3.

1 STERILIZATION
Contents

 WHAT IS STERILIZATION
 METHODS OF STERILIZATION

 MERITS, DEMERITS AND APPLICATIONS

OF DIFFERENT METHODS OF
STERILIZATION
 PHARMACEUTICAL IMPORTANCE OF

STERILIZATION
WHAT IS STERILIZATION?

•Sterilization can be defined as any

process that effectively kills or
eliminates transmissible agents (such
as fungi, bacteria, viruses and
prions) from a surface, equipment,
foods, medications, or biological
culture medium.
 METHODS OF STERILIZATION

 The various methods of sterilization are:

 1. Physical Method
a. Thermal (Heat) methods
b. Radiation method
c. Filtration method
 2. Chemical Method
a. Gaseous method
 PHYSICALMETHODS:

1. HEAT STERILIZATION:
 heat sterilization is the most widely used and reliable
method of sterilization, involving destruction of
enzymes and other essential cell constituents.
 this method of sterilization can be applied only to the
thermostable products and moisture- sensitive materials.
i) Dry Heat (160-180˚C)
Sterilization for thermo stable products
ii) Moist heat (121-134 ˚C)
sterilization is used for moisture- resistantmaterials.
 The efficiency with which heat is able to inactivate
microorganisms is dependent upon
i) the degree of heat, the exposure time and
ii) the presence of water.
 The action of heat will be due to induction of lethal
chemical events mediated through the action of
water and oxygen.
 In the presence of water much lower temperature
time exposures are required to kill microbe than in
the absence of water.
 THERMAL (HEAT) METHODS
Thermal methodsincludes:
i) Dry Heat Sterilization
Ex:1. Incineration
2. Red heat
3. Flaming
4. Hot air oven
ii) Moist Heat Sterilization
1.Dry saturated steam – Autoclaving
2.Boiling water/ steam at atmospheric
pressure
3. Hot water below boiling point
 Dry Heat Sterilization

 It employs higher temperatures in the range of 160-

180˚C and requires exposures time up to 2 hours,
depending upon the temperature employed.
 The benefit of dry heat includes good penetrability
and non-corrosive nature which makes it applicable
for sterilizing glass wares and metal surgical
instruments.
 It is also used for sterilizing non- aqueous thermo
stable liquids and thermo stable powders.
 Dry heat destroys bacterial endotoxins (or
pyrogens) which are difficult to eliminate by other
means and this property makes it applicable for
sterilizing glass bottles which are to be filled
aseptically
Physical methods for Sterilization

Sunlight:
 The microbicidal activity of sunlight is mainly due to the
presence of ultra violet rays in it.
 It is responsible for spontaneous sterilization in natural
conditions.
 In tropical countries, the sunlight is more effective in
killing germs due to combination of ultraviolet rays and
heat.
 By killing bacteria suspended in water, sunlight provides
natural method of disinfection of water bodies suchas
tanks and lakes.
 Heat:
 Heat is considered to be most reliable method of
sterilization of articles that can withstand heat.
 Heat acts by oxidative effects as well as
denaturation and coagulation of proteins.
 Those articles that cannot withstand high
temperatures can still be sterilized at lower
temperature by prolonging the duration of
exposure.
Factors affecting sterilization by heat:

 Nature of heat: Moist heat is more effective than dry heat

 Temperature and time: temperature and time are inversely
proportional. As temperature increases the time takendecreases.
 Number of microorganisms: More the number of microorganisms,
higher the temperature or longer the duration required.
 Nature of microorganism: Depends on species and strain of
microorganism, sensitivity to heat may vary. Spores are highly
resistant to heat.
 Type of material: Articles that are heavily contaminated require
higher temperature or prolonged exposure. Certain heat sensitive
articles must be sterilized at lower temperature.
 Presence of organic material: Organic materials such as protein,
sugars, oils and fats increase the time required.
Action of heat:
 Dry heat acts by protein denaturation, oxidative
damage and toxic effects of elevated levels of
electrolytes.
 The moist heat acts by coagulation and denaturation of
proteins.
 Moist heat is superior to dry heat in action.
 Temperature required to kill microbe by dry heat is
more than the moist heat.
 Thermal death time is the minimum time required to kill
a suspension of organisms at a predetermined
temperature in a specified environment.
DRYHEATSTERILIZATION
Red heat

