STERILIZATION

 Sterilization is defined as the process of destruction or removal of all viable

microorganisms from an object or from a particular environment.
 Sterilization can be achieved by physical, chemical and physiochemical means.
 Chemicals used as sterilizing agents are called chemisterilants.
 Disinfection is the process of elimination of most pathogenic microorganisms (excluding
bacterial spores) on inanimate objects.
 Sanitization is any cleansing technique that mechanically removes microorg. To reduce
the level of contaminants / microbial population to a safe level as determined by public
health standards.

FACTORS AFFECTING STERILIZATION

1. Population size: Because an equal fraction of a microbial population is killed during each
interval, a larger population requires a longer time to die than a smaller one.

2. Population composition. The effectiveness of an agent varies greatly with the nature of
the organisms being treated because microorganisms differ markedly in susceptibility.
Bacterial endospores are much more resistant to most antimicrobial agents than are
vegetative forms, and younger cells are usually more readily destroyed than mature
organisms. Some species are able to withstand adverse conditions better than others.

3. Concentration or intensity of an antimicrobial agent: the more concentrated a chemical

agent or intense a physical agent, the more rapidly microorganisms are destroyed.
However, agent effectiveness usually is not directly related to concentration or intensity.
Over a short range a small increase in concentration leads to an exponential rise in
effectiveness; beyond a certain point, increases may not raise the killing rate much at all.
Sometimes an agent is more effective at lower concentrations. For example, 70% ethanol
is more effective than 95% ethanol because its activity is enhanced by the presence of
water.

4. Duration of exposure. The longer a population is exposed to a microbicidal agent, the

more organisms are killed. To achieve sterilization, an exposure duration sufficient to
reduce the probability of survival to 10–6 or less should be used.

5. Temperature. An increase in the temperature at which a chemical acts often enhances its
activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used
at a higher temperature.
STERILIZATION METHODS

PHYSICAL METHOD STERILIZATION

SUNLIGHT
 The microbicidal activity of sunlight is mainly due to the presence of ultra violet rays
and heat in it.
 It is responsible for spontaneous sterilization in natural conditions.
 By killing bacteria suspended in water, sunlight provides natural method of
disinfection of water bodies such as tanks and lakes.
 Sunlight does not have sporicidal effect.
 Not used for the sterilization of pharmaceutical product.
HEAT

 Heat is most reliable source of method of sterilization.

 Heat acts by oxidative effects as well as denaturation and coagulation of proteins.

Mode of action of heat sterilization

 Heat causes the damage of the cell membrane, cytoplasmic membrane, RNA
by oxidation.
 Denaturation and coagulation is due to the hydrolytic process

There are two type of heat sterilization:-

1. Dry heat sterilization
2. Moist heat sterilization
Dry Heat Sterilization

 The lethal (death) effects of dry heat on microorganisms are due largely to oxidative
processes.
 This causes the vegetative cells whereas the spores are much resistant to it.
 This can by
 Red hot
 Flaming
 Incineration
 Hot air oven
Red heat:

 Articles such as bacteriological loops, straight wires, tips of forceps and searing spatulas.
 They are sterilized by holding them in Bunsen flame till they become red hot.

Flaming:

 This is a method of passing the article over a Bunsen flame, but not heating it to redness.
 Articles such as scalpels, mouth of test tubes, flasks, glass slides and cover slips are
passed through the flame a few times.
 Vegetative cells are killed but spores not.
 This method too is limited to those articles that can be exposed to flame.
Incineration
 This is a method of destroying contaminated material by burning them in incinerator.
 Articles such as soiled dressings; animal carcasses, pathological material and bedding etc
should be subjected to incineration.
 This technique results in the loss of the article, hence is suitable only for those articles
that have to be disposed.

Hot air oven

 Principle
In dry heat sterilization the chief cause of death is oxidation. Sterilization by dry heat is
usually carried out in an apparatus called hot air oven. In which heat is transferred from
its source to the load by radiation, convection and to the lesser extend conduction.
Exposure time and temperature for hot air oven is 60 minutes at 160◦ C
 Construction
o Dry heat sterilization is usually carried out in a hot air oven.
o It consist of insulated polished stainless steel chamber, with a usual capacity of up
to 250 litres, surrounded by an outer case containing electric heaters located in
positions to prevent cool spots developing inside the chamber.
o A fan is fitted to the rear of the oven to provide circulating air, thus ensuring more
rapid equilibration of temperature.
o Shelves within the chamber are perforated to allow good airflow.
 o Thermocouples can be used to monitor the temperature of both the oven air and
articles contained within.
o A fixed temperature sensor connected to a chart recorder provides a permanent
record of the sterilization cycle.
o Appropriate door-locking controls should be incorporated to prevent interruption
of a sterilization cycle once begun.
Diagram important

