,.

STERILIZATION
PROCESS

Department of Pharmaceutics
Definition :
 Sterilization is a process that eliminates (removes) or kills all forms of
life, including transmissible agents (such as fungi, bacteria ,viruses, spore
forms, etc.) present on a surface, contained in a fluid, in medication, or in a
compound such as biological culture media.

 Sterilization can be achieved by applying the proper combinations of heat,

chemicals, irradiation, high pressure, and filtration.
A . Physical methods:
1.Heat sterilization:
a . Dry heat sterilization
b . Moist heat sterilization
2 . Sterilization by radiation
a . Use of ultra violet rays
b . Ionising radiation
B . Chemical methods:
a . Sterilization by Gases
C . Mechanical methods
a . Ceramic filters
b . Seitz filter
c . Sintered glass filters
d . Sintered meta filters
e . Membrane filters
A . PHYSICAL(THERMAL ) METHODS :
The lethal effectiveness of heat on microorganism depends on
 Degree of heat
 Exposure period
 Moisture present
Within the range of sterilizing temperatures, the time required to produce a
lethal effect is inversely proportional to temperature employed.
Eg.,
Dry heat at 170°C - 1 hour
at 140°C - 3 hour
Mechanism:
Coagulation of the protein of the living cell
DRY HEAT STERILIZATION :
 Substances that resist degradation at temperatures above 140° C may be
rendered sterile by means of dry heat.
 Two hours exposure to a temperature of 180°C or 45min at 260°C
normally can be expected to kill spores as well as vegetative forms of all
microorganisms .
FACTORS IN DETERMINING CYCLE TIME:
The cycle time normally includes a
 Reasonable lag time for the substance to reach the sterilizing temperature
of the oven chamber assuming both start at room temperature.
 Hold period at the maximum temperature
 Cooling time
The time required for all of the materials to catch up with the temperature of
the chamber is longer with
 Larger quantities of the material
 Poor thermal conductance of the material
 Lower heat capacity
 Hold time may be shown by the sensors detecting the temperature at its
coolest spot.
 Better indication of the thermal condition is obtained by sensing, usually
with a thermocouple the coolest spot of the material to be sterilized.
 When such a location is used and when the coolest spot is known from
previous studies the timing required for sterilization is programmable.
 Thermal stability of the material to be sterilized must be known and the
optimum method of sterilization selected to achieve effective sterilization.

STERILIZER TYPES:
 Natural Convection type
 Forced Convection type
NATURAL CONVECTION TYPE :
 Natural convection depends on the currents produced by the rise of hot air
and fall of cool air.
 Circulation can be easily blocked with containers resulting in poor heat
distribution efficiency.
 Difference in temperature of 20°C or may be found .

FORCED CONVECTION TYPE:

 Provides a blower to circulate the heated air around the material.
 Efficiency is improved over natural convection.
 Temperature differences at various locations on the shelves may be reduced
to as low as +/- 1°C.
 The lag times of the load material are greatly reduced because fresh hot air
is circulated rapidly around the materials.
 Another type of sterilizer is the tunnel unit with a moving belt designed to
thermally sterilize glass bottles and similar item as they move through the
tunnel.
EFFECT ON MATERIALS:
 Elevated temperatures have adverse effect on many substances .
 Eg.,
Cellulose materials such as paper and cloth begin to char at a temperature of
about 160°C.
 At these temperatures many chemicals are decomposed ,rubber is rapidly
oxidized, thermoplastic materials melt.
 So this method of sterilization is applicable for Glass ware , metal ware ,
anhydrous oils and chemicals .
Advantages of Dry heat sterilization :
1 . It is used for sterilization of those substances which get spoiled during
moist heat sterilization.
EXAMPLE : Oily materials and powders.
2 . It is not damaging to glass and metal equipment as moist heat.
Disadvantages :
1 . Not suitable for surgical dressings .
2 . Not suitable for most of the medicaments , rubber and plastic good .
Applications :
1 . It is mainly used for sterilization of glass wares.
2 . It is used for sterilization of powders such as ,sulphacetamides , talc etc.,
3 . Injections where fixed oil I used as vehicle are sterilised by the dry heat
method .
EXAMPLE : Injections of progesterone , testosterone propionate
MOIST HEAT STERILIZATION :
 It is more effective than dry heat sterilization.
 It causes coagulation of cell proteins at lower temperature than dry heat .
 Thermal capacity of steam much greater than the hot air .
 Therefore when saturated steam strikes a cool object and is condensed , it
librates approximately 500 times the amount of energy librated by an
equal weight of hot air .
AIR DISPLACEMENT :
 Steam when enters an Autoclave chamber rises to the top displacing the
air downwards due to its lower density.
 Objects must be placed in the chamber with adequate circulation space
around each object and so arranged that air can be displaced downward
and out of the exhaust line.
 Any trapped air in containers prevent penetration of steam to these areas
and prevent sterilization.
 Air trapped in this manner is heated to the temperature of the steam, but
hot air at a temperature of 120°C requires a cycle time of 60 hours to
ensure lethal effect on spores.
FACTORS DETERMINING CYCLE TIME:
Spores and vegetative forms of bacteria may be effectively destroyed in an
autoclave employing
 steam under pressure during an exposure time of 20min at 15pounds
pressure or 3min at 27pounds pressure
 In case of bottles of solutions the heat must be conducted through the
wall of the container to raise the temperature of the solution
 Therefore a significant lag time is involved before the solution reaches the
sterilizing temperature.

AIR STEAM MIXTURES:

 These have lower temperature and lower thermal capacity than that of
pure steam. Air may be utilized to control the pressure in the chamber
when flexible walled containers of products are being sterilized.
APPROACHES TO REDUCTION OF CYCLE TIME:
 Prolonged heating of most objects may cause the deterioration of the material.
 Therefore the total cycle time should be controlled so that the heating period is
not unnecessarily prolonged.
Usually this is accomplished by shortening the cooling period.
SPRAY COOLING METHOD :
 By spraying the containers of the solution with gradually cooling water while
the pressure in the chamber is concurrently reduced.
 Cooling time for a load of 200 one liter bottles of solution may be reduced
from 20 hrs to 20 min
ACCELERATED COOLING METHOD :
 Employs short pulses of high pressure steam introduced into the loaded
chamber .
 As the steam expands in the chamber it extracts heat from the containers of
the solution.
 The steam is exhausted from the chamber at a rate that provides for a gradual
reduction of the pressure concurrent with the temperature reduction.
 PRECYCLE VACUUM METHOD :
 In a specially designed autoclave a recycle vacuum of atleast 20mm Hg
is drawn.
 More recent studies have shown that a double vacuum drawn in a
sequence prior to the heating cycle remove air more effectively from
porous materials.
 The subsequent introduction of steam permits rapid penetration and load
heating with complete elimination of air pockets ,total heating period is
reduced leading to less deleterious effects on materials.
 Particularly suited to operating room packs in hospitals where the total
cycle time for large packs has been reduced from 78min to 14min.
LOWER TEMPERATURE STERILIZATION :
 Moist heat also is used for lower temperature sterilization procedures.
 Temperatures of 100°C are lower are used for MARGINAL and
FRACTIONAL methods.
MARGINAL METHOD :
 For substances that must be processed by a thermal method but cannot
withstand high temperatures.
 Assurance of sterility is low.
FRACTIONAL METHOD :
 These processes are normally performed by 2 or 3 exposures to moist heat
alternated with intervals during which material is held at room or
incubated temperature.
 Methods such as TYNDALLIZATION and INSPISSATION are effective
in reducing the number of vegetative forms of microorganism but
unreliable against spores.
 Effectiveness can be improved by inclusion of bacteriostatic agent.
WRAPPING MATERIALS :
 Wrappings for equipment and supplies and permits easy penetration of
steam and escape of air.
 Will not tear or burst during the process .
 Provide an efficient bacterial barrier so that equipment remain starial after
sterilization.
 Maintenance of sterility depends on
a . Coverage of the contents of the pack.
b . Drying of the wrapping after the process .
c . Static air state.
INDICATORS USED FOR EVALUATING STERILIZATION PROCESS :
 Indicators should be used to check the conditions of proven validated
process.
 Most widely used indicator is THERMOCOUPLE.
 For autoclave sterilization , a variety of other indicators also are used.
 These include WAX or CHEMICAL PELLETS that melt at 121° C .
 Paper strips that are impregnated with chemicals that change color under
the influence of moisture and heat.
 BIOLOGIC INDICATORS: Resistant bacterial spores in sealed
ampoules or impregnated in dry paper strips are used.
Advantages of Autoclaving :
 Destroys micro organism more efficiently than dry heat.
 Used for sterilization of large number of official injections .
 Large quantity of material can be sterilized in one batch using a big
autoclave .
Disadvantages :
 Unsuitable for sterilization of powders and oils.
 Cannot be used for sterilization of injections and articles such as plastics
which get spoiled at 115-116°C for 30min .
Applications :
 Used for sterilization of surgical dressings and surgical instruments.
 Containers and closures are sterilized by autoclaving.

HOT AIR OVEN AUTOCLAVE

 STERILIZATION BY RADIATION
( NON THERMAL METHODS ) :
1 .USE OF U.V RAYS :
U.V rays are primarily used for their germicidal effect on surfaces or for
their penetrating effect through clean air and water .
LETHAL ACTION :
 When U.V light passes through matter energy liberated is absorbed by
atoms within the molecules of microorganism causing excitation of
electrons and altering the reactivity.
 When such a excitation and alteration of activity of essential atoms occurs
the organism dye or unable to reproduce.
 Germicidal effectiveness of U.V radiation is a function of intensity of
radiation and time of exposure.
 To maintain maximum effectiveness U. V LAMP must be kept free from
dust , grease , and scratches.
 They must be replaced when emission levels decreased substantially.
Disadvantages :
 Have low penetrating power so it is not applicable for sterilization of
packed pharmaceuticals.
 Less effective against organism in the atmosphere .
Applications :
• Used for sterilization of air to prevent cross contamination.
• Used for sterilization of thermolabile substances before packing.
2 .IONISING RADIATION :
 Radiations emitted from radio active isotopes such as COBALT 60
(GAMMA RAYS ) or produced by mechanical acceleration of electrons
to very high velocities and energy ( BETA RAYS )
 Gamma rays have high penetration power but are relatively expensive.
Two types of electron accelerators are present .
A . Linear accelerator
B . Van de Graff accelerator
LETHAL ACTION :
 Destroy microorganisms by stopping reproduction as a result of mutation
brought about by transfer of radiation beam energies to receptive
molecules.
 Mutations are also brought about by indirect action in which water
molecule are transformed into hydrogen and hydroxyl ions.
 These bring about energy changes in nucleic acids thus eliminating their
availability for the metabolism of the bacterial cell.
Advantages :
 No aseptic precautions are required.
 Suitable for all type of materials such as dry , moist and frozen.
 Exposure time is very short .
Disadvantages :
 Plant used is very costly.
 Radiation are very harmful to workers.
Applications :
 Used for sterilization of plastic syringes , hypodermic needles , scalpels .
 Used for sterilization of bone and tissue transplant , plastic tubing ,
catheters.
B . CHEMICAL METHODS :
Sterilization by Gases :
Formaldehyde and sulphur dioxide are commonly used.
These gases are highly reactive chemicals but difficult to remove from many
materials after exposure .
Therefore their usefulness is limited.
Ethylene oxide and beta- propiolactone have fewer disadvantages than
HCHO and SO2 agents.
 Ethylene oxide gas is commonly used to sterilize objects sensitive to
temperatures greater than 60 °C and / or radiation such as plastics, optics
and electrics.
 It can kill all known viruses, bacteria and fungi, including bacterial spores
and is compatible with most materials (e.g. of medical devices), even
when repeatedly applied.
 However, it is highly flammable, toxic and carcinogenic.
 A typical process consists of a preconditioning phase, the actual
sterilization run and a period of post-sterilization aeration to remove toxic
residues, such as ethylene oxide residues.
 The two most important ethylene oxide sterilization methods are:
(1) the gas chamber method
(2) the micro-dose method.
 MECHANISM :
• Exerts its effect by alkylation essential metabolites effecting the
reproductive process.
• Alkylation occurs by replacing an active hydrogen on sulfhydryl , amino ,
carboxyl , or hydroxyl groups with a hydroxyethylradicals.
• The altered metabolites are not available to the micro organism and so it
dies without reproducing.

BETA PROPIOLACTONE:
 Non flammable liquid at room temperature.
 Has low vapor pressure.
 Bactericidal against a wide variety of organisms at relatively low
concentrations.
 It is an alkylating agent similar to that of ethylene oxide.
 Vapor concentrations of approximately 2-4mg/l of space are effective at a
temperature not below 24 C and a relative humidity of atleast 70% with an
exposure period of atleast 2hrs.
 C . MECHANICAL METHODS :
Sterilization by Filteration :
 Filteration may be used for the removal of particles including micro organism
from solutions and gases without the application of heat.
 Ideally filters should not alter the solution or gas.
 Clear liquids that would be damaged by heat, irradiation or chemical
sterilization can be sterilized by membrane filtration.
 Currently used filters for parenteral solutions are of membrane type.
 Main phenomenon involved is adsorption occurs during the first portion of
filtration until the surface of the filter is saturated with adsorbed molecule.
 Membrane filters usually composed of plastic polymers including cellulose
acetate and nitrate , nylon , polyvinyl chloride , and Teflon .
 Membranes are usually rendered hydrophilic by the treatment with a surface
active agent for filtration of aqueous solutions.
 Non aqueous solvents as ethanol and inert gases the membrane is left in its
hydrophobic form.
 FUNCTIONS :
 Membrane filters function primarily by sieving or by screening particles
from a solution or gas thus retaining them on the filter surface.
 They also function in some instances by electrostatic attraction
particularly to the filtration of dry gases in which electrostatic charges
tend to increase because of the frictional effect of the flowing gas.
CERAMIC FILTERS:
 Also called filter candles.
 Made of porcelain or kieselguhr and are available in a range of pore size .
 Kieselguhr filters are usually softer than the porcelain type.
MECHANISM :
 The candle is placed in the solution to be sterilized and its opening is
attached to the vacuum system.
 When vacuum is applied the pressure inside the candle is decreased.
 Due to the difference in pressure between the outside and inside of the
candle the solution moves into the candle.
 The filtrate is collected in sterile container.
 SEITZ FILTER :
• It consists of perforated discs and asbestos sheet which is made up of
asbestos fibers but may also contain cellulose and alkaline earth metals.
• Due to fibrous nature of asbestos pads it may shed fiber into filtrate .
• Hence a few ml of filtrate should always be rejected .
• CERAMIC FILTER SEITZ FILTER
 SINTERED GLASS FILTERS :
 Made from Borosilicate glass.
 Glass is finely powdered and particles of the required size are separated
and then packed into disc moulds.
 These moulds are heated until a suitable adhesion ha taken place between
granules.
 These discs are fused to funnels of suitable shape and size.
 Available in different pore size and are numbered accordingly.
 It does not change the pH of the solution and also does not shed fibres.
 SINTERED META FILTERS :
 These are the metallic counter part of the sintered glass filters usually
made of stainless steel.
 Have greater mechanical strength but are liable to attack by the solutions
passing through them.
Advantages :
1 . The method is suitable for sterilization of thermolabile medicaments,
such as blood products , insulin and enzymes .
2 . All type of bacteria i.e., living as well as dead , are removed from the
preparation.
Disadvantages :
1. The method is not reliable one and therefore a sterility test is necessary.
2 . The suspensions and oily preparation cannot be sterilized by this
method.
3 . Aseptic technique is necessary .
Applications :
The method is useful for sterilization of parenteral solutions containing
thermolabile substances with out any decomposition.
EXAMPLE : Insulin and blood stream
REFERENCES :
• The theory and practice of industrial pharmacy by Leon Lachmann and
Herbert A .Liebermann
• CDC-Guideline for Disinfection and Sterilization in Healthcare
Facilities, 2008 Prions, p. 100. Retrieved July 10, 2010
Thiel, Theresa (1999).
"http://www.umsl.edu/~microbes/pdf/tyndallization.pdf" (PDF). Science
in the Real World.
http://www.umsl.edu/~microbes/pdf/tyndallization.pdf. Retrieved 2007-
03-06.
" Alberta Health and Wellness". Health.gov.ab.ca.
http://www.health.gov.ab.ca/resources/publications/PersonalServicesPt1
.pdf
. Retrieved 2010-06-25.
THANK YOU