 Articles suchas bacteriological loops, straight wires,

tips of forceps and searing spatulas are sterilized
by holding them in Bunsenflame till they become
red hot.
 This is a simple method for effective sterilization of
such articles, but is limited to those articles that can
be heated to rednessin flame.
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.
 Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short
exposure.
 This method too is limited to those articles that can be
exposed to flame. Cracking of the glassware may
occur.
Incineration:
 This is a method of destroying contaminated
material by burning themin 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.
 Burning of polystyrene materials emits dense smoke,
and hence they should not be incinerated.
Hot air oven:
 Hot air oven:
 This method was introduced by Louis Pasteur.
 Articles to be sterilized are exposed to high
temperature (160°C) for duration of one hour in an
electrically heated oven.
 Since air is poor conductor of heat, even distribution of
heat throughout the chamber is achieved by a fan.
 The heat is transferred to the article by radiation,
conduction and convection.
 The oven should be fitted with a thermostat control,
temperature indicator, meshed shelves and must have
adequate insulation.
 Hot air oven:
 Articles sterilized:
Metallic instruments (like forceps, scalpels, scissors), glass wares (such as
petri-dishes, pipettes, flasks, all-glass syringes), swabs, oils, grease,
petroleum jelly and some pharmaceutical products.
 Sterilization process:

 Articles to be sterilized must be perfectly dry before placing them

inside to avoid breakage.
 Articles must be placed at sufficient distance so as to allow free
circulation of air in between.
 Mouths of flasks, test tubes and both ends of pipettes must be
plugged with cotton wool.
 Articles such as petri dishes and pipettes may be arranged inside
metal canisters and then placed.
 Individual glass articles must be wrapped in Kraft paper or aluminum
foils.
 Hot air oven:
 Sterilization cycle:
 This takes into consideration the time taken for the articles to reach
the sterilizing temperature, maintenance of the sterilizing
temperature for a defined period (holding time) and the time taken
for the articles to cool down.
 Different temperature-time relations for holding time are
 60 minutes at 160°C,
 40 minutes at 170°C and
 20 minutes at 180°C.
 Increasing temperature by 10 degrees shortens the sterilizing time
by 50%.
 The hot air oven must not be opened until the temperature inside has
fallen below 60°C to prevent breakage of glassware.
Hot air oven:
 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 inside envelopes 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 37°C for 3-5 days.
 Proper sterilization should kill the spores and there should not
be any growth.
 Hot air oven:
 Advantages:
 It is an effective method of sterilization of heat stable
articles.
 The articles remain dry after sterilization.
 This is the only method of sterilizing oils and powders.
 Disadvantages:
 Since air is poor conductor of heat, hot air has poor
penetration.
 Cotton wool and paper may get slightly charred.
 Glasses may become smoky.
 Takes longer time compared to autoclave.
MOIST HEATSTERILIZATION
 Moist Heat Sterilization

 Moist heat sterilization involves the use of steam in

the range of 121-134˚C. Steam under pressure is
used to generate high temperature needed for
sterilization. Saturated steam acts as an effective
sterilizing agent.
 Autoclave
 Autoclaves use pressurized steam to destroy
microorganisms, and are the most dependable
systems available for the decontamination of
laboratory waste and the sterilization of laboratory
glassware, media, and reagents. For efficient heat
transfer, steam must flush the air out of the autoclave
chamber.
 Generally the conditions employed are
Temperature upto121-134˚C for 15-20 min under
15 lbs pressure,based ontype of metiral used.
 Autoclave
 Sterilization can be effectively achieved at a temperature
above 100°C using an autoclave.
 Water boils at 100°C at atmospheric pressure, but if pressure is
raised, the temperature at which the water boils also increases.
 In an autoclave the water is boiled in a closed chamber. As the
pressure rises, the boiling point of water also raises.
 At a pressure of 15 lbs inside the autoclave, the temperature is
said to be 121°C.
 Exposure of articles to this temperature for 15 minutes sterilizes
them.
 Todestroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times
are used; 135°C or 121°C for at least one hour are
recommended.
 Autoclave

 Advantages of steam:
 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.
 Different types of autoclave:
 Simple “pressure-cooker type” laboratory autoclave,
 Steam jacketed downward displacement laboratory autoclave
 High pressure pre-vacuum autoclave
Construction And Operation Of Autoclave:
Construction And Operation Of Autoclave:
 A simple autoclave has vertical or horizontal cylindrical body with a heating
element, a perforated tray to keep the articles, a lid that can be fastened
by screwclamps, a pressure gauge, a safety valve and a discharge tap.
 The articles to be sterilized must not be tightly packed.
 The screw caps and cotton plugs must be looselyfitted.
 Thelid isclosed but the discharge tap is kept open and the water is heated.
 Asthe water starts boiling, the steamdrives air out of the discharge tap.
 When all the air is displaced and steam start appearing through the discharge
tap, the tap is closed.
 Thepressure inside is allowed to rise up to 15 lbs per square inch.
 At this pressure the articles are held for 15 minutes, after which the heating is
stopped and the autoclave is allowed to cool.
 Once the pressure gauge shows the pressure equal to atmospheric pressure, the
discharge tap isopened to let the air in.
 Thelid isthen opened and articles removed.
 Articles sterilized:
 Culture media, dressings, certain equipment, linen etc.
 Precautions:
 Articles should not be tightly packed, the autoclave must not be overloaded.
 Air discharge must be complete and there should not be any residual air trapped
inside
 Caps of bottles and flasks should not be tight.
 Autoclave must not be opened until the pressure has fallen or else the contents
will boil over.
 Articles must be wrapped in paper to prevent drenching, bottles must not be
overfilled.
 Advantage: Very effective way of sterilization, quicker than hot air oven.
 Disadvantages:
 Drenching and wetting or articles may occur,
 trapped air may reduce the efficacy,
 takes long time to cool
Radiation Sterilization