Operation
 Preparation of Articles
All containers should be dry before loading. It is better if containers are oven dried at
100oC prior to sterilization.Articles to be sterilized must be wrapped or enclosed in
containers of sufficient strength and integrity to provide good post-sterilization protection
against contamination. Suitable materials are paper, cardboard tubes or aluminium
containers.
 Loading the hot air oven
Articles should be loaded in such a manner that there is no obstacle in the air to be
circulated between and around them.
 Holding of articles
Hold the article till the temperature of the sterilizer becomes 160◦C and wait for 60
minutes after attaining 160◦C. After 60 minutes, switch off the sterilizer.
 Cooling of sterilizer
Following sterilization,the chamber temperature is usually allowed to fall to around 40°C
before removal of sterilized articles; this can be accelerated by the use of forced cooling
with filtered air.
 Unloading
The material sterilized is transferred to the aseptic area.

For destruction of vegetative cells

160°c for 1 hr
170°C for 45 minutes
180°C for 30 minutes
For destruction of spores:
180°C for 2 hrs
260°C for 45 minutes

Advantages
 o It is used for the sterilisation of those substances which gets spolied during moist
heat sterilisation. Eg: oily materials and powders.
o The method is suitable for sterilisation of assembled equipments such as all glas
syringes due to expose to high temeprature for a long time.
o It is not so damaging to glass and metals equipments as moist heat.
o Dry heat will not corrode or rust instruments or needles.
o Dry heat will sterilize instruments containing many parts that can not be
disassembled

Disadvantages
o It is used for the sterilisation of those substances which gets spolied during moist heat
sterilisation. Eg: oily materials and powders.
o The method is suitable for sterilisation of assembled equipments such as all glas
syringes due to expose to high temeprature for a long time.
o It is not so damaging to glass and metals equipments as moist heat.
o Dry heat will not corrode or rust instruments or needles.
o Dry heat will sterilize instruments containing many parts that can not be disassembled
Applications
o It is used for the sterilization of glasswares such as petridishes, flasks, pipettes,
test tubes etc.
o It is used in the sterilization of dry powders such as sulphacetamides,
sulphadiazenes, kaolin, zinc oxide, starch etc..
o Injections where fixed oil is used as a vehicle are sterilized by dry heat
sterilization eg: injections of progestrone, injection of testosterone propionate and
injections of oestradiols dipropionate.
o It is also used for the sterilization of scalpels, scissors, spatula etc..

Sterilization control
Three methods exist to check the efficacy of sterilization process, namely physical, chemical
and biological
 Physical: Temperature chart recorder and thermocouple.
 Chemical: Browne’s tube No.3 (green spot, color changes from red to green)
 Biological: 106 spores of Bacillus subtilis var niger or Clostridium tetani on paper strips are
placed insideenvelopes and then placed inside the hot air oven. Upon completion of
sterilization cycle, the strips are removed and inoculated into thioglycollate broth or cooked
meat medium and incubated at 37oC for 3-5 days. Proper sterilization should kill the spores
and there should not be any growth.

Moist Heat Sterilization

Moist heat sterilization produces lethal effect by the denaturation and coagulation of protein. The
advantage of steam lies in the latent heat liberated when it condenses on a cooler surface, raising
the temperature of the surface.
Advantages of steam sterilization over dry heat sterilization
It has more penetrative power than dry air, it moistens the spores (moisture is essential for
coagulation of proteins), condensation of steam on cooler surface releases latent heat,
condensation of steam draws in fresh steam.
Types of moist heat sterilization
 At temperature below 100°C
o Vaccine bath(60°C for 1 hour)
o Pasteurization of milk(63°C for 30 mts and 72°C for 20 sec)
o Inspissator(80-85°C, 3 days)
 At temperature of 100°C
o Boling at 100°C ( 10-30 minutes, vegetative forms)
o Steam at atmospheric pressure at 100°C for 90 minutes( sugar media, Arnold or
Koch sterilizer)
o Fractional sterilization (Tyndallization)
 At temperature above 100°C
o Autoclave

Vaccine Bath
 This is used for the sterilization of vaccines.
 This method is used to kill the organism which will affect the stability of the antigen
present.
 The vaccines prepared are thermo liable and is stable up to temperature 60°C.
 This temperature is derived from thermal dead time.
 Usually below 60 °C for 1 hour.
 This is carried by using a thermostatically controlled water bath at set temperature.
 The container must be immersed entirely because organism on the surface above the
water level may not be killed.

