : It is the process by which an article, surface or media are freed of all the micro-organisms either in vegetative or spore state. Disinfections: It is the process of removing the micro-organisms which are liable to produce infections.

3. Classification: 1. Physical methods A. Heat B . Filtration C. Radiation 2. Chemical methods. Various chemicals

4. HEAT • Factors affecting by heat: • Nature of heat – dry or moist • Temperature & time • No. of organisms present • Characteristics of the organism • Type of material Two methods • Dry heat • Moist heat

5. DRY HEAT Mechanism: Kills by oxidation, protein denaturation & toxic effect of elevated levels of electrolyte Types: 1. Flaming 2. Incineration 3. Hot air oven

6. 1. Flaming • Temp of flame 2500C – 3000C

7. Points of forceps & Inoculation loops – heat in bunsen flame till red hot • Slow passage through flame to destroy vegetative bacteria on surface of scalpel blade, glass slides, mouths of test tubes

8. 2. INCINERATION “ Complete burning of hospital waste until it becomes ash” Eg: Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

10. 3. HOT AIR OVEN

11. Holding temp & time: 1600C for 1 hr • Used for glassware, forceps, swabs, water impermeable oils, waxes & powders • Before placing in hot air oven A. Dry glassware completely B. Plug test tubes with cotton wool C. Wrap glassware in Kraft papers D. Don’t over load the oven E. Allow free circulation of air between the material

12. Sterilization controls: 1. Chemical controls: Browne’s tubes • Color change from red to green 2. Thermocouples 3. Biological controls: paper strips containing106 spores of Clostridium tetani • Place strips in oven along with other material for the sterilization • Later culture the strips in thioglycollate broth or RCM at 370C for 5 days • Growth in medium indicates failure of sterilization

14. MOIST HEAT Mechanism: Lethal effect due to coagulation & denaturation of proteins Methods: • Temp below 1000C • Temp at 1000C • Temp above 1000C

15. TEMP BELOW 1000C 1. Pasteurization • 630C – 30 min (Holder method) • 720C – 15-20 sec (Flash method) Eg Salmonella, Brucella and Mycobacteria 2. Vaccine baths - 600C – 60 min • For vaccines of non-sporing bacteria 3. Water bath - 560C – 60 min – 3 days consecutive days • For serum / body fluids containing coagulable proteins 4. Inspissation – 80-850C – 30 min – 3 days • For media containing egg or serum – LJ, LSS

17. TEMP AT 1000C 1. Boiling (1000C): • Kills all vegetative bacteria • Water should be soft, deionized or distilled • 2% sodium bicarbonate promotes the process • Kills vegetative bacteria, hepatitis virus & some spores • Steaming (free steam): Arnold /Koch steamer • 90 minutes at a stretch. • Tyndallisation(intermittent sterilization) - 1000C, 30 min, 3 days • Nutrient media & media containing sugars or gelatin • I day all vegetative bacteria are killed. On II & III day spores that germinate are killed

18. TEMP ABOVE 1000C Autoclave (steam under pressure):

20. Uses: Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media. • Sterilization control • Thermocouples • Browne’s tube (red-green). • 106spore of B stearothermophilus. Incubate at 550C for 5 days

21. FILTRATION • Aqueous liquids may be sterilized by forced passage through a filter of porosity small enough to retain any microorganisms present in them. • Used to sterilize serum, sugar soln., filtrates of toxins & bacteriophages, in water bacteriology, in examination of Schistosoma eggs

22. Types of filters: 1. Earthenware candles- Unglazed ceramic & diatomaceous earthen filters Eg. Chamberland filters, Doulton filters 2. Asbestos filter- Seitz, Carlson 3. Sintered glass filter 4. Membrane filters – cellulose nitrate, cellulose acetate, polycarbonate, polyester filters 5. HEPA (high efficiency particle air)filters – for large volumes of air.

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27. RADIATION Types: 1. Non ionising – • Infra Red radiation ( rapid mass sterilization of syringes, etc) • Ultra Violet radiation (enclosed areas) 2. Ionising – Gamma, X ray, cathode ray (plastics)

28. Disinfection • Strong disinfectants – for inanimate object • Mild disinfectant (antiseptic) – superficial application on living tissue

29. Categories of disinfectants • Alcohol • Aldehyde • Phenolics • Halogens • Dyes • Surface active agents • Gases • Metallic salts

30. Alcohols: • Ethanol, isopropyl alcohol have optimal bactericidal activity in aqueous solution at 70% conc. • Have limited activity against Mycobacteria and nonsporicidal. Action against viruses is good. • Due to volatile activity widely recommended as disinfectant of skin and surfaces. • Their efficacy in organic matter is low. • Methyl alcohol – to treat cabinets / incubators affected by fungal spores

31. Aldehydes: 1. Formaldehyde: • In aq. Soln (10%) is virucidal, bactericidal, sporicidal • Used in preservation of Anatomical specimens. • Has pungent smell, irritant to skin, eyes, mucus memb & toxic when inhaled 2. Glutaraldehyde– • less toxic, less irritant. • Used rubber endotrachealtubes, face masks metal instruments, polythene tubing.

32. Phenolics: • Carbolic acid – 5% • Powerful microbicidal. • General purpose disinfectant in hospital Eg- Cresol, lysol • Chloroxylenol, chlorophenol, hexachlorophane – less toxic, less irritant, less active, more readily inactivated by organic matter

33. HALOGENS Mechanism: Kills by oxidation • Iodine – 2.5% in 70% alcohol, Skin antiseptic • Iodophores (iodine + non-ionic surface active agent) – betadine – non staining, less irritant, less toxic • Chlorine – disinfect water supplies, swimming pools • Sodium hypochlorite – 1% for HIV • Organic chloramines – antiseptic for wound dressings

34. DYES Mechanism: Combine with nucleic acids 1. Aniline dyes Eg- Brilliant green, malachite green, crystal violet 2. Acridinedyes Eg-Proflavine, acriflavine, euflavine, aminacrine Skin & wound antiseptics (Bacteristatic)

35. SURFACE ACTIVE AGENTS • Disrupt cell membrane • 4 main groups- 1. Anionic surfactants – strong detergent action, weak antimicrobial action 2. Non-ionic surfactants 3. Cationic surfactants – quaternary ammonium compounds – cetrimide 4. Amphotericsurfactants – both detergent & antimicrobial properties – Tego comps

36. GASES • Ethylene oxide: • It is a colourless gas highly inflammable and explosive. Mixed with carbon dioxide to eliminate its explosiveness. • Effective against all organisms including spores. Used to sterilize heart lung machines, respirators dental equipments etc. • Not suitable for fumigation.

37. 2. Formaldehyde gas: Generated by adding pottacium permanganate to fomaldehyde solution. Room to be closed for 48 hrs. 3. Betapropiolactone (BPL): More effective than formaldehyde gas, suitable for fumigation of OT.

38. METTALIC SALTS • Mercuric salts – ointments • Silver salts – AgNO3 – to prevent infection of burns, ophthalmianeonatorum • Copper salts – antifungal – water reservoirs, swimming pools

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