 Many types of radiation are used for sterilization like

electromagnetic radiation (e.g. gamma rays and UV light),
particulate radiation (e.g. accelerated electrons).The major
target for these radiation ismicrobialDNA.
 Radiation sterilization with high energy gamma rays or
accelerated electrons has proven to be a useful method for the
industrial sterilization of heat sensitive products.
 Two types of radiation are used, ionizing and 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".
 In some parts of Europe, fruits and vegetables are
irradiated to increase their shelf life up to 500
percent.
 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 nmbeing most effective.
 UV rays are generated using a high-pressure mercury vapor lamp.
It is at this wavelength that the absorption by the microorganisms is
at its maximum, which results in the germicidaleffect.
 UV rays induce formation of thymine-thymine dimers, which
ultimately inhibits DNA replication.
 UV readily induces mutations in cells irradiated with a non-lethal
dose.
 Microorganisms such as bacteria, viruses, yeast, etc. that are
exposed to the effective UV radiation are inactivated within
seconds.
Non-ionizing rays:
 SinceUVrays don’t kill spores, they are considered to be of usein
surface disinfection.
 UVrays are employed to disinfect hospital wards, operation
theatres, virus laboratories, corridors, etc.
 Disadvantages of using uv rays include low penetrative power,
limited life of the uvbulb, some bacteria have DNArepair
enzymes that can overcome damage caused by uv rays, organic
matter and dust prevents its reach, rays are harmful to skin and eyes.
 It doesn't penetrate glass, paper or plastic.
Ionizing rays:
 Ionizing rays are of two types, particulate and
electromagnetic rays.
 Electron beams are particulate in nature while gamma
rays are electromagnetic in nature.
 High speed electrons are produced by a linear
accelerator from a heated cathode.
 Electron beams are employed to sterilize articles like
syringes, gloves, dressing packs, foods and
pharmaceuticals.
 Sterilization is accomplished in few seconds. Unlike
electromagnetic rays, the instruments can be switched
off.
Ionizing rays:
 Disadvantage includes poor penetrative power and requirement of
sophisticated equipment.
 Electromagnetic rays suchas gamma rays emanate from nuclear
disintegration of certain radioactive isotopes (Co60, Cs137).
 They have more penetrative power than electron beam but require longer
time of exposure. These high-energy radiations damage the nucleic acid of
the microorganism.
 A dosage of 2.5 megarads kills all bacteria, fungi, viruses and spores. It is
used commercially to sterilize disposable petri dishes, plastic syringes,
antibiotics, vitamins, hormones, glasswares and fabrics.
 Disadvantages include; unlike electron beams, they can’t be switched off,
glasswares tend to become brownish, loss of tensile strength in fabric.
 Gamma irradiation impairs the flavour of certain foods. Bacillus pumilus
E601 is used to evaluate sterilization process.
 FILTRATION
STERILIZATION
 Filtration does not kill microbes, it separates them out.
 Membrane filters with pore sizes between 0.2-0.45 μm are commonly
used to remove particles from solutions that can't be autoclaved.
 It is used to remove microbes from heat labile liquids suchas serum,
antibiotic solutions, sugar solutions, ureasolution.
 Various applications of filtration include removing bacteria from
ingredients of culture media, preparing suspensions of viruses and
phages free of bacteria, measuring sizes of viruses, separating toxins
from culture filtrates, counting bacteria, clarifying fluids and purifying
hydrated fluid.
 Filtration is aided by using either positive or negative pressure using
vacuum pumps.
 The older filters made of earthenware or asbestos are called depth
filters.
Different types of filters are:

Earthenware filters:
These filters are made up of diatomaceous earth or
porcelain. They are usually baked into the shape of
candle.
Different types of earthenware filters are:
a. Pasteur-Chamberland filter:
These candle filters are from France and are made up of
porcelain (sand and kaolin).
Similar filter from Britain is Doulton. Chamberland filters
are made with various porosities, which are graded as L1,
L1a, L2, L3, L5, L7, L9 and L11.
Doulton filters are P2, P5 and P11.
b. Berkefeld filter:
These are made of Kieselguhr, a fossilized diatomaceous
earth found in Germany.
They are available in three grades depending on their
porosity (pore size); they are V (veil), N (normal) and W
(wenig).
Quality of V grade filter is checked using culture
suspension of Serrtia marcescens (0.75 μm).
c. Mandler filter:
This filter from America is made of kieselguhr, asbestos
and plaster of Paris.
Asbestos filters:
 These filters are made from chrysotile type of
asbestos, chemically composed of magnesium
silicate.
 They are pressed to form disc, which are to be used
only once.
 The disc is held inside a metal mount, which is
sterilized by autoclaving.
 They are available in following grades; HP/PYR
(for removal of pyrogens), HP/EKS (for absolute
sterility) and HP/EK (for clarifying).
 Sintered glass filters:
 These are made from finely ground glass that are
fused sufficiently to make small particles adhere to
each other.
 They are usually available in the form of disc fused
into a glass funnel.
 Filters of Grade 5 have average pore diameter of
1-1.5 μm.
 They are washed in running water in reverse
direction and cleaned with warm concentrated
H2SO4 and sterilized by autoclaving.
Membrane
filters:
 These filters are made from a variety of polymeric
materials such as cellulose nitrate, cellulose diacetate,
polycarbonate and polyester.
 The older type of membrane, called gradocol (graded
colloidion) membrane was composed of cellulose
nitrate.
 Gradocol membranes have average pore diameter of
3-10 μm.
 The newer ones are composed of cellulose diacetate.
These membranes have a pore diameter ranging from
0.015 μm to 12 μm.
 These filters are sterilized by autoclaving.
 Membrane filters are made in two ways, the capillary
pore membranes have pores produced by radiation
while the labyrinthine pore membranes are produced
by forced evaporation of solvents from cellulose esters.
 The disadvantages of depth filters are migration of
filter material into the filtrate, absorption or retention
of certain volume of liquid by the filters, pore sizes are
not definite and viruses and mycoplasma could pass
through.
 The advantages of membrane filters are known
porosity, no retention of fluids, reusable after
autoclaving and compatible with many chemicals.
However, membrane filters have little loading capacity
and are fragile.
 Air Filters:
 Air can be filtered using HEPA(High Efficiency Particle Air)
filters.
 They are usually used in biological safety cabinets.
 HEPAfilters are at least 99.97% efficient for removing
particles >0.3 μm in diameter.
 Examples of areas where HEPAfilters are used include
rooms housing severely neutropenic patients and those
operating rooms designated for orthopedic implant
procedures.
 HEPAfilter efficiency is monitored with the dioctylphthalate
(DOP) particle test using particles that are 0.3 μm in
diameter.
SONIC AND ULTRASONIC
VIBRATIONS:
 Sound waves of frequency >20,000 cycle/second kills
bacteria and some viruses on exposing for onehour.
 Microwaves are not particularly antimicrobial in
themselves, rather the killing effect of microwaves are
largely due to the heat that they generate.
 High frequency sound waves disrupt cells.
 They are used to clean and disinfect instruments as well
as to reduce microbial load.
 This method is not reliable since many viruses and
phages are not affected by these waves.
CHEMICAL STERILIZATION METHOD
GASEOUS METHOD
 The chemically reactive gases such as formaldehyde,
(methanol, HCHO) and ethylene oxide (CH2)2O
possess biocidal activity. Ethylene oxide is a
colorless, odorless, and flammable gas.
 The mechanism of antimicrobial action of the two
gases is assumed to be through alkylations of
sulphydryl, amino, hydroxyl and carboxyl groups on
proteins and amino groups of nucleic acids.
 The concentration ranges (weight of gas per unit
chamber volume) are usually in range of 800-
1200 mg/L for ethylene oxide and 15-100 mg/L
for formaldehyde with operating temperatures of
45-63°C and 70-75°C respectively.
 Both of these gases being alkylating agents are
potentially mutagenic and carcinogenic. They also
produce acute toxicity including irritation of the skin,
conjunctiva and nasal mucosa
MERITS, DEMERITS
AND APPLICATIONS OF
DIFFERENT METHODS
OF STERILIZATION
S.no METHOD MECHANISM MERITS DEMERITS APPLICATIONS

Most widely Destroys Can be Dry heat is

1 Heat used and bacterial endo applied applicable
sterilization toxins for sterilizing glass
reliable only to the
method of thermo wares and metal
sterilization, stable surgical instruments
involving products and moist heat is
destruction the most
of enzymes dependable method
and other for decontamination
essential of laboratory waste
cell and the sterilization
constituents of laboratory
glassware, media,
and reagents.
S.no METHOD MECHANISM MERITS DEMERITS APPLICATIONS

1 Gaseous Alkylation Penetrating Gases being Ethylene oxide gas has

sterilization ability of gases. alkylating agents been used widely to
are potentially process heat-sensitive
mutagenic and devices.
carcinogenic.

