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
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

Dont 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: Brownes 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 100 C

0

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 100 C

0

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 100 C

0

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 7 edn Page 27)
th

Sterilization control
Thermocouples Brownes 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 m 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
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. 2. 3. 4. 5. 6. 7. 8. 9. 10. 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

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 Bacillus subtilis Clostridium sporogenes MYCOBACTERIA M. tuberculosis var bovis NON LIPID OR SMALL VIRUSES Polio virus, Rhino virus FUNGI Trichophyton, Candida, Cryptococcus VEGETATIVE BACTERIA Pseudomonas, Staphylococcus, VRE LIPID OR MEDIUM SIZED VIRUSES HBV, HIV, HSV, HCV, EBOLA, CMV

Sterilisation High Level Disinfection Intermediate Level disinfection

Low Level Disinfection

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 alcohols
bacteria including M. tuberculosis var bovis, all fungi and most viruses - phenolics, iodophores, chlorine compounds,

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