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

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

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

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

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

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

Flaming

Dry heat - INCINERATION 8700C - 9800C Complete burning to ashes Used for soiled dressings, animal carcasses, pathological material, disposables, non-reusable soiled bedding

Incineration

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

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

Dry heat – Hot air oven

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

Moist heat - Temp below 1000C Pasteurization 630C – 30 min (Holder method) 720C – 15-20 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 – 80-850C – 30 min – 3 days For media containing egg or serum – LJ, LSS

Inspissator Water bath

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) – 30-60 min in Arnold /Koch steamer For heat labile media – DCA, TCBS 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

Moist Heat - Temp above 1000C Autoclave (steam under pressure) - 1210C, 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

Moist Heat - Autoclave

Moist Heat - Autoclave

Steam Jacketed Horizontal Autoclave

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

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

Asbestos Filter holder Earthenware filter

Sintered glass filter

Membrane filters

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

STERILANT GASES Ethylene oxide Formaldehyde Betapropiolactone More efficient than HCHO

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

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

Categories of disinfectants Alcohol Aldehyde Ethylene oxide Dyes Halogens Phenolics Surface active agents Metallic salts Diguanides Amides

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

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

ETHYLENE OXIDE Highly inflammable, mixed with inert gases – CO2, N Especially for heart lung machines, respirators, sutures, syringes, dental equipments

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

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

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

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

METTALIC SALTS Mercuric salts – ointments Silver salts – AgNO3 – to prevent infection of burns, ophthalmia neonatorum Copper salts – antifungal, antialgae – water reservoirs, swimming pools

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

AMIDES Propamide Dibromopropamide Antiseptic cream, eye ointments

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

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

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

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

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

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

Sterilizing agents include Cyclohexidene Ethylene oxide Diethyl ether Gallamine

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

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

Sterilization of serum containing media Autoclaving Filtration Gamma radiation Incineration

Sterilizing agents include Ether Alcohol Chlorhexidine Dry heat Ethylene oxide