Pasteurization
 This is the process used to make food safe and improve its property by reducing the
viable count to 97% to more than 99% but does not kill the spores.
 The use of pasteurization to kill pathogenic bacteria has helped reduce the transmission
of diseases, such as typhoid fever, tuberculosis, scarlet fever, polio, and dysentery.
 This is done to kill bovine tuberculosis caused by tuberculi.
HOLDING TIME: here the milk is heated in a tank at 63 °C for 30 minutes.
During the process it is agitated and cooled rapidly.
FLASH METHOD : the milk is paased through heat exchanger rapidly to 72 °C
for 15 mts.
 This method is suitable to destroy most milk borne pathogens like Salmonella,
Mycobacteria,
 Streptococci, Staphylococci and Brucella, however Coxiella may survive pasteurization.
 Efficacy is tested by phosphatase test and methylene blue test.
Inspissation
 This is a technique to solidify as well as disinfect egg and serum containing media.
 The medium containing serum or egg are placed in the slopes of an inspissator and heated
at 80-85°C for 30 minutes on three successive days. On the first day, the vegetative
bacteria would die and those spores that
  germinate by next day are then killed the following day. The process depends on
germination of spores in
 between inspissation. If the spores fail to germinate then this technique cannot be
considered sterilization.

Steam at atmospheric pressure at 100°C

 Boiling water (100°C) kills most vegetative bacteria and viruses immediately.
 Certain bacterial toxins such as Staphylococcal enterotoxin are also heat resistant. Some
bacterial spores are resistant to boiling and survive; hence this is not a substitute for
sterilization.
 The killing activity can be enhanced by addition of 2% sodium bicarbonate.
 When absolute sterility is not required, certain metal articles and glassware can be
disinfected by placing them in boiling water for 10-20 minutes.

Tyndallization
 Sugar and gelatin in medium may get decomposed on autoclaving, they can withstand the
temperature of free-flowing steam (100°C).
 Hence they are exposed to free steaming for 20 minutes for three successive days. This
process is known as tyndallisation or fractional sterilization or intermittent sterilization.
 The vegetative bacteria are killed in the first exposure and the spores that germinate by
next day are killed in subsequent days. The success of process depends on the
germination of spores.
 Arnold’s and Koch’s steamers were used.
 A steamer is a metal cabinet with perforated trays to hold the articles and a conical lid.
The bottom of steamer is filled with water and heated. The steam that is generated
sterilizes the articles when exposed for a period of 90 minutes.
 Use: Used to sterilize media such as TCBS, DCA and selenite broth

Autoclave
  This is designed to use steam under regulated pressure for sterilization.
 Also known by Steam sterilizers
Principle
The principle behind the autoclave and moist heat sterilization is that water boils when
vapour pressure becomes equal to atmospheric pressure. Hence when the pressure inside a
closed vessel increases the temperature at water boils also increases. Saturated steam has
greater penetration power. When this comes into contact with the cooler surface it condenses
to water and gives up the latent heat to that surface. The larger reduction in the volume sucks
more steam to the area and the process continues till the temperature of that surface is raised
to that of the steam. The condensed water ensures moist condition for killing the microbes
present.
Design
They fabricated with stainless steel vessels to withstand the steam pressures employed in
sterilization.
They are of two type :- portable sterilizer and large scale sterilizer.
Steam sterilizers are constructed with either cylindrical or rectangular chambers, with
preferred capacities ranging from 400 to 800 litres. They can be sealed by either a single door
or by doors at both ends (to allow through passage of processed materials). During
sterilization the doors are held closed by a locking mechanism which prevents opening when
the chamber is under pressure and until the chamber has cooled to a pre-set temperature,
typically 80°C.
In the larger sterilizers the chamber may be surrounded by a steam-jacket which can be used
to heat the autoclave chamber and promote a more uniform temperature throughout the load.
The same jacket can also be filled with water at the end of the cycle to facilitate cooling and
thus reduce the overall cycle time. The chamber floor slopes towards a discharge channel
through which air and condensate can be removed. Temperature is monitored within the
opening of the discharge channel and by thermocouples in dummy packages; jacket and
chamber pressures are followed using pressure gauges.
Operation
The stages of operation are as follows.
1. Air removal and steam admission.
 Air can be removed from steam sterilizers either by downward displacement with
steam, evacuation or a combination of the two.
 Downward displacement means the heavier cool air is forced out of the discharge
channel by incoming hot steam.
 This has the benefit of warming the load during air removal, which aids the heating-
up process. This is used for the sterilization of bottled fluids where bottle breakage
may occur under the combined stresses of evacuation and high temperature.
 For more air-retentive loads (i.e. dressings), the removal of air is by mechanical
evacuation. This can either be to an extremely high level (e.g. 2.5kPa) or can involve
a period of pulsed evacuation and steam admission, the latter approach improving air
extraction from dressings packs.
 The removal of air is for effective penetration of steam into the load.

