Sterilization & Disinfection

Tukaram Prabhu KAsst ProfessorMicrobiologyPCMS & 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– 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 holderEarthenware

filter

• Sintered glass filter

Membrane filters

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

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

1. Alcohol2. Aldehyde3. Ethylene oxide4. Dyes5. Halogens6. Phenolics7. Surface active agents8. Metallic salts9. Diguanides10. 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– 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%– 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