PHYSICAL METHODS OF STERILISATION

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PHYSICAL METHODS OF STERILISATION Dr. Saurabh Singh, MDS (Oral n Maxillofacial

Transcript of PHYSICAL METHODS OF STERILISATION

PHYSICAL METHODS OF

STERILISATION

By :: Dr. Saurabh Singh, MDS (Oral n Maxillofacial Surgery)

The process by which an article, surface or

medium is freed of all living microorganisms

either in the vegetative or spore state.

“ STERILISATION ”

NEED FOR STERILISATION

• Microorganisms are constantly present in the

external environment and on the human body.

• Microorganisms are capable of causing

contamination and infection.

HOW CAN MICROORGANISMS BE KILLED

• Denaturation of proteins

• Oxidation

• Filtration

• Interruption of DNA synthesis/repair

• Interference with protein synthesis

• Disruption of cell membranes

Most Resistant

Least Resistant

Endospores

Mycobacteria

Fungal spores

Small non-enveloped viruses (polio, rota virus, rabies)

Vegetative fungal cells

Enveloped viruses (Herpes, Hepatitis B and C, HIV)

Vegetative bacteria

HOW STERILISATION WORKS

Disruption of cell wall cannot prevent cell from

bursting due to osmotic effects.

Damage to cytoplasmic membrane causes cellular

contents to leak out.

Damage to viral envelope interrupts viral

replication.

• DISINFECTION – The process of destruction or removal of all

pathogenic organisms, or organisms capable of giving rise to

infection.

• ASEPSIS – The avoidance of pathogenic organisms involving

the methods that prevents contamination of wounds and

other sites by ensuring that only sterile objects and fluids

come into contact them and risk of air-borne contamination

is minimized. For eg- no touch technique.

OTHER TERMINOLOGIES

• ANTISEPSIS – The procedure or application of antiseptic

solution or agent which inhibits growth of microorganisms

while remaining in contact with them. For eg- scrubbing up.

Antiseptic solution is betadiene.

PRINCIPLES OF STERILISATION

1. Thorough cleaning of instruments before

sterilisation.

2. Contact of sterilizing agent with all surfaces of each

item for specified period of time at specified

temperature.

3. Regular service and maintenance of sterilizing

equipment.

PHYSICAL METHODS CHEMICAL METHODSSTERILISATION

Sunlight

Drying

Heat •Dry heat•Moist heat

Filtration

Radiation

Alcohols

Aldehydes

Dyes

Halogens

Phenols

Surface-active agents

Metallic salts

Gases

SUNLIGHT Possesses bactericidal activity.

Action – Content

• UV rays,

• most oil which are screened out by glass

• presence of ozone in outer regions of

atmosphere.

DRYING

Most of the bacteria grows in moist

environment and thus 4/5th of their weight is

attributed to water.

DRYING

HEAT

Most reliable method.

Can be used either in dry form or moist form.

Factors influencing sterilisation by heat :

• Nature of heat – dry or moist

• Temperature and time

• No of microorganisms present

• Characteristics of organisms i.e. their species, strain and

sporing capacity

• Type of material from which organisms have to be eradicated

DRY HEAT STERILISATION Principle - Killing effect is due to

• protein denaturation

• oxidative damage

• toxic effect of elevated levels of electrolytes.ADVANTAGES DISADVANTAGES

Can be used for sharp instruments, glasswares, water impermeable oils,

waxes and powders.

Cannot be used for water containing culture media,plastic and rubber items.

Instruments do not rust Process is time consuming.

FLAMING

Bunsen flame is used.

Uses – Scalpel blades, inoculation wires and

loops, glass slides, cover slips.

INCINERATION

Excellent method.

Materials are reduced to ashes by burning.

For contaminated and pathological materials

at a high temperature.

HOT AIR OVEN

Employs dry heat that kills by dehydration and

oxidation.

Devices in Hot air oven

• heating elements in the wall of chamber

• fan

• temperature indicator

• control thermostat

• timer

• open mesh shelving

• Door interlocks

Time-temperature combinations

TEMPERATURE HOLDING TIME

160 oC 120 minutes

170 oC 60 minutes

180 oC 30 minutes

Measurements to quantify the killing power of heat –

• DRT (Decimal Reduction Time) / D value

Measures the rate of kill at a given temperature required to

reduce the no of viable organisms by 90%.