2. Heating-up and exposure.

  When the sterilizer reaches its operating temperature and pressure the sterilization stage
begins.
 The duration of exposure may include a heating-up time in addition to the holding time
and this will normally be established using thermocouples in dummy articles.
3. Drying or cooling.
 Dressings packs and other porous loads may become dampened during the sterilization
process and must be dried before removal from the chamber. Drying is achieved by the
application of a vacuum.
 Followed by the restoration of the atmospheric pressure by admission of sterile filtered
air.
 For bottled fluids the final stage of the sterilization process is cooling, and this needs to
be achieved as rapidly as possible to minimize thermal degradation of the product and to
reduce processing time. Cooling is done by circulating water in the jacket that surrounds
the chamber or by spray-cooling with retained condensate delivered to the surface of the
load by nozzles fitted into the roof of the sterilizer chamber.
 This is often accompanied by the introduction of filtered, compressed air to minimize
container breakage due to high internal pressures (air ballasting).
 Containers must not be removed from the sterilizer until the internal pressure has dropped
to a safe level, usually indicated by a temperature of <80°C.
 The material sterilized is transferred to a as
 The material sterilized is transferred to a aseptic room.
 Temperature Holding time Pressure

115°C 30 min 10 psi

121°C 15 min 15 psi

126°C 10 min 20 psi

134°C 3 min 30 psi

Advantages
 Autoclaving destroys microorganisms more effectively than dry heat sterilization and
therefore shorter exposure at a lower temperature is required.
 It can be used for large proportion of the official injection.
 Dry saturated steam is used for the sterilization of porous materials for preventing the
damage.
 Equipments or components of rubber and certain plastic such as nylon and PVC will
withstand the condition.
Disadvantages
 It is unsuitable for anhydrous materials such as powders and oils.
 It cannot be used for injection and articles such as plastic that detoriates at 115◦C.
 Repeated exposure of glass at high temperature can cause clouding as well as alkali
extraction.
Applications
1. Typical loads include laboratory glassware, other equipment and waste, surgical
instruments and medical waste.
2. Sterilization is the total destruction of all forms of life, including bacterial spores. It is
best done with heat, either dry heat in an oven, or steam under pressure.
3. It is used in sterilizing of surgical dressings and surgical instruments.
4. It is used to sterilize the containers and closures.
5. It is used in the sterilizing the majority of official injections which can withstand the
pressure of 15 lb per square inch for 30 mins.
6. A notable growing application of autoclaves is the pre-disposal treatment and sterilization
of waste material, such as pathogenic hospital waste.
7. A medical autoclave is a device that uses steam to sterilze equipment and other objects.
This means that all bacteria, viruses, fungi and spores are inactivated. However, such as
those associated with the disease, may not be destroyed by autoclaving at the typical 134
⁰C for three minutes or 121 ⁰C for 15 minutes
Sterilization control:
 Physical method includes automatic process control, thermocouple and temperature chart
recorder.
 Chemical method includes Browne’s tube No.1 (black spot) and succinic acid (whose
melting point is 121oC) and Bowie Dick tape. Bowie Dick tape is applied to articles
being autoclaved. If the process has been satisfactory, dark brown stripes will appear
across the tape.
 Biological method includes a paper strip containing spores of Geobacillus
stearothermophilus.

RADIATION

 Two types of radiation are used:-

o ionizing
o non-ionizing.
 Non-ionizing rays are low energy rays with poor penetrative power while ionizing rays
are high-energy rays with good penetrative power.
 Since radiation does not generate heat, it is termed "cold sterilization".

Non-ionizing rays:
Rays of wavelength longer than the visible light are non-ionizing.
 Microbicidal wavelength of UV rays lie in the range of 200-280 nm, with 260 nm being
most effective.

Mode of Action
 This will denature the bacterial protein
 Leads the production of H2O2 and other toxic substances
 Will block DNA replication
 Thereby causes the death of micro organism
Application
 Irradiation of internal or incoming air or to sterilize the filling area of the antibiotic
plant.
 To prevent closed infection in hospital
 To improve bacteriological capacity of water used for non-sterile preparation.

Ionizing rays:
 Powerful agent than non-ionising agent.
 Gamma and accelerating electron.

Mode of action

The radiation causes excitation as well as ionization of particles

Sterilization capacity may be due to direct or indirect action
Direct Action (Target Theory)-the ionization radiation will reach the target region and will cause
lethal mutation. Thereby will causes cessation of reproduction and alteration of metabolism,
thereby causes the destruction of organism and sterility is achieved.
Indirect Action(Chemical Theory)- ionization radiation will be absorbed by the water molecule
which is present in the cell or surrounding cells and produces free radical generation and causes
oxidation/ reduction and causes the damage of cell and hence produces sterility.

Application
 To sterilize articles like syringes, gloves, dressing packs, foods and pharmaceuticals.
Advantages
 Insignificant temperature rise
 No need of aseptic handling
 Reliable and accurately controlled method
 Can sterilize vaccines without losing antigensity
 No degradation of media during sterilization, thus it can be used for thermally labile
media
 Leaves no chemical residue
 Administration of precise dosage and uniform dosage distribution
 Immediate availability of the media after sterilization
Disadvantages
 Poor penetrative power and requirement of sophisticated equipment.

FILTRATION STERILIZATION
  This method is used for the sterilization of thermoliable substance, by passing the
solution through the bacteria proof filter and the sterile filtrate is collected in sterile
receiver.
 Commonly used bacteria proof filters are:
o Membrane filter
o Ceramic filter
o Seitz filter
o Sintered metal filter
o Sintered glass filter

Precaution Required for Sterilization

1. Whole apparatus must be sterile.

2. An aseptic technique should be followed to prevent contamination.
3. There should be minimum exposure of filtrate to atmosphere.
4. The filter should be very fine to trap all bacteria.
Steps Involved in Filtration Sterilization
1. The filtration set up should be previously sterilized before carrying out the filtration
process.
2. Aseptic technique should be followed to minimize the risk of contamination.
3. Filtration solution should be passed through the previously sterilized bacteria proof
filter.
4. Aseptically transfer the filtrate into the sterile final container.
5. Testing the sample for sterility.
Mechanism of Filtration
The mechanism whereby particles are retained by a filter is significant only in initial stages of
filtration.

 Straining
Similar to sieving, i.e., particles of larger size can’t pass through smaller pore size
of filter medium.
 Impingement
Solids having the momentum move along the path of streaming flow and strike
(impinge) the filter medium.
 Entanglement
Thus the solids are retained on the filter medium • Particles become entwined
(entangled) in the masses of fibres (of cloths with fine hairy surface or porous felt) due to
smaller size of particles than the pore size. Thus solids are retained within filter medium.

Advantages/Applications
 1. Suitable for the sterilization of heat sensitive medicaments and substances like blood
product, insulin, enzymes.
2. All types of bacteria are removed.
3. Both clarification and filtration process is done side by side.

Disadvantages

1. The method is not reliable.

2. Aseptic technique is necessary.
3. Suspension and oily substance cannot be sterilized

Filtration Sterilization are of Two Type

1. Filtration sterilization of liquid
2. Filtration sterilization of gas

FILTRATION STERILIZATION OF LIQUID

1. Membrane Filter
 These are made of cellulose acetate or cellulose nitrate.
 The membrane filter is with a pore size of 0.22-0.45µ.
Advantages
 The method is suitable for the sterilization of thermo liable substances.
 Both clarification and filtration occurs simultaneously.
 All types of bacteria i.e. living and dead are removed from the preparation.
Disadvantages
 The method is not a reliable one and therefore a sterility test is necessary.
 The suspension and oily preparation cannot be sterilized.
 There are chances of adsorption of medicaments from a solution by the filter.
 Aseptic technique is necessary.
Application
 It is used for the sterilization of parenteral solution like insulin, blood products etc..
 It is also used for the sterilization of ophthalmic, biological, hormones and enzymes.
2. Ceramic Filter
 There are also called as filter candles.
 These are made of porcelain or kieselguhr.
 The candles are placed in the solution to be sterilized and the opening is attached to the
vacuum system. When vacuum is applied the pressure inside the candle is decreased.
Due to the difference in the pressure inside and outside , the solutions moved into the
candle. The filtrate is collected in sterile container.
Advantages
  The method is suitable for the sterilization of thermo liable substances.
 All types of bacteria i.e. living and dead are removed from the preparation.
Disadvantages
 This has a tendency to absorb the material from aqueous solution.

3. Seitz Filter
 A filter made of compressed asbestos fibres used in microbiological work to sterilize
fluids such as serum and to filter off bacteria but permit the passage of viruses.
 It has two parts :- the lower part is fitted with a perforated plate over which a
compressed asbestos pad is placed which act as filtering media.
 The upper part has a valve through which pressure is applied. The two parts i.e. upper
and lower are joined together by means of winged nuts. The solution to be filtered is
filled in the apparatus, pressure is applied and the solution is collected at the bottom.
Advantages
 The pads are meant for single use.
 The apparatus is very simple to use.
 For viscous solution they are more suitable than ceramic or glass filters.
Disadvantages
 Asbestos pads may shed loose fibres which makes the solution unsuitable.
 It imparts alkalinity to the filtrate which may be sufficient to precipitate the alkaloids
from solution of their salts.
 The pads may absorb sufficient amount of medicaments.
4. Sintered Glass Filter
 These are made from borosilicate glass.
 Sintered glass filter is available in various pore size and for bacterial filtration Grade 5
having the maximum pore size of 2µm is used.
Advantages
 They are used for filtering small as well as large volumes.
 Very little amount of medicaments may be absorbed.
 Volume of filtrate retained in the medium is negligible.
Disadvantages
 They are very costly
 The medium is unsuitable for large volume filtration because fore this purpose large
discs are required which is mechanically weak.

FILTRATION STERILIZATION OF GASES

The principal application for filtration sterilization of gases is in the provision of sterile air to
aseptic manufacturing suites, hospital isolation units and some operating theatres.
High Efficiency Particulate Air (Hepa) Filters
The high efficiency particulate air (HEPA) filters can remove up to 99.997% of particles
>0.03µm in diameter and thus are acting as depth filters. The filter will filter off the majority of
bacteria are found associated with dust particles and only the larger fungal spores are found in
the free state. Air is forced through HEPA filters by blower fans, and prefilters are used to
remove larger particles to extend the lifetime of the HEPA filter. The operational efficiency and
integrity of a HEPA filter can be monitored by pressure differential and airflow rate
measurements, and dioctylphthalate smoke particle penetration tests.
Other applications of filters include sterilization of venting or displacement air in tissue and
microbiological culture (carbon filters and hydrophobic membrane filters); decontamination of
air in mechanical ventilators (glass fibre filters); treatment of exhausted air from microbiological
safety cabinets (HEPA filters); and the clarification andsterilization of medical gases (glass wool
depth filters and hydrophobic membrane filters)

CHEMICAL STERILIZATION
There are two methods :-
1. Sterilization by heating with a bactericide.
2. Gaseous sterilization
Sterilization by heating with a bactericide
 This method is used for the sterilizing aqueous preparation which are unstable at higher
temperature.
 In this process the medicament is dissolved or suspended in a suitable solution of
bactericide and then transferred into final containers which are sealed and heated at 98-
100 oC for 30 minutes by heating it in boiling water.
 IP permit following bacteriocides:-
o For injections:
 Chlorocresols 0.1%w/v
o For eye drops
 Benzalkonium chloride:0.01%
 The bactericide should possess
o It should be non-toxic.
o Compatible with medicaments.
o Stable and active at various pH.
o Stable during heating and storage.
Applications:-
1. It is used for the sterilization of injections reasonably thermostable at 115-116 oC
GASEOUS STERILIZATION
Ethylene oxide
Mode of Action:
 This will the microorganism by the process of alkylation of essential substance present in
the protein.
Application:
 It is a highly effective chemisterilant, capable of killing spores rapidly. It is highly
inflammable so it is combined with CO2 and produce good penetration in the
presence of humidity.
 It is used to sterilize heat liable articles such as bedding, textiles, rubber, plastic,
syringes, disposable petrididsh.
Advantages
 Thermoliable substance can be sterilized at room temperature or at a slightly raised
temperature.
 It can be used for the prepacked drugs because of its good penetration power
Disadvantages
It is highly toxic, irritating to eyes, skin, highly flammable, mutagenic and
carcinogenic.

Sterilization Cycle
The typical sterilization cycle consists of six phases:
1. presterilization conditioning,
2. sterilization,
3. evacuation,
4. air wash,
5. chamber exhaust,
6. aeration
Presterilization conditioning:- After the products have been loaded into the chamber
and the airtight door is sealed. Successful operation of the sterilizer requires removal of air from
the chamber by evacuation, humidification and conditioning of the load by passage of
subatmospheric pressure steam.
Sterilization:- The sterilant, which is supplied as a liquid, is vaporized and introduced
into the chamber to achieve the desired concentration of EO. The chamber pressure, which
depends on the type of sterilant gas used, is maintained for about 4 to 6 hours.
Evacuation:- Following sufficient exposure time, the sterilant gas is evacuated from the
chamber with a vacuum pump. This postcycle vacuum phase typically lasts about 10 minutes.
Air wash: The pressure in the chamber is bought to atmospheric pressure by
introducing either air, nitrogen, or CO2 (depending on the flammability of the sterilant gas
mixture). The combination of evacuation and air wash phases is repeated from four to six times
to remove as much of the EO from the product as possible
Chamber Exhaust: The chamber exhaust is an exhaust system that evacuates EO-laden air from
the chamber prior to unloading and while the chamber is being unloaded (and reloaded).
Aeration: Following their removal from the sterilization chamber, the sterile products
are placed in an aeration room and kept there for several hours or days depending on the product.
Sterilizer for ethylene oxide
Formaldehyde

Mode of action
Acts through alkylation of amino-, carboxyl- or hydroxyl group, and probably damages
nucleic acids. It kills all microorganisms, including spores.

Application:
 40% Formaldehyde (formalin) is used for surface disinfection and fumigation of
rooms, chambers, operation theatres, biological safety cabinets, wards, sick rooms
etc.

Disadvantages:
 Vapors are irritating (must be neutralized by ammonia), has poor penetration,
leaves non-volatile residue, activity is reduced in the presence of protein.

Beta-propiolactone (BPL):
Mode of action: It is an alkylating agent and acts through alkylation of carboxyl- and
hydroxyl- groups.
Application: It is an effective sporicidal agent, and has broad-spectrum activity. 0.2% is
used to sterilize biological products. It is more efficient in fumigation that formaldehyde.
It is used to sterilize vaccines, tissue grafts, surgical instruments and enzymes
Disadvantages: It has poor penetrating power and is a carcinogenic.