Radiation It is a useful Radiation sterilization is

Ionization of Undesirable
2 sterilization method for
changes occur in
generally applied to articles
nucleic acids the industrial in the dry state; including
irradiated
sterilization surgical instruments,
products,an
of heat sutures, prostheses, unit
example is
sensitive dose ointments, plastics
aqueous solution
products
where radiolysis
of water occurs.
S.n METHOD MERITS DEMERITS APPLICATIONS
o MECHANISM

1 Filtration Does not destroy It is used for both Does not This method is Sterilizing
sterilization but removes the the clarification and differentiate grade filters are used in
microorganisms sterilization of between viable the treatment of heat
liquids and gases and non viable sensitive injections and
as it is capable of particles ophthalmic solutions,
preventing the biological products and
passage of both air and other gases for
viable and non supply to aseptic areas
viable particles
UNIT 3
Equipment employed in Large scale sterilisation
Autoclave as short note
 Autoclave
 Autoclaves use pressurized steam to destroy
microorganisms, and are the most dependable
systems available for the decontamination of
laboratory waste and the sterilization of laboratory
glassware, media, and reagents. For efficient heat
transfer, steam must flush the air out of the autoclave
chamber.
 Generally the conditions employed are
Temperature upto121-134˚C for 15-20 min under
15 lbs pressure,based ontype of metiral used.
 Autoclave
 Sterilization can be effectively achieved at a temperature
above 100°C using an autoclave.
 Water boils at 100°C at atmospheric pressure, but if pressure is raised,
the temperature at which the water boils also increases.
 In an autoclave the water is boiled in a closed chamber. Asthe pressure
rises, the boiling point of water also raises.
 At a pressure of 15 lbs inside the autoclave, the temperature is said to
be 121°C.
 Exposure of articles to this temperature for 15 minutes sterilizes them.
 Todestroy the infective agents associated with spongiform
encephalopathies (prions), higher temperatures or longer times are
used; 135°C or 121°C for at least one hour are recommended.
 Autoclave
• Advantages of steam:
• 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.
• Different types of autoclave:
• Simple “pressure-cooker type” laboratory autoclave,
• Steam jacketed downward displacement laboratory autoclave
• High pressure pre-vacuum autoclave
Construction And Operation Of Autoclave:
Construction And Operation Of Autoclave:
• A simple autoclave has vertical or horizontal cylindrical body with a heating
element, a perforated tray to keep the articles, a lid that can be fastened by screw
clamps, a pressuregauge, a safety valve and a discharge tap.
• The articles to be sterilized must not be tightly packed.
• The screw caps and cotton plugs must be looselyfitted.
• Thelid isclosed but the discharge tap iskept open and the water is heated.
• Asthe water starts boiling, the steamdrives air out of the discharge tap.
• When all the air isdisplaced and steamstart appearing through the discharge
tap, the tap is closed.
• Thepressureinside isallowed to rise up to 15 lbs per square inch.
• At this pressure the articles are held for 15 minutes, after which the heating is stopped
and the autoclave isallowed to cool.
• Once the pressure gauge shows the pressure equal to atmospheric pressure, the discharge
tap isopened to let the air in.
• Thelid isthenopened and articles removed.
• Articles sterilized:
• Culture media, dressings, certain equipment, linen etc.
• Precautions:
• Articles should not be tightly packed, the autoclave must not be overloaded.
• Air discharge must be complete and there should not be any residual air trapped inside
• Caps of bottles and flasks should not be tight.
• Autoclave mustnot be opened until the pressure has fallen or else the contents will boil
over.
• Articles must be wrapped in paper to prevent drenching, bottles must not be
overfilled.
• Advantage: Very effective way of sterilization, quicker than hot air oven.
• Disadvantages:
• Drenching and wetting or articles may occur,
• trapped air may reduce the efficacy,
• takes long time to cool
3.2 EVALUATION OF STERILIZATION
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Introduction
Sterilization means killing of microbes.
The main reasons for controlling the microorganisms are:
1. To prevent the contamination in sterile products.
2. To prevent decomposition and spoilage of food and food products.
3. To prevent contamination of unwanted microbes in pure cultures and
other microbiological experiments performed for research studies.
4. To prevent unwanted microbial contamination in antibiotics, enzymes,
vitamin fermentation and other industrial processes.
5. To prevent contamination in aseptic areas which are used for the
preparation of sterile dosage form.
Sterilization methods
1. Physical methods
a) Dry heat sterilization
b) Moist heat sterilization/steam sterilization
c) Radiation/Cold sterilization
d) Filtration/Mechanical method
2. Chemical methods
a) Gaseous sterilization
b) By using disinfection
1. Physical Method
a) Dry heat sterilization
• Heat is the most reliable and rapid method of sterilization.
• The killing effect of dry heat is due to protein denaturation.
• The time required for the sterilization is inversely proportional to the
temperature of exposure.
• Micro organisms are more resistant to dry heat as compared to moist
heat and therefore this process requires higher temperature and
longer exposure times.
Sunlight and drying
• Sunlight possesses ultraviolet rays which along with the heat raysare
responsible for appreciable germicidal activity.
• This is the natural method for sterilization of water in tanks, riversand
lakes.
• Spores are unaffected by drying. Hence it is very unreliable method.
Red hot
• It is used to sterilize metallic objects by holding them on the flame till they
are red hot. Eg : inoculating wires, needles, forceps etc.
Flaming
• The article is passed over the flame without allowing it to become red hot.
E.g.: mouth of culture tube, glass slides, needles, coverslips etc.
b) Moist heat sterilization
• Sterilization by moist heat means killing of microorganisms with hot
air or steam. The lethal effect of moist heat is due to denaturation
and coagulation of proteins.
• Moist heat sterilization is divided into three forms.
1. Temperature below 1000C
2. Temperature at 1000C
3. Temperature above 1000C
1. Temperature below 1000C:
• Heat liable fluids may be disinfected by heating at temperature below
1000C.
2. Temperature at 1000C:
• Boiling at 1000C for 10 to 30 minutes kills all vegetative bacteria and
some bacterial spores.
3. Temperature above 1000C:
• Saturated steam is a more efficient sterilizing agent.eg: Autoclave.
C) Radiation/Cold sterilization:
• Use of ultra-violet rays.
• Ionising radiations: X-rays, gamma rays,beta-rays
D) Filtration/Mechanical Method
• This is a non thermal method of sterilization used widely in
pharmaceutical industries where heat labile solutions are to be
sterilized.
• The following types of filters have been used for sterilization
1. Asbestos filter ( seitz filter)
2. Sintered glass filter (Morton filter)
3. Filter candles (ceramic filter)
4. Membrane filter (Millipore/ultra filter)
2. Chemical Method
a. Gaseous sterilization:
Eg: Formaldehyde, Ethylene oxide etc.
b. By using disinfectants
Eg: Cresol, phenol etc.
Equipment's involved in large scale sterilization

