sterilization

Presented by:- arun patel m.sc abt 4nd sem. md university rohtak

DEFINITIONS

Sterilization – process by which an article, surface or medium is freed of all living microorganisms either in the vegetative or spore state.

Disinfection – destruction or removal of all pathogenic organisms. (sanitization w.r.t. Food processing and catering)

The process of sterilization is used in microbiology for preventing contamination by

extraneous organisms, in surgery for maintaining asepsis, in food and drug manufacture for ensuring safety

from contaminating organisms.

The methods of sterilization employed depend on

the purpose for which it is carried out, the material which has to be sterilized and the nature of the microorganisms that are to be

removed or destroyed.

PHYSICAL AGENTSSunlight

Drying

Dry heat – flaming, incineration, hot air.

Moist heat – pasteurization, boiling, steam under normal pressure, steam under pressure.

Filtration – candles, asbestos pads, membranes.

Radiation

Ultrasonic and sonic vibrations.

CHEMICAL AGENTS

Alcohols – ethyl, isopropyl, trichlorobutanol

Aldehydes – formaldehyde, glutaraldehyde

Dyes

Halogens

Phenols

Surface active agents

Metallic salts

Gases – ethylene oxide, formaldehyde, betapropiolactone.

Sunlight: Action primarily due to UV rays.

Eg: In tropical country, the germicidal effect is better than seasoned countries.

Bacteria in water are readily destroyed by sunlight.

Drying: Moisture is essential for growth of bacteria. Drying in air has deleterious effect on many bacteria. However, spores are unaffected.

Therefore, it is not really unreliable.

Physical methods of Sterilization

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Heat: Most reliable method of sterilization and should be the method of

choice.

The factors influencing sterilization by heat: Nature of heat-dry or moist Temperature and time Number of microorganisms present Characteristics of organisms –species, strain, sporing capacity Type of material from which organism have to be eliminated. Killing effect is due to protein denaturation, oxidative damage and

toxic effect of elevated level of electrolytes. Killing effect of moist heat due to denaturation and coagulation of

proteins.

Dry Heat & Moist Heat.

Dry heatFlaming:-Heating over fire, till they become red hot.Instruments like: Point of Forceps, Spatulas,

Inoculating loops and Wires.(Inoculating loop is better dipped in disinfectant

first before flaming to prevent spattering)

Incineration: It is a process that involves the combustion

of organic substances contained in waste materials. Items: contaminated cloth, animal carcasses and

pathological material.

(However, polystyrene will emit black smoke. Hence should be autoclaved in appropriate container.)

Hot air oven Hot air ovens are electrical devices used in sterilization. The oven uses dry heat to sterilize articles. Generally, they can be operated from 50 to 300 °C (122 to 572 °F) . There is a thermostat controlling the temperature. This is the most widely used method of sterilization by dry heat.

Items: glassware, forceps, scissors, scalpels, all-glass syringes,

swabs, liquid paraffin, dusting powder, fats, grease.

(Materials should be properly arranged to allow free circulation of air )

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Temperature (c)

Holding time(in minutes

160 45

170 18

180 7.5

190 1.5

Precautions: Glass wares should be dry.Oven should not be over loaded.Articles are to be arranged in a manner to allow free circular of air.Door of the Oven should be opened after it cools down (2Hours).

Advantages & Disadvantages:They do not require water and there is not much pressure

build up within the oven, unlike an autoclave, making them safer to work with.

Suitable to be use in a laboratory environment.They are much smaller than autoclaves but can still be as

effective. As they use dry heat instead of moist heat, some

organisms like prions, may not be killed by them every time.

Moist heat:Moist heat can be categorized into 3 groups: Temperature below 100°C Temperature at 100°C Temperature above 100°C

Pasteurisation of milk

Holding period: 63°C, 30 minutes (holder method) ; or 72°C, 15-20 minutes followed by cooling quickly to 130c or lower.

Target: all nonsporing pathogens

Temperature at 100°C:

BoilingNot recommended for sterilising but used for disinfection.sterilization may be promoted by addition of 2% sodium bicarbonate to the water.Holding period: 10-30 minutes. 

Steam at atmospheric pressure (100°C)Used to sterilize culture media.This is an Inexpensive methodHolding period: 100°C, 20 minutes on three succesive days

Principle: first exposure kills vegetative bacteria and then the next exposure will kill vegetative bacteria that matures from the spore.

Steam under pressure Autoclave/steam sterilizer--: autoclave is a device that

uses steam to sterilize equipment and other objects. This means that all bacteria, viruses, fungi, and spores are

inactivated. However, prions may not be destroyed by autoclaving at the typical

134 °C for 3 minutes or 121 °C for 15 minutes.

Principle:

Water boils when its vapour pressure equals the surrounding atmosphere. Thus, when pressure inside closed vessels increases, the temperature at which water boils increases too.

Holding period: varies.

Temperature: between 108°C and 147°C.

Items: dressings, instruments, laboratory ware, media and pharmaceutical products.

filtrationFiltration helps to remove bacteria from large volumes of fluid,

especially fluid containing heat-labile components such as sera, solution of sugars and antibiotics.

A pore size of 0.2 m is effective because filters act not only mechanically but by electrostatic adsorption of particles to their surface.

The filters are of two types (deep and membrane).

FILTERS

DEEP FILTERS

Made up of Examples Comments and Uses

Candle filters 1.Diatomaceous earth2.Unglazed porcelain

Berkefeld filtersChamberlain filters

They are available in different porosity.Mostly used for the purification of drinking water and industrial uses.

Asbestos filters Asbestos (magnesium silicate)

Seitz and Sterimat filters

They have high adsorbing capacity and tend to alkalinize the filtered fluid.These are disposable , single use discs available in different grades.Asbestos is carcinogenic hence its use is discouraged.

Sintered glass filters

Fusing finely powdered glass particles

They are expensive and brittle.They have low absorptive property.

MEMBRANE FILTERS

Cellulose estersOther polymers

They are routinely used for water purification and analysis, sterilization, sterility testing and solutions of parenteral use.Wide range of average pore diameters(APD) are available but 0.2mm size is widely used.

RADIATION

Non Ionisizing radiation:

Types Produced by Mode of action Uses Comments

Low energy (Infrared and ultraviolet rays)

UV lamps DNA damage Infrared rays:Sterilization of prepacked items such as syringes and catheters.Ultraviolet rays:Used for disinfecting operation threatres and laboratories.

Use of UV light is limited by penetration and hazardous.

Ionisizing radiation:

High energy ionizing type (Gamma rays and high energy electrons such as X-rays and cosmic rays)

Cobalt-60 based instruments

DNA damage For the sterilization of antibiotics, hormones and other prepacked disposable items such as catheters, gloves, syringes, infusion sets, oils, animal feeds, etc.

They are expensive.They are very effective due to high penetrative power.

Ultrasonic and sonic vibration:

High frequency sound waves beyond the sensitivity of human ears are called as ultrasonic waves.

They have the property to disrupt the cells but the results have been variable.

Gram negative rods are more sensitive to ultrasonic vibration whereas Gram positive cocci, spores of fungi and bacteria are resistant to the vibration.

Ultrasonic devices are used in dental. However, most sonic machines are not reliable for routine use.

Chemical Agents: Properties of Ideal antiseptics or disinfectants should

• Effective against all microorganisms and have a wide spectrum of activity.• Have speedy action.• Be effective in presence of organic matter.• Be effective in varying pH(acidic and alkaline medium).• Be active in presence of organic matter.• Be stable.• Also compatible with other antiseptics and disinfectants.• Have high penetrating power.• Not corrode metals.• Not cause local irritation or sensitization.• Not interfere with healing.• Not be toxic if absorbed into circulation.• Be cheap and safe.

Factors influencing antiseptics or disinfectants:

Time of action.Temperature.Concentration of the substance.pH of the medium.Nature of the organisms.Presence of foreign material.

Mode of action

Protein coagulation.Disruption of cell membrane.Removal of free sulphydryl groups which is essential for the functioning of the enzymes.Substrate competition.

alcoholsEthyl alcohol, isopropyl alcohol, and n-propanol exhibit

rapid, broad-spectrum antimicrobial activity against vegetative bacteria, viruses, and fungi but are not sporicidal.

Activity is optimal when they are diluted to a concentration of 60–90% with water.

Protein slows its action whereas 1% mineral acid or alkali enhances the action.

aldyhyde

• Formaldehyde is bactericidal, sporicidal, and virucidal. • It is active against the amino group in the protein

molecule.• Formaldehyde gas is used for sterilizing instruments and

heat sensitive catheters and for fumigating wards, sick rooms and laboratories.

• Glutaraldehyde is used for low-temperature disinfection and sterilization of endoscopes and surgical equipment.

• It is normally used as a 2% solution to achieve sporicidal activity.

dyesTwo groups of dyes are there1.Aniline dyes eg: brilliant green, malachite green, crystal violet

2.Acridine dyes eg: proflavine, acriflavine, euflavine and aminacrine.They are used as skin and wound antiseptics.They are more active against Gram positive organisms

than Gram negative organisms

halogensIodine is an effective disinfectant that acts by iodinating

or oxidizing essential components of the microbial cell.Iodine is rapidly bactericidal, fungicidal, tuberculocidal,

virucidal, and sporicidal. 2% iodine in 50% alcohol (tincture), which kills more

rapidly and effectively than alcohol alone.The most important types of chlorine-releasing agents

are sodium hypochlorite, chlorine dioxide, and sodium dichloroisocyanurate, which are oxidizing agents that destroy the cellular activity of proteins

Phenolic compounds:

The use of phenolic compounds are introduced by the Lister.The mode of action is due to their capacity to disrupt the cell membrane.Phenol(carbolic acid) is a potent protein denaturant and bactericidal agent. It is used as disinfectants for various hospital purposes.Cresol and Lysol are active against a wide range of organisms but they are toxic

and corrosive.They are used to disinfect surgical instruments, contaminated materials and

floors.Halogenated diphenyl compounds such as hexachlorophene and

chlorohexidine.Hexachlorophene is toxic and should be handled with care.Chlorohexidene is nontoxic, hence used as skin antiseptic which is more

effective against Gram positive organisms than Gram negative organisms.

gasesEthylene oxide is an inflammable and potentially explosive gas. By mixing with inert gases like CO2 and N2 to concentration of

10% its explosive tendency is eliminated.It is an alkylating agent that inactivates microorganisms by

replacing labile hydrogen atoms on hydroxyl, carboxy, or sulfurhydryl groups, particularly of guanine and adenine in DNA.

Under controlled condition it is used to sterilize materials such as glass, artificial heart valves, respirators, lung machines, equipments and clothing.

Formaldehyde gas and Betapropiolactone(BPL) are used for fumigation of operation theatres and other rooms.

BPL is more efficient than formaldehyde gas.

Surface active agentsThese compounds have two regions in their molecular structures, one a water-

repelling (hydrophobic) group and the other a water-attracting (hydrophilic) group.

They are classified into four groups; anionic, cationic, nonionic and amphoteric.Cationic detergents, particularly the quaternary ammonium compounds

(“QACs") such as acetyl trimethyl ammonium bromide are highly bactericidal.They are effective against Gram +ve organisms than Gram –ve organisms.They are more active in alkaline pH.They are inactive against spores and viruses, tubercle bacilli.Anionnic detergents Eg; common soap.Amphoteric or ampholytic compounds or tego compounds are active against

Gram+ve organisms than Gram –ve organisms.

Mettalic salts

Salts of mercury, copper and silver are used as disinfectant.

Mercuric chloride is highly toxic. Hence organic compounds such as mercurochrome, phenyl mercury nitrate and thiomersal are used as antiseptics(less toxic).

Copper salts are used as fungicides.Silver salts in aqueous solution have a limited use.

Thank you