SATURATED STEAM VS SUPERHEATED STEAM

When the boiling point of water is reached, the water’s temperature ceases to rise and stays the
same until all the water is vaporized. The water goes from a liquid state to a vapour state and
receives energy in the form of “latent heat of vaporization”. As long as there’s some liquid water
left, the steam’s temperature is the same as the liquid water’s. Steam is then called saturated
steam.
When all the water is vaporized, any subsequent addition of heat raises the steam’s temperature.
Steam heated beyond the saturated steam level is called superheated steam.

STERILIZATION INDICATOR

Validation is a process of establishing documentary evidence demonstrating that a procedure,

process, or activity carried out in production or testing maintains the desired level of compliance
at all stages.
3 principles involved in the validation process
 To build sterility into a product
  To demonstrate maximum level of probability that sterilization methods have
established sterility to all units of batch
 To provide greater assurance of the results of the end product sterility test

Physical indicators

Heat sterilization.
 heat distribution and penetration within a sterilizer can be gained by the use of
thermocouples placed at selected sites in the chamber or inserted directly into test packs
or bottles.
 Pressure in steam sterilization is measured by the use of pressure gauge.

Gaseous Sterilization

 Gaseous sterilization procedures, elevated temperatures are monitored for each

sterilization cycle by temperature probes, and routine leak tests are performed to ensure
gas-tight seals.
 Gas concentration is measured independently of pressure rise, often by reference to
weight of gas used.

Radiation sterilization
 radiation sterilization, a plastic (often perspex) dosimeter which gradually darkens in
proportion to the radiation absorbed gives an accurate measure of the radiation

Filtration sterilization
 Integrity of the filter bed is studies by the bubble point pressure test, which is a technique
employed for determining the pore size of filters, and may also be used to check the
integrity of certain types of filter device. The principle of the test is that the wetted filter,
in its assembled unit, is subjected to an increasing air or nitrogen gas pressure
differential. The pressure difference recorded when the first bubble of gas breaks away
from the filter is related to the maximum pore size. When the gas pressure is further
increased slowly, there is a eruption of bubbles over the entire surface. The pressure
difference here is related to the mean pore size. A pressure differential below the
expected value would signify a damaged or faulty filter.

Efficiency testing of HEPA filters

DOP (dioctylphthalate) test

 DOP testing is a very quick process that tests the integrity of the HEPA (High Efficiency
Particulate Air) filter using DOP solutions in their operational conditions. These
solutions generate a gas type smoke and then generate gas particles that will be greater
than .03 microns. The test pass if HEPA the air filter must remove at least 99.97% of all
airborne particles greater than 0.3 micrometers in diameter from the air that passes
through it.

Chemical Indicator
 Chemical monitoring of a sterilization process is based on the ability of heat, steam,
sterilant gases and ionizing radiation to alter the chemical and/or physical characteristics
of a variety of chemical substances
 Properties of a chemical indicator
(i) those chemical indicators which integrate several sterilization parameters (i.e.
temperature, time and
(ii) those which measure only one parameter and consequently can only be used to
distinguish processed from unprocessed articles.

Sterilization Principle Device Parameter

Parameters

Autoclaving or heat Temperature Browne’s Tube Temperature , time

sterilization sensitive coloured Sealed tubes filled
solution with a solution which
changes colour at
elevated temperature;
rate colour change is
proportional to
temperature

Heating in an Steam sensitive Bowie-Dick test Saturated steam

autoclave chemicals Organic
chemicals(printed
ink base) is
impregnated into a
carrier material. A
combination of
moisture and heat
produces a darkening
of the ink.

Gaseous Reactive chemicals Indicator paper Gas concentration,

sterilization impregnated with a temperature and
Ethylene oxide reactive chemical time
which undergoes a
distinct colour
change on reaction
with ethylene oxide
in the presence of
heat and moisture.
 Radiation Radiation dosimeter Acidified ferric Accurate measure of
sterilization ammonium sulphate radiation dose
solution respond to
irradiation by dose
related changes in
their optical density

Biological indicators
 Biological indicators (BIs) for use in thermal, chemical or radiation sterilization
processes consist of standardized bacterial spore preparations which are usually in the
form either of suspensions in water or culture medium or of spores dried on paper,
aluminium or plastic carriers.
 The spores are of the particular bacterial species are kept in the specified sterilization
process during the sterilization cycle and removed and transferred to fresh media and
evaluated to the growth of microorganism

Dry Heat Sterilization

Physical indicator: In this process temperature record chart is made of each sterilization cycle with dry
heat sterilization. This chart forms the batch documentation and is compared against a master temperature
records. The temperature should be taken as the coolest part of the loaded sterilizer, further information
on heat distribution and penetration within sterilizer can be gained by the use of thermocouple place at
selected site in the chamber or injected into test packs or bottles.

