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.

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.

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

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

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

1. Flaming Temp of flame 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

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

3. 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 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

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

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

Eg Salmonella, Brucella and Mycobacteria 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

Tyndallisation (intermittent sterilization) - 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

TEMP ABOVE 1000C Autoclave (steam under pressure):

Uses: Used for rubber articles, dressings, sharp instruments, infectious medical waste, culture media. Sterilization control Thermocouples 106 spore of B. stearothermophilus. Incubate at 550C for 5 days

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

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

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

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

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

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 endotracheal tubes, face masks metal instruments, polythene tubing.

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

Mechanism: Kills by oxidation 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

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

1. Anionic surfactants – strong detergent 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. Amphoteric surfactants – both detergent & antimicrobial properties – Tego comps

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.

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.

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

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