Autoclave Koch or Arnold steam

sterilizer

Hot air oven

 1. Autoclave
• The laboratory autoclave or pressure cooker type
autoclave consist of a vertical or horizontal
cylinder of gun metal or stainless steel in a
supporting frame or case.
• The autoclave lid is provided with pressure
gauge.
• The autoclave is provided with control for
adjusting the pressure and temperature and a
safety valve to avoid explosions.
• Water is added on the bottom of the autoclave
and the articles to be sterilized are placed in a
perforated shelf.
• The lid is closed, discharge tap is opened and
safety valve is adjusted to the required
pressure.
Autoclave conditions
(temperature/time/pressure relationship)
Temperature0C Steam Holding
pressure time
(lb/sq.inch) (minutes)
115-118 10 30
121-124 15 15
126-129 20 10
135-138 30 3
Advantages
• Microorganisms are destroyed more efficiently than the dry heat
because of the greater penetration power of the steam under
pressure.
• The method is applicable to the wide variety of materials and for large
load.
• Spores are easily destroyed by moist heat sterilization.
Disadvantages
• During autoclaving the pH of aqueous solution gets changed due to
water loss.
• Oils do not get sterilized in autoclave as they are hydrophobic in
nature and they don’t allow steam to penetrate them.
• Autoclave sterilization is not used for thermolabile substances,
powders and plastic that melts.
Applications
• To sterilize aqueous solution, saline solution, bacteriological media
like nutrient broth etc.
• To sterilize surgical dressings, rubber gloves, plastic fibrics and surgical
instruments.
• Sterilize different glassware like pipettes, petri plates, flasks and
different metal instruments.
2. Hot air oven
• This is the most widely used method of
sterilization by dry heat.
• The hot air oven consist of a doubled walled
chamber of aluminium or stainless steel
separated from the outer case by a thick layer
of insulation made of fibre glass.
• An oven is based on the principle where
sterilization is accomplished by dry heat or hot
heat.
• Dry heat is less effective as compered to moist
heat because in the presence of moisture,
proteins are easily coagulated and moist heat
has greater penetration power than the dry
heat.
• By the process of denaturation and oxidation,
microbes are destroyed by dry heat.
Temperature-Time relationship for hot air oven
• Substances that are not heat-labile and can tolerate temperature
upto 2500C may be sterilized by hot air oven.
• For normal sterilization work, the oven should be operated at 1600C
for 2 hours.
Temperature (0C) Time (hr)
140 3
150 2.5
160 2
170 1
180 0.5
 Application
• Hot air oven is used to sterilize glassware, forceps, scissors, syringes,
petri-dishes, test tubes, pipettes etc.
• Hot air oven is used for sterilization of fixed oils, powders, glycerin,
liquid paraffin, propylene glycol, waxes and other articles that are
either spoiled or not effectively sterilized by the moist heat.
3. Koch or Arnold steam sterilizer
3. Koch or Arnold steam sterilizer
• A Koch or Arnold steam sterilizer is usually used in industries.
• This steam sterilizer consists of a vertical metal cylinder with removable
conical lid having a small opening for the escaping steam.
• Water is added on the bottom and a perforated shelf above the water level
is present.
• Single exposure to steam for 90 minutes ensures complete sterilization but
media containing sugar and gelatin, which may get decomposed on long
heating. Hence such materials may be exposed at 1000C for 20 minutes on
three successive days. This is known as tyndallisation or intermittent or
fractional sterilization.
• First exposure to steam kills all vegetative bacteria and at second exposure
all spores germinate in a favourable medium and are killed on subsequent
occasions.
Evaluation of efficiency of sterilization/ Sterility
testing
Sterility tests can be carried out using following methods.
1. Method A: Membrane filtration.
2. Method B: Direct Inoculation.
Membrane Filtration
Membrane Filtration
• First clean the membrane filter unit and sterilize the unit and membrane
filter separately by moist heat sterilization (Autoclave).
• Transfer the unit on laminar air flow bench or aseptic area and place the
membrane filter in the unit as shown in fig.
• Pass all the solution through filter under vacuum.
• After filtration, the membrane is removed aseptically and cut into two
parts using sterile scissors.
• One half part of membrane filter is placed in 100ml of fluid thioglycollate
medium (FTM) and incubate at 30-350C for not less than 7 days.
• Other half part of membrane filter is placed in soyabean casein digest
medium (SCDM) and incubate at 20-250C for not less than 7 days.
Membrane Filtration