• Z value / Thermal death point

Measures the thermal resistance of the spore to the process

of measured as the no of degrees centigrade required to

produce a 10-fold change in thermal death time.

Uses – Glassware, forceps, scissors, scalpels,

all glass syringes, swabs, pharmaceutical

products such as liquid paraffin.

Disadvantages

• It does not penetrate grease, oil and

powders, so equipments containg these

substances can not be sterilised by hot air

oven.

• High temperature damages fabrics and melts

rubber.

STERILISATION CONTROL

Spores of nontoxigenic strain of Clostridium tetani

MOIST / STEAM HEAT STERILISATION

Employs the steam generated by heating water.

MICROBIAL INACTIVATION BY MOIST HEAT

IN SPORULATING BACTERIA IN NON-SPORULATING BACTERIA

Denaturation of spore enzyme Damage to cytoplasmic membrane

Impairment of germination Breakdown of RNA

Damage to membrane Coagulation of proteins

Increased sensitivity to inhibitory agents

Damage to bacterial chromosome

Structural damage

Damage to chromosomes

MOIST / STEAM HEAT STERILISATION

TEMPERATURE BELOW 100o C (PASTEURISATION)

Uses – for serum or other body

fluids containing proteins.

HOLDER METHOD – Heating at

63o C for 30 minutes.

FLASH PROCESS – Heating at

72o C for 15-20 seconds.

Vegetative bacteria are killed at 90-100 o C

Requires immersion in water and boiling for

10-30 minutes

Promoted by addition of 2% sodium

bicarbonate

TEMPERATURE AT 100o C (BOILING)

STEAM AT ATMOSPHERIC PRESSURE (TYNDALLISATION / INTERMITTENT STERILISATION)

Uses free steam at normal atmospheric

pressure i.e. 760 mmHg for 60 minutes.

PRINCIPLE - The first exposure kills all vegetative

bacteria and spores which survived the

heating process will germinate and are killed

in subsequent exposure.

TEMPERATURE ABOVE 100O C (AUTOCLAVES)

Most reliable method of sterilisation.

PRINCIPLE - Water boils when its vapour

pressure is equal to the vapour pressure of

surrounding atmosphere. Hence, when pressure

inside a closed vessel increases, temperature at

which water boils also increases.

3 major factors required for effective autoclaving

• Pressure - 1 kpa = 0.145 psi

• Temperature - 121o C

• Time - a minimum of 20 minutes after reaching full

temperature and pressure.

Sterilisation hold time

Heat penetration time

Various combinations of temperature, pressure

and holding times are used for sterilisation with

PURE, DRY, SATURATED steam.

i.e. free from admixture with air or other non-condensable gas

i.e. free from suspended droplets of condensed water

i.e. in free molecular balance with water from which it is formed

TEMPERATURE

ABOVE ATMOSPHERIC PRESSURE

HOLDING TIME

(oC) (psi) (bar) (min)

115-118 10 0.7 30

121-124 15 1.1 15

134-138 30 2.2 3

Higher temperatures and greater pressures shorten the time required for sterilization.

Functioning

• Preparation of load

• Condensation of steam – 1600ml steam at

100o C and at atmospheric pressure

condenses into 1ml of water and releases 518

calories of heat.

Importance of steam condensation into

water.

• Wetting the microorganisms

• Liberation of latent heat of steam

• Contraction in the volume of the steam

1670 volumes of steam at 1 bar pressure will

contract to form only 1 condensate.

Steam quality is IMPORTANT

.Saturated steam – 98% Steam

2% Water vapour

Dry steam – Superheated

Wet steam – Supersaturated

X

X

SUPERHEATED STEAM

Superheating may be caused

• by overheating of the jacket.

• by too great reduction in pressure.

• by processing too dry load of textiles.

SUPERSATURATED STEAM

Moisture content of steam 1 1

dryness fraction

Dryness fraction measures the proportion of

latent heat still available in it.

• Air removal – all air should be removed from

the chamber before holding time.

• If air-steam mixture is left then the total

pressure in the chamber will consist of sum of

pressure of air and pressure of steam

according to DALTON’S LAW.

Reasons for presence of air in chamber

during holding time includes :

• Insufficient time

• Leak in the chamber

• Contamination of steam supply

Drying the load – promoted by the

application of a vacuum before opening the

autoclave

SIMPLE LABORATORY AUTOCLAVE

Small, simple, portable autoclave.

Operates like domestic pressure cooker.

Sterilisation of small metal or glass

instruments.

Devices

• metal tank

• a lid with a gasket

• Manually operated tap

• pressure gauge

• pressurestat

• pressure-regulated (safety) valve

• thermal cut-out device

DOWNWARD DISPLACEMENT LABORATORY AUTOCLAVE

Removes air from the chamber and loads

efficiently.

Devices that

• assist the drying of wrapped and porous loads

• prevent the door from opening while chamber is

under pressure

• brings out the automatic control of the process

MULTI-PURPOSE LABORATORY AUTOCLAVE

Efficient assisted air removal and assisted

cooling.

AUTOCLAVES

Device for air removal

from chamber and

porous loads

Device for drying of

wrapped and porous

loads

Device for safe handling

SIMPLE LABORATORY

None None None

DOWNWARD DISPLACEMENT LABORATORY

Balanced pressure steam

trap

Condenser or low

vacuum venturi

Door interlock Thermocouple in

chamber

MULTI-PURPOSE LABORATORY

Vacuum pulsing High vacuum pump

Door interlock Thermocouple in

chamber

STERILISATION CONTROL

Bacterial spores – Bacillus stearothermophilus

Thermocouple

Brown’s test

Autoclave tape

FILTRATION Forced passage through a filter of porosity

small enough to retain any microorganisms

contained in them.

CANDLE FILTERS Hollow ‘Candle’ form

Principle – Fluid is forced by suction or

pressure from the inside to outside or vice

versa.

2 types-

• Unglazed ceramic filters eg- Chamberland

and Doulton filter

• Diatomaceous earth filters eg- Berkfeld and

Mandler filter

Cleaning – When they become clogged with

organic matter they should be heated to

redness in a furnace and allowed to cool

slowly

ASBESTOS FILTERS Disposable, single use discs with

high adsorbing capacity.

Discarded – Cariogenic potential

After use the disc is discarded.

Examples – Seitz and Sterimat filter.

SINTERED GLASS FILTERS Made from finely ground glass fused

sufficiently to make small particles adhere

Cleaning – After use, they are washed with

running water in reverse direction and cleaned

with warm, strong sulphuric acid.

MEMBRANE FILTERS

Made up of variety of polymeric materials

such as cellulose nitrate, cellulose diacetate,

polycarbonate and polyester.

Membranes are made in 2 ways-

• Capillary pore membranes

• Labyrinthine pore membranes

SYRINGE FILTERS

Fitted in syringe

Fluid is forced through the filter by pressing

down the piston.

Uses – solutions of heat-labile sugars

AIR FILTERS

Mainly HEPA (High Efficiency Particle Arresters) filter is used.

RADIATION

IONISING RADIATION

• Lethal action – breakdown of single stranded

or sometimes double-stranded DNA and effect

on other vital cell components.

• Cold sterilisation.

• X-rays, gamma rays and beta rays

NON-IONISING RADIATION

Electromagnetic rays with wavelengths

longer than those of visible light are used.

Ultraviolet and infrared rays

Ultraviolet rays kills microorganisms by

chemical reaction.

Low penetrating capacity

Infrared rays have no penetrating capacity.

REFERENCES• Textbook of Microbiology (7th edition) - by C K J Paniker• Textbook of Oral and Maxillofacial Surgery - by Prof Dr Neelima Anil Malik• Practical Medical Microbiology (13th edition) - by J. G. Collee, J. P. Duguid, A.G. Fraser, B. P.Marmion• Essentials of Medical Microbiology (3rd edition) - by Rajesh Bhatia, Rattan Lal Ichhpujani