Chemical indicator: It is based on the ability of heat to alter the chemical or physical characteristics of
variety of chemical substances. This change should take place only when satisfactory condition for
sterilization prevails. Thus conforming that sterilization cycle has been successfully completed. Chemical
indicators generally undergo melting or colour change.

E.g;- Browne’s Tube

Sealed tubes filled with a solution which changes colour at elevated temperature; rate colour
change is proportional to temperature.

Biological indicator: The biological indicators are the standardized bacterial spore(bacillus subtilis)
preparations which are usually in the form of suspension in water or culture medium or of spore dried on
paper or plastic carriers, they are placed in sterilizer.

After the sterilization process the aqueous suspension /spores are on carriers are aseptically transferred to
an appropriate nutrient medium, which is then incubated and occasionally seen for the growth.
Clostridium species is generally used for dry heat sterilization indicator.

Moist Heat Sterilization

Physical Indicator: In this process temperature record chart is made of each sterilization cycle with dry
heat sterilization. This chart of the batch documentation is compared against a master temperature
records. The temperature should be taken as the coolest part of the loaded sterilizer, further information
on heat distribution and penetration within sterilizer can be gained by the use of thermocouple place at
selected site in the chamber or injected into test packs or bottles.

Chemical Indicator: It is based on the ability of heat to alter the chemical or physical characteristics of
variety of chemical substances. This change should take place only when satisfactory condition for
sterilization prevails. Thus conforming that sterilization cycle has been successfully completed chemical
indicator generally undergoes melting or colour change.

E.g;_ Bowie-Dick test

Organic chemicals(printed ink base) is impregnated into a carrier material. A combination of

moisture and heat produces a darkening of the ink.
Biological Indicator: Spores of B.steareothermophylus in sealed ampoules of culture medium are used
for moist heat sterilization monitoring and these may be incubated directly at 55°C, thus may eliminate
the need of aseptic transfer.

Gaseous Sterilization

Physical Indicator: Gas concentration is measured independently of pressure rise, often by reference to
weight of gas used.

Chemical Indicator: The chemical indicator used here are Royach Sacket, the indicator paper impregnated
with reactive chemical which undergoes a distinct colour change on reaction. Chemical indicators are
valuable monitors of the condition prevailing at the coolest of most in accessible part of a sterilizer.

Biological Indicator: As with chemical indicator they are usually packed in dummy packs located at
strategic sites in the sterilizer. Alternatively for gaseous sterilization, these may also be placed in tubular
helix device. The species of bacteria generally used for gaseous sterilization are B.subtilis var.niger and
B.subtilis var.golbigii

Radiation Sterilization

Physical Indicator: In radiation sterilization a plastic or perspex dosimeter which gradually darkens in
proportion to the radiation it absorbs give an accurate measure of the radiation dose and is considered to
be the best technique currently available for the radiation sterilization process.

Chemical Indicator: Chemical dosimeter acidified with cerric ammonium sulphate or cerric sulphate
solution .These responds to irradiation by dose change in the applied density. Those are considered best
and accurately measure relation dose.
Biological Indicator: These consist of standardized bacterial spore preparation which are usually in the
form of suspension in water or culture medium or of spore dried on paper or plastic carriers, they are
placed in sterilizer. After the sterilization process the aqueous suspension /spores are on carriers are
aseptically transferred to an appropriate nutrient medium, which is then incubated and periodically
observed for the growth. Clostridium species is generally used for dry heat sterilization indicator

Filtration Sterilization

Physical Indicator: Sterilizing filters are subjected to a bubble point pressure test. This is a technique for
determining the pore size of a filter, and may also be used to check the integrity of certain types of filters.
The principle of the test is that the wetted filter in its assembled unit is subjected to an increasing air or
nitrogen gas pressure difference. The pressure difference recorded when the first bubble of gas breaks
away from the filter is related to maximum pore size. When the gas pressure is further increased slowly
there is general eruption of bubble over the entire surface. The pressure difference here is related to the
mean pore size. Pressure difference below the expected value would signify a damage or faulty filter.

Biological Indicator: Filtration sterilization requires a different approach from biological monitoring, the
test effectively measure in the ability of a filter to produce a sterile filtrate from a culture of suitable
organism S. marcesence, a small gram negative rod shape bacterium. B. diminuta used as a biological
indicator having a dimension 0.5 micrometres and 0.3 micrometre respectively has been used for filters of
0.45 micrometre and 0.22 micrometre. The extent of the passage of this organism through membrane
filter is enhanced by increasing the filtration pressure. Thus successful sterile filtration depends markedly
on the challenge condition. Such tests are used as the part of filter manufacture characterization and
quality assurance process, and user’s initial validation procedure.