Observe the turbidity in the medium by comparing with the standard tube. If it has
no turbidity in fluid thioglycollate medium and soyabean casein digest medium
means it is free from microorganisms and suitable for use.
Direct Inoculation
• Cotton or prefilled syringe is transferred directly to culture media using sterile instruments such as
sterile foreceps.
• Incubate sample containing fluid thioglycollate medium (FTM) at 30-350C for not less than 7 days
and soyabean casein digest medium (SCDM) at 20-250C for not less than 7 days.

Observe the turbidity in the medium by comparing with the standard tube. If it has no turbidity in
fluid thioglycollate medium and soyabean casein digest medium means it is free from microorganisms
and suitable for use.
EVALUATION OF EFFICIENCY OF STERILIZATION METHODS
I. Evaluation of the effectiveness of the chemical agents used for sterilization:
a. The phenol coefficient

By taking phenol as a standard disinfectant, the activity of other disinfectants is measured and
compared with the activity of phenol. Any disinfectant which has phenol coefficient equals to
1 is having same activity as that of phenol, while having phenol coefficient less than 1.0, the
activity of that disinfectant is said to be lesser than that of phenol and vice-versa.
For the determination of the phenol coefficient, two organisms, namely, Staphylococcus
aureus and Salmonella typhi are used. In two culture samples, phenol and disinfectant are
mixed separately and the activity can be measured after a certain time for phenol and the
disinfectant used against the organisms. Phenol coefficient can be similar or different for
different microorganism.
It can be understood by the example of Lysol. Lysol shoes no growth at 10 minutes when used
as 1:450 dilution, while phenol shows no growth at 10 minutes when used as 1:90 dilutions.
Therefore, the phenol coefficient for Lysol is 5, that means, Lysol is 5 times more effective
than phenol.
b. Filter paper method
In this method, a small disk of filter paper is soaked into the disinfectant and
then placed on the agar plate cultured with test microorganism. A zone of
inhibition is visible after some time. Disinfectant having a wider zone of
inhibition is said to be more active for that test sample. The activity of
disinfectant can differ with the test microorganism.
c. Use dilution test
In this, the broth culture of the test microorganism is coated over the
stainless steel cylinder and is allowed to dry. These cylinders are then dipped
in the test tubes filled with broth and having different dilutions of the
disinfectant. The effectiveness of the disinfectant can be measured by the
greatest dilution at which the growth of microorganism is inhibited.
II. Sterilization controls
For the evaluation of the efficiency of the sterilization by the moist heat, the following
methods can be used:
a. Thermocouples: Thermocouples can be used for the determination of the
temperature inside the autoclave. Sterilization is considered to be proper if a
particular standard temperature is reached inside the autoclave.
b. Brown tubes: These tubes are placed inside the autoclave along with the articles.
These tubes are red in color, which turns green when the temperature inside the
autoclave reaches 121o this helps in determining the proper sterilization of the
articles.
c. Bacillus stearothermophilus spores: It requires exposure to 121oC for 12 minutes to
kill these spores. The paper strips having 106 spores are placed inside an envelope and
then in the autoclave. After the autoclaving of the articles, these strips are then
inoculated in culture media. Proper sterilization is determined if there is no growth of
these spores in the culture media.
d. Autoclave tape: It is lead carbonate based tape, which changes its color when
exposed to 121oC in the autoclave.
 Sterility testing of products
(solids, liquids, ophthalmic and
other sterile products)
according to IP, BP and USP
 STERILIZATION
 Sterilization is the complete removal of microorganisms from an object or surfaces.
 Sterilization is obtained when microorganisms are subjected to antimicrobial agents
for sufficient time and at optimum conditions.
Some physical methods associated with sterilization are explained below
PHYSICAL METHODS OF STERILIZATION
HEAT STERILIZATION
 Heat sterilization is the most effective and widely used method of sterilization, where
the bactericidal activity results through the destruction of enzymes and other essential
cell constituents.
 The effects of heat sterilization occur more rapidly in a fully hydrated state, as it requires
a lower heat input, with low temperature and less time, under high humidity conditions
where the denaturation and hydrolysis reactions are predominant, rather than in the dry
state where oxidative changes take place.
 Under circumstances where thermal degradation of a product is possible, it can usually
be minimized by adopting a higher temperature range, as the shorter exposure times
generally result in a lower partial degradation.
 This method of sterilization is applicable to thermostable products. Still, it can be
applied to both moisture-sensitive and moisture-resistant products, for which dry (160–
180°C) and moist (121–134°C) heat sterilization procedures are respectively used.
A) DRY HEAT STERILIZATION
 Dry sterilization is the process of removing microorganisms by applying moisture- free
heat which is appropriate for moisture-sensitive substances.
 The dry heat sterilization process is based on the principle of conduction; that is the
heat is absorbed by the outer surface of an item and then passed onward to the next
layer. Ultimately, the entire item reaches the proper temperature needed to achieve
sterilization.
 Dry moisture-less heat destroys microorganisms by causing denaturation of proteins
and also lyses the proteins in many organisms, causes oxidative free radical damage,
causes drying of cells, and can even burn them to ashes, as in incineration
 Dry heat sterilization is used for the sterilization of materials which are difficult to
sterilize by moist heat sterilization for several reasons.
 Substances like oil, powder, and related products cannot be sterilized by moist heat
because moisture cannot penetrate into deeper parts of oily materials, and powders are
destroyed by moisture.
 Similarly, laboratory equipment like Petridishes and pipettes are challenging to sterilize
by moist heat due to the penetration problem.
 The lethal effects of dry heat on microorganisms are primarily due to oxidative
processes which are less effective when compared to the hydrolytic damage that results
from exposure to steam in moist heat sterilization.
 Thus, in dry heat sterilization usually higher temperatures in the range 160–180°C are
employed and also require exposure times of up to 2 hours depending upon the
temperature employed.
 This principle is used in instruments like hot air oven and incineration, which generates
very hot moisture-free air.
 The primary industrial application of dry heat sterilization is in the sterilization of glass
bottles which are to be filled aseptically.
  In addition to the fact that this method achieves an adequate sterility assurance level,
this method also destroys bacterial endotoxins (which are the products of Gram-
negative bacteria also called pyrogens, which cause fever when injected into the body)
which are difficult to eliminate through other sterilization techniques.
 For the purposes of depyrogenation of glass, temperatures of approximately 250°C are
used.
 There are different types of dry heat sterilization which are explained below:

Table 1:(Temperature time relationship in hot air oven)

Working
● The most common time –temperature relationships for sterilization with hot
sterilizers are
● 170°C(340°F) for minutes,
● 160°C(320°F) for 60 minutes, and
● 150°C(300°F) for 150 minutes or longer depending up the volume

RED HEAT
 Red heat sterilization is the process of instant sterilization by holding the instruments
in a Bunsen flame till they become red hot.
 This method is based on dry heat sterilization is commonly used for sterilization of
instruments like incubation loops, wires, and points of forceps.
 This process ensures effective sterilization; however, it is only limited to substances
that can endure heating until redness in flame.

FLAMING
 Flaming is a type of dry sterilization that involves exposure of metallic objects to flame
for some time where the flame burns microbes and other dust presents in the instrument.
 In the case of flaming, the instrument is dipped in alcohol or spirit before burning it in
a gas flame.
 This process doesn’t ensure sterility and is not as effective as red heat sterilization.

INCENERATION
 Incineration is the process of sterilization along with a significant reduction in the
volume of the wastes.
 It is usually conducted during the final disposal of the hospital or other residues.
 The scraps are heated till they become ash which is then disposed of later.
 This process is conducted in a device called incinerator.

HOT AIR OVEN

 Hot air oven is a method of dry heat sterilization which allows the sterilization of objects
that cannot be sterilized by moist heat.
 It uses the principle of conduction in which the heat is first absorbed by the outer surface
and is then passed into the inner layer.
 A hot air oven consists of an insulated chamber that contains a fan, thermocouples,
temperature sensor, shelves and door locking controls.
 The commonly-used temperatures and time that hot air ovens need to sterilize materials
are 170°C for 30 minutes, 160°C for 60 minutes, and 150°C for 150 minutes.
  These ovens have applications in the sterilization of glassware, Petri plates, and even
powder samples.

Fig.1:(HOT AIR OVEN)

B) MOIST HEAT STERILIZATION

 Moist heat sterilization is one of the most effective method of sterilization where the
steam under pressure acts as a bactericidal agent.
 Moist heat sterilization usually involves the use of steