This document discusses various methods of sterilization. It defines key terms like sterilization, disinfection, and antisepsis. It explains how sterilization works by disrupting the cell walls, membranes, and envelopes of microorganisms. The two main methods of sterilization discussed are physical methods like heat and radiation, and chemical methods using various agents. Heat sterilization can be achieved through dry heat methods like flaming and hot air ovens, or moist heat methods like steam under pressure in an autoclave. Proper packaging, steam penetration, air removal and drying are important for effective sterilization in an autoclave.

1. SEMINAR 1 – PHYSICAL METHODS OF STERILISATION
NEED FOR STERILISATION?
• Microorganisms are constantly present in the external environment and on the human body.
• Microorganisms are responsible for contamination and infection.
HOW STERILISATION WORKS?
 Cell wall maintains integrity of cell
• When disrupted, cannot prevent cell from bursting due to osmotic effects
 Cytoplasmic membrane contains cytoplasm and controls passage of chemicals into and out of cell
• When damaged, cellular contents leak out
 Viral envelope responsible for attachment of virus to target cell
• Damage to envelope interrupts viral replication
STERILISATION – The processs by which an article, surface or medium is freed of all living
microorganisms either in vegetative or spore state.
DISINFECTION – The 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 in which the operator doen not touch any
instruments with bare hands i.e. without cleaning hands and getting gloved.
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 butadiene.
PRINCIPLES OF STERILISATION
 All instruments should be thoroughly cleaned in such a way that all deposits of blood and debris are
removed before sterilisation.
 Sterilizing agent such as steam, heat, or gas should come in contact with each surface of all itemsto be
sterilised for specified period of time at specified temperature.
 All sterilizing equipments should me regularly serviced and maintained.
METHODS OF STERILISATION
PHYSICAL
 Sunlight
 Drying – freeze drying, cold shock, osmotic shock
 Heat
1. Dry heat – flaming, incineration, hot air
2. Moist heat – pasteurisation, boiling, steam at atmospheric pressure, steam under pressure
 Filtration – candles, asbestos pads, membranes
 Radiation
CHEMICAL
 Alcohols- ethyl, isopropyl, trichlorobutanol
 Aldehydes – formaldehyde, glutaraldehyde
 Dyes
 Halogens
 Phenols
 Surface-active agents
 Metallic salts
 Gases – ethylene oxide, formaldehyde, beta propiolactone
SUNLIGHT – It possesses bactericidal activity. The action is due to its content of UV rays, most oif which
are screened out by glass and presence of ozone in outer regions of atmosphere.

2. DRYING – Most of the bacteria grows in moisture environment and thus 4/5th of their weight is attributed to
water. So, drying in air has deleterious effect on many bacteria.
1. FREEZE DRYING – At reduced temperature bacterial growth slows down and finally stops. Some
bacteria like psychrophiles can grow even at 0o celcius, thus, for such bacterias freeze drying is
employed which involves rapid freezing with subsequent drying. Freezing inactivates bacteria by
damaging their outer membrane.
2. COLD SHOCK – The process in which organisms are suddenly chilled without freezing.
It kills gram +ve and gram –ve bacteria but yeasts survive.
3. OSMOTIC SHOCK – In this bacteria are suspended in hypertonic solution of sucrose with EDTA and
then it suspended in ice cold magnesium chloride solution.
It kills the bacteria but some of the periplasmic enzymes are released.
Drying is not reliable method of sterilisation as bacterial spores remain unaffected by it.
HEAT – It is the most reliable method. It can be used either in dry form or moist form. Factors that influence
sterilisation by heat are-
a. Nature of heat – dry or moist
b. Temperature and time
c. No of microorganisms present
d. Characteristics of organisms i.e. their species, strain and sporing capacity
e. Type of material from which organisms have to be eradicated.
DRY HEAT STERILISATION
The killing effect is due to protein denaturation, oxidative damage and the toxic effect of elevated levels of
electrolytes.
It is suitable for glasswares, instruments specially sharp and paper wrapped articles, water impermeable oils,
waxes and powders. But it cannot be used for water containing culture media.
ADVANTAGES DISADVANTAGES
Can be used for sharp instruments. Cannot be used for water containing culture media,
plastic and rubber items.
Instruments do not rust Process is time consuming.
1. FLAMING – Bunsen flame is used to destroy vegetative organisms present on the surface of
instrument.
It can be used for scalpel blades, glass slides, coverslips, mouths of culture tubes and bottle,
inoculation wires and loops. Care is taken not to crack the glass.
2. INCINERATION – It is an excellent method for safe destruction of materials such as contaminated and
pathological materials at a high temperature Eg- contaminated cloth, animal carcasses . Materials are
reduced to ashes by burning. Instrument used is incinerator.
3. HOT AIR OVEN – It employs dry heat that kills by dehydration and oxidation. The oven is usually
heated by electricity, with heating elements in the wall of chamber. A fan is fitted to ensure circulation
and even distribution of air between the objects being sterilised and elimination of air pockets. In case
there is no fan, the holding time has to be doubled for that particular temperature. Oven also has a
temperature indicator, control thermostat and timer, open mesh shelving. Door interlocks are also
fitted to ensure that heating cycle does not starts until door is shut and also that doors does not opens
after the cycle until chamber temperature is fallen below 60o C. This method depends on combination
of time and temperature. Various time-temperature combinations are:
TEMPERATURE HOLDING PERIOD
160o C 120 minutes
170o C 60 minutes
180o C 30 minutes

3. Several measurements have been defined to quantify the killing power of heat.
 DRT (Decimal Reduction Time) / D value – It measures the rate of kill at a given temperature
required to reduce the no of viable organisms by 90%.
 Z value / Thermal death point – It 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, dusting powder, fats and Greece.
Disadvantages: 1. It does not penetrate grease, oil and powders, so equipments containg these
substances can not be sterilised by hot air oven as hot air is a poor conductor of heat and thus has
poor penetrating capacity.
3. High temperature damages fabrics and melts rubber.
STERILSATION CONTROL – Spores of nontoxigenic strain of Clostridium tetani are used as
microbiological test for efficacy of dry heat sterilisation. Paper strips impregnated with 106 spores are
placed in paper envelops and inserted into suitable packs. After sterilisation, strips are removed and
inoculated into a thioglycollate or cooked meat media and incubated for sterility test under strict
anaerobic conditions at 37o C for 5 days.
MOIST / STEAM HEAT STERILISATION
It employs the steam generated by heating water. Steam is very efficient means of sterilisation as –
 It has high penetrating capacity
 It gives up a large amount of heat to the surface with which it comes into contact and on which it
condenses as water.
MICROBIAL INACTIVATION BY MOIST HEAT
IN SPORULATING BACTERIAL 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
1. (PASTEURISATION) TEMPERATURE BELOW 100o C – Heat labile fluids are disinfected by holder
method or flash process and then are allowed to cool quickly to 13o C.
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.
Serum or body fluids containing coagulable proteins can be sterilised by heating for 1 hour at 56o C in
a water bath on several successive days.
Media such as Lowenstein-Jensen and Loeffler’s serum are rendered sterile by heating at 80-85o C for
30 minutes on 3 successive days in an inspissator.
2. (BOILING) TEMPERATURE AT 100 o C – Vegetative bacteria are killed almost immediately at 90-
100 o C. The material, for which boiling is considered adequate, should be immersed in water and
boiled for 10-30 minutes. The lid of steriliser should not be opened during sterilisation. Sterilisation is
promoted by addition of 2% sodium bicarbonate to the water. Hard water should not be used.
Disadvantages – It is not recommended for sterilisation of instruments used for surgical procedures.

4. 3. (TYNDALLISATION / INTERMITTENT STERILISATION) STEAM AT ATMOSPHERIC PRESSURE –
Free steam at normal atmospheric pressure i.e. 760 mmHg for 60 minutes is used to sterilize culture
media and kills all organisms except viruses and the spores of most resistant mesophilic and
thermophilic bacteria.
The steamer consists of tinned copper cabinet with the walls suitably lagged. The conical lid enables
drainage of condensed steam and a perforated tray fitted above the water level ensures that the material
placed on it is surrounded by steam. Steaming at 100o C is done for 20 min each on 3 successive days.
PRINCIPLE- The first exposure kills all vegetative bacteria and spores which survived the heating
process will germinate and are killed in subsequent exposure.
4. (AUTOCLAVES) TEMPERATURE ABOVE 100O C – Autoclaving is the most reliable method of
sterilization. It can be used for all materials that are water-containing, permeable or wettable and not
liable to be damaged by the process.
The principle of autoclave or steam steriliser is that 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.
There are three major factors required for effective autoclaving:
 Pressure
 Temperature
 Time
Pressure is expressed in terms of psi (pounds per square inch) or kpa (kilo pascals) 1 kpa = 0.145 psi
Temperature must be reached and maintained at 121o C to achieve required pressure.
Time wrapped loads require a minimum of 20 minutes after reaching full temperature and pressure.
Sterilization hold time – time for which entire load requires to be exposed to pure dry saturated steam
at effective temperature to ensure sterilization.
Heat penetration time – additional time required before the start of sterilization hold time to allow
the penetration of heat into articles and heat them up to the effective temperature.
Various combinations of temperature, pressure and holding times are used for sterilisation with PURE,
DRY, SATURATED steam.
PURE i.e. free from admixture with air or other non-condensable gas
DRY i.e. free from suspended droplets of condensed water
SATURATED 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 : Materials to be sterilised should be packaged in permeable wrappings of cloth,
plastic, or kraft paper so that they are protected from contamination with environmental bacterial after
their removal from autoclave. Autoclave should not be packed too fully so as to provide space between
articles to permit a free passage of steam between them.
Condensation of steam : When steam makes contact with cooler articles in autoclave, it condenses into
water over their surface and in their interstices. 1600ml steam at 100o C and at atmospheric pressure
condenses into 1ml of water and releases 518 calories of heat. This condensation of steam into water is
of critical importance.
 It wets the microorganisms on the articles and so provides essential condition for their killing
by moist heat.
 It liberates a large amount of latent heat of steam, so, heat up the articles very rapidly to
sterilizing temperature.
 It causes greatest contraction in the volume of the steam and so promotes the ingress of fresh
steam.

5. As the steam condenses into water, its contraction in volume is very great. Thus 1670 volumes of
steam at 1 bar pressure will contract to form only 1 condensate. The negative pressure created by the
contraction draws fresh steam on to the surface and into the interstices of the article. The cycle of
condensation, liberation of latent heat and drawing in of fresh steam is then repeated until article is
heated upto steam temperature. Effective condensation takes place only if the steam is on the boundry
between vapour and liquid phases and it must be pure, dry and saturated.
3 types of steam-
Saturated steam – 98% Steam, 2% Water vapour
Dry steam – Superheated
Wet steam – Supersaturated
:
 Superheated steam – Steam above the phase boundry i.e. at a temperature too high for its
pressure, behaves as a dry gas. Being unsaturated it fails to condense over the articles and thus
moisturising and latent heat effects are lost. Moreover, temperature and pressure are no longer
related so temperature cannot be controlled by regulation of pressure.
Superheating may be caused
i) by overheating of the jacket of a jacketed autoclave or
ii) by too great reduction in pressure as steam enters the autoclave or
iii) by processing too dry load of textiles which will absorb an excessive amount of steam.
 Supersaturated steam – If the steam is fed to autoclave by a long, poorly insulated, poorly
drained or low velocity supply lines, it is liable to become wet due to condensation, forming
suspended droplets of water as latent heat is lost before sensible heat. Wet steam delays the air
removal from autoclave chamber.
Moisture content of steam is inversely proportional to dryness fraction which measures the
proportion of latent heat still available in it.
Air removal : Autoclave must contain pure steam free from admixture with residual air throughout the
holding time of sterilization. Before holding time all air should be removed from the chamber. 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. Thus steam’s pressure will be less and temperature
will also be less than expected from pure steam. Moreover, bubbles of air trapped on article will form a
insulating layer over solid surfaces and thus, will inhibit condensation and heat transfer.
Reasons for presence of air in chamber during holding time includes :
i) Allowance of insufficient time for steam penetration into and removal of air from the load
ii) Occurrence of leak in the chamber
iii) Contamination of steam supply with air or other non condensable gas.
Drying the load : The articles of load will be moist at the end of their exposure to steam under
pressure. They tend to dry by evaporation when the pressure of the steam is reduced (i.e. after holding
time) and autoclave is opened. Drying may be promoted by the application of a vacuum before
opening the autoclave that is done by the use of steam ejector working on venture principle.
When the vacuum is used, air is reintroduced into the chamber but it passes through a filter which will
hold back airborne bacteria that may contaminate the load.
TYPES OF AUTOCLAVES
1. Simple laboratory autoclave
2. Larger simple autoclaves
3. Downward displacement laboratory autoclaves
4. Porous load autoclaves
5. Multi-purpose laboratory autoclaves
6. High-security autoclaves
SIMPLE LABORATORY AUTOCLAVE – It is small, simple portable autoclave that operates like
domestic pressure cooker for sterilisation of small metal or glass instruments.
It consists of an upright metal tank heated below by electricity or gas and have a lid with a gasket
enabling it to be fitted hermetically. The lid bears a manually operated tap for the discharge of air and

6. steam, a pressure gauge and a pressurestat. A pressure-regulated (safety) valve is fitted that can be set
to open when the pressure of steam rises above the chosen temperature for sterilization and to close
when it falls below it, thus, pressure and temperature are held automatically at required levels. Before
the holding period, air is removed by turbulence and entraining in steam discharged freely through
manually opened valve. A thermal cut-out device is fitted to shut off the source of heat in the event of
autoclaving boiling dry.
LARGE SIMPLE AUTOCLAVE – It is the larger version of simple, pressure-cooker type autoclave. It
is often horizontal with door at one end.
The size of the chamber is too large to permit the effective removal of air, so the working temperature
is not reached at the bottom of the chamber where the cooler air is layered and this failure goes
undetected as thermometer is not fitted in a gravity discharge drain.
DOWNWARD DISPLACEMENT LABORATORY AUTOCLAVE – It is more sophisticated type of
autoclave that removes air from the chamber and loads efficiently and also has other useful devices
such as
 Device that assist the drying of wrapped and porous loads
 Device that prevent the door from opening while chamber is under pressure
 Device that brings out the automatic control of the process
Pure steam is introduced from an external source into the upper part of chamber. It percolates
downwards through the load and expels dense and air-steam mixture out through a discharge channel
at the foot. A thermostatic steam trap is set on this channel to to open on arrival of air, air-steam
mixture and close on arrival of pure steam at correct temperature. By this repeated cycle of opening
and closing, the trap discharges a lot of air scavenged from the load and completes the removal of free
air from the chamber in heating-up and air displacement period of about 10 min. A thermometer is
fitted in the discharge line above the steam trap that indicates the temperature effectively in the lowest
and coolest part of the chamber. When it shows the sterilizing temperature, sterilizing period begins.
At the end of sterilizing period, the steam supply to chamber is cut off and the chamber with its load
begins to cool through its door in jacketed autoclave and through its walls in unjacketed autoclave.
Drying of load is effected by vapourizing the condensate by means of a vacuum drawn by venture
steam injector or condenser. Drying takes 20-30min depending on nature of load.
MULTI-PURPOSE LABORATORY AUTOCLAVE – It has a essential feature of efficient assisted
air removal and assisted cooling, door interlocks are provided to ensure that steam cannot enter the
chamber until door is locked and door does not open if chamber gauge pressure exceeds 0.2 bar or
until chamber temperature has fallen below 80o C.
