PHYSICAL METHODS OF

STERILISATION
 “ STERILISATION ”
The process by which an article, surface or
medium is freed of all living microorganisms
either in the vegetative or spore state.
 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
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

Least Resistant
 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.
 OTHER TERMINOLOGIES
• 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.
• 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 S CHEMICAL METHODS

Sunlight T Alcohols
E Aldehydes
Drying R
I Dyes
Heat •Dry heat
•Moist heat
L Halogens
I Phenols
Filtration S
A Surface-active agents

Radiation T Metallic salts

I
O Gases

N
 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, Cannot be used for water

glasswares, water impermeable oils, containing culture media,
waxes and powders.
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 Damage to bacterial chromosome
agents
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.
TEMPERATURE AT 100o C (BOILING)

 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
 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
 ABOVE ATMOSPHERIC HOLDING
TEMPERATURE PRESSURE 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
X steam – Superheated

Wet
X steam – Supersaturated
 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

ᴕ
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.
 Device for air Device for Device for safe
removal drying of handling
AUTOCLAVES from chamber wrapped and
and porous
porous loads loads
SIMPLE None None None
LABORATORY

DOWNWARD Balanced Condenser or Door interlock

DISPLACEMENT pressure steam low Thermocouple in
LABORATORY trap vacuum venturi chamber

MULTI-PURPOSE Vacuum pulsing High vacuum Door interlock

LABORATORY pump 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.