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Sterilization & Disinfection

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Presentation on theme: "Sterilization & Disinfection"— 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 Two methods 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 2500C – 3000C 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

8700C 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: 1600C 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 Don’t 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: Browne’s tubes Color change from red to green Thermocouples 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

12 Dry heat – Hot air oven

13 MOIST HEAT Lethal effect due to denaturation & coagulation of proteins
Temp below 1000C Temp at 1000C Temp above 1000C

14 Moist heat - Temp below 1000C
Pasteurization 630C – 30 min (Holder method) 720C – sec (Flash method) 1320C – 1 sec (Ultra high temp) Vaccine baths - 600C – 60 min For vaccines of non-sporing bacteria Water bath - 560C – 60 min – 3 days For serum / body fluids containing coagulable proteins Inspissation – C – 30 min – 3 days For media containing egg or serum – LJ, LSS

15 Inspissator Water bath

16 Moist Heat - Temp at 1000C Boiling - 1000C 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) – min in Arnold /Koch steamer For heat labile media – DCA, TCBS Tyndallisation (intermittent sterilization) 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 1000C
Autoclave (steam under pressure) C, 15 min, 15 lbs Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media Principle – (refer Ananthanarayan & Paniker 7th edn Page 27) Sterilization control Thermocouples Browne’s tube (red-green), Bowie & Dick tape (white-brown) 106 spore of B stearothermophilus. Incubate at 550C for 5 days

18 Moist Heat - Autoclave


20 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: – 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 – Ionising – Gamma, X ray, cathode ray
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
Alcohol Aldehyde Ethylene oxide Dyes Halogens Phenolics Surface active agents Metallic salts Diguanides 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 – CO2, N
Especially for heart lung machines, respirators, sutures, syringes, dental equipments

35 DYES Combine with nucleic acids Aniline dyes Acridine 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% Cresol, lysol
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 – AgNO3 – 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

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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