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Sterilization & Disinfection Tukaram Prabhu K Asst Professor Microbiology PCMS & RC.

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Presentation on theme: "Sterilization & Disinfection Tukaram Prabhu K Asst Professor Microbiology PCMS & RC."— Presentation transcript:

1 Sterilization & Disinfection Tukaram Prabhu K Asst Professor Microbiology PCMS & RC

2 Lecture conducted for II yr MBBS (2008-2009 session) The following lecture is complied from different textbooks and is for teaching - learning use only This lecture is not a substitute for classroom lecture but an adjunct for the same This lecture may be useful for BSc Nursing, BMLT & DMLT students too

3 DEFINITION The process of freeing an article or a surface from all living microorganisms including viruses & bacterial spores Various methods –Heat –Filtration –Radiation –Sterilant gases

4 HEAT Factors affecting –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 Kills by oxidation, protein denaturation & toxic effect of elevated levels of electrolyte Types of processes –Flaming –Incineration –Hot air oven

6 Dry heat - FLAMING 250 0 C – 300 0 C 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

7 Flaming

8 Dry heat - INCINERATION 870 0 C - 980 0 C Complete burning to ashes Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

9 Incineration

10 Dry heat - HOT AIR OVEN Holding temp & time: 160 0 C for 1 hr Used for glassware, forceps, swabs, water impermeable oils, waxes & powders Before placing in hot air oven –Dry glassware completely –Plug test tubes with cotton wool –Wrap glassware in Kraft papers Dont over load the oven Allow free circulation of air between the material

11 Dry heat - HOT AIR OVEN Sterilization controls: to check whether the equipment is working properly –Chemical controls: Brownes tubes Color change from red to green –Thermocouples –Biological controls: paper strips containing10 6 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 37 0 C for 5 days Growth in medium indicates failure of sterilization

12 Dry heat – Hot air oven

13 MOIST HEAT Lethal effect due to denaturation & coagulation of proteins –Temp below 100 0 C –Temp at 100 0 C –Temp above 100 0 C

14 Moist heat - Temp below 100 0 C Pasteurization 63 0 C – 30 min (Holder method) 72 0 C – 15-20 sec (Flash method) 132 0 C – 1 sec (Ultra high temp) Vaccine baths - 60 0 C – 60 min For vaccines of non-sporing bacteria Water bath - 56 0 C – 60 min – 3 days For serum / body fluids containing coagulable proteins Inspissation – 80-85 0 C – 30 min – 3 days For media containing egg or serum – LJ, LSS

15 InspissatorWater bath

16 Moist Heat - Temp at 100 0 C Boiling - 100 0 C for 10 min 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) – 30-60 min in Arnold /Koch steamer –For heat labile media – DCA, TCBS –Tyndallisation (intermittent sterilization) - 100 0 C, 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

17 Moist Heat - Temp above 100 0 C Autoclave (steam under pressure) - 121 0 C, 15 min, 15 lbs –Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media –Principle – ( refer Ananthanarayan & Paniker 7 th edn Page 27 ) Sterilization control –Thermocouples –Brownes tube (red-green), Bowie & Dick tape (white-brown) –10 6 spore of B stearothermophilus. Incubate at 55 0 C for 5 days

18 Moist Heat - Autoclave



21 Steam Jacketed Horizontal Autoclave

22 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, carbohydrates soln, filtrates of toxins & bacteriophages, in water bacteriology, in examination of Schistosoma eggs

23 FILTRATION Types of filters –Earthenware candles Unglazed ceramic & diatomaceous earth filters Eg. Chamberland filters, Doulton filters –Asbestos filter – Seitz, Carlson, Sterimat –Sintered glass filter –Membrane filters – cellulose nitrate, cellulose acetate, polycarbonate, polyester filters –Pore size: 0.015 – 12 μm HEPA filters – for large volumes of air Sterilization control – bubble pressure test

24 Asbestos Filter holder Earthenware filter

25 Sintered glass filter

26 Membrane filters

27 RADIATION 2 types –Non ionising – Infra Red radiation ( rapid mass sterilization of syringes, etc) Ultra Violet radiation (enclosed areas) –Ionising – Gamma, X ray, cathode ray (plastics, syringes, oil, metal foils) –Sterilization controls Dosimeter – measures radiation dose Colored discs M radiodurans, B pumilus

28 STERILANT GASES Ethylene oxide Formaldehyde Betapropiolactone –More efficient than HCHO

29 DISINFECTION The process of freeing an article or a surface from all or some of the living microorganisms but not necessarily bacterial spores Strong disinfectants – for inanimate object Mild disinfectant (antiseptic) – superficial application on living tissue

30 Factors affecting DISINFECTION Conc of disinfectant Time of action pH of the medium Temperature Nature & number of organisms Presence of extraneous material Others – hardness of water, relative humidity

31 Categories of disinfectants 1.Alcohol 2.Aldehyde 3.Ethylene oxide 4.Dyes 5.Halogens 6.Phenolics 7.Surface active agents 8.Metallic salts 9.Diguanides 10.Amides

32 ALCOHOL Ethanol, isopropyl alcohol –Skin antiseptics at 70% –Less sporicidal & virucidal activity –Denature bacterial proteins –Isopropyl alcohol better fat solvent, more bactericidal and less volatile –Methyl alcohol – to treat cabinets / incubators affected by fungal spores –Others – benzyl alcohol, chlorbutol, phenylethanol

33 ALDEHYDE Formaldehyde - 10% used –In aq. soln is virucidal, bactericidal, sporicidal –Used to fumigate wards, sick rooms, labs –Expose to ammonia to remove residual formaldehyde –Has pungent smell, irritant to skin, eyes, mucus memb & toxic when inhaled Glutaraldehyde – less toxic, less irritant –Endotracheal tubes, metal instruments, polythene tubing Β propiolactone (BPL) – condensation product of ketane & formaldehyde –More efficient for fumigation but is carcinogenic –0.2% generally used

34 ETHYLENE OXIDE Highly inflammable, mixed with inert gases – CO 2, N Especially for heart lung machines, respirators, sutures, syringes, dental equipments

35 DYES Combine with nucleic acids Aniline dyes –Brilliant green, malachite green, crystal violet Acridine dyes –Proflavine, acriflavine, euflavine, aminacrine Skin & wound antiseptics Bacteriostatic, more active against GP bacteria

36 HALOGENS 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

37 PHENOLICS Carbolic acid – 2-5% –Powerful microbicidal, very corrosive –General purpose disinfectant in hospital Cresol, lysol Chloroxylenol, chlorophenol, hexachlorophane – less toxic, less irritant, less active, more readily inactivated by organic matter

38 SURFACE ACTIVE AGENTS Disrupt cell memb, 4 main groups Anionic surfactants – strong detergent action, weak antimicrobial action Non-ionic surfactants Cationic surfactants – quaternary ammonium compounds – cetrimide, benzalkonium chloride - bacteriostatic Amphoteric surfactants – both detergent & antimicrobial properties – Tego comps

39 METTALIC SALTS Mercuric salts – ointments Silver salts – AgNO 3 – to prevent infection of burns, ophthalmia neonatorum Copper salts – antifungal, antialgae – water reservoirs, swimming pools

40 DIGUANIDES Chlorhexidine – burns, skin disinfection Picloxydine –hospital equipment, floors

41 AMIDES Propamide Dibromopropamide Antiseptic cream, eye ointments

42 TESTS FOR DISINFECTANTS To determine efficacy of disinfectants –Phenol Co-efficient method Rideal Walker test Chick Martin test –Compares disinfectant with phenol –Kelsey & Sykes Capacity test –Determines dilution of disinfectant to be used –Kelsey & Mauer In-use test (stability test) –Checks end result of disinfection

43 PRIONS BACTERIAL SPORES Sterilisation High Level Disinfection Bacillus subtilis Clostridium sporogenes Intermediate Level disinfection MYCOBACTERIA M. tuberculosis var bovis NON LIPID OR SMALL VIRUSES Low Level Disinfection Polio virus, Rhino virus FUNGI Trichophyton, Candida, Cryptococcus VEGETATIVE BACTERIA Pseudomonas, Staphylococcus, VRE LIPID OR MEDIUM SIZED VIRUSES HBV, HIV, HSV, HCV, EBOLA, CMV

44 Categories Sterilisation High level disinfection (HLD) kills all microorganisms except high number of bacterial spores - aldehydes, hydrogen peroxide, ortho-phthaldehyde Intermediate level disinfection (ILD) kills all vegetative bacteria including M. tuberculosis var bovis, all fungi and most viruses - phenolics, iodophores, chlorine compounds, alcohols Low level disinfection (LLD) kills most vegetative bacteria but not M. tuberculosis var bovis, some fungi and some viruses - Hospital type germicides- quaternary ammonium compounds

45 Plasma technology Glow discharge or low temperature plasmas Plasma has sufficient energy to disrupt molecular bonds Direct current, radiofrequency, microwave power is used to produce the plasma Used in disinfection of OTs

46 A known HIV positive patient is admitted in an isolation ward after an abdominal surgery following an accident. The resident doctor who changed his dressing the next day found it to be soaked in blood. Which of the following would be the right method of choice of discarding the dressings : a) Pour 1% hypochlorite on the dressing material and send it for incineration in a appropriate bag b) Pour 5% hypochlorite on the dressing material and send it for incineration in a appropriate bag c) Put the dressing material directly in an appropriate bag and send for incineration d) Pour 2% Lysol on the dressing material and send it for incineration in a appropriate bag

47 Heat labile instruments for use in surgical procedures can be best sterilized by Absolute alcohol Ultra violet rays Cholorine releasing compound Ethylene oxide gas

48 Sterilizing agents include –Cyclohexidene –Ethylene oxide –Diethyl ether –Gallamine

49 Sharp instruments are not sterilized by Dry heat Boiling 2% glutaraldehyde Autoclaving

50 Which is a form of cold sterilization Gamma rays Beta rays IR rays Autoclave

51 Sterilization of serum containing media Autoclaving Filtration Gamma radiation Incineration

52 Sterilizing agents include Ether Alcohol Chlorhexidine Dry heat Ethylene oxide

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