Devices that are present in different autoclaves for different purpose
AUTOCLAVES Device for air removal
from chamber and
porous loads
Device for drying of
wrapped and porous
loads
Device for safe
Handling
SIMPLE None None None
DOWNWARD
DISPLACEMENT
Balanced pressure steam
trap
Condenser or low
vacuum venturi
Door interlock
Thermocouple in
Chamber
MULTI-PURPOSE Vacuum pulsing High vacuum pump Door interlock
Thermocouple in
Chamber
STERILISATION CONTROL – Spores of Bacillus stearothermophilus are used as test organism for
determining the efficacy of moist heat sterilisation. It is an thermophilus organism with an optimum
growth temperature of 55-60 oC and its spores requires an exposure of 12 minutes to be killed at 121
oC. Paper strips impregnated with 106 spores are dried at room temperature and placed in paper
envelops. These envelops are inserted in different parts of load. After sterilisation, strips are inoculated
into a suitable recovering medium and incubated for sterility tests at 55o C for 5 days.

7. FILTRATION – It helps to remove bacteria from heat labile liquids by forced passage through a filter of
porosity small enough to retain any microorganisms contained in them. It can also be used for purification of
water.
There are variety of filters-
1. Candle filters
 Unglazed ceramic filters
 Diatomaceous earth filters
2. Asbestos filters
3. Sintered glass filters
4. Membrane filters
5. Air filters
6. Syringe filters
CANDLE FILTERS
They are made in the form of hollow candle and the fluid is forced by suction or pressure from the inside to
outside or vice versa. They are manufactured in different grades of porosity. When they become clogged with
organic matter they should be heated to redness in a furnace and allowed to cool slowly. They are of 2 types-
Unglazed ceramic filters eg- Chamberland and Doulton filter
Diatomaceous earth filters eg- Berkfeld and Mandler filter
ASBESTOS FILTERS
These are disposable, single use discs with high adsorbing capacity and tends to alkalinise filtered liquids. It
has cariogenic potential due to which its use has been discarded. After use the disc is discarded. Eg- Seitz and
Sterimat filter.
SINTERED GLASS FILTERS
These are made from finely ground glass fused sufficiently to make small particles adhere. They have low
absorptive property and are brittle and expensive. After use they are washed with running water in reverse
direction and cleaned with warm, strong sulphuric acid.
MEMBRANE FILTERS
These membranes are made up of variety of polymeric materials such as cellulose nitrate, cellulose diacetate,
polycarbonate and polyester. They come in wide range of average pore diameter, 0.22 being most widely
used. They are sterilised by autoclaving.
These membranes are made in 2 ways-
1) Capillary pore membranes in which pores are produced by radiation that results in transparent
membranes less than 10 microm thick with smooth surface and high tensile strength.
2) Labyrinthine pore membranes which are produced by forced evaporation of solvents from cellulose
esters that results in thicker membrane and has 100-200 microm depth.
SYRINGE FILTERS
They are commonly fitted in syringe with the membrane of 13mm and 25mm diameter and fluid is forced
through the filter by pressing down the piston. Uses – solutions of heat-labile sugars.
AIR FILTERS
A principal use of this filtration is to render safe, the air withdrawn from an exhaust-ventilated safety cabinet
which is being used for working with dangerous pathogens.
Also it is used to decontaminate the air input into laminar flow cabinet used for work.
Mainly HEPA (High Efficiency Particle Arresters) filter is used.

8. RADIATION – Radiation used for sterilisation is of 2 types-
1. Ionizing radiation
2. Non-ionizing radiation
IONIZING RADIATION
It is effective for heat labile items and it has great penetrating properties. Its lethal action is due to its effect on
DNA of nucleus i.e. breakdown of single stranded or sometimes double-stranded DNA and on other vital cell
components. It is also called as Cold Sterilisation as there is no appreciable increase in temperature. Ionizing
radiation includes X-rays, gamma rays and beta rays. The efficacy of ionizing radiation is influenced by
certain factors-
 Type of organisms
 Pre-irradiation treatment
 Oxygen effect
 Stage of sporulation
NON-IONIZING RADIATION
In this type of radiation, electromagnetic rays with wavelengths longer than those of visible light are used.
Non-ionizing radiation includes ultraviolet and infrared rays.
Ultraviolet rays – They are absorbed by proteins and nucleic acid and kills microorganisms by chemical
reactions that they set up in bacterial cell. They has low penetrating capacity.
Infrared rays – It is another method of dry heat sterilisation. Infrared rays have no penetrating capacity.