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.
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.
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.
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
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.
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.
• 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.
ᴕ
• 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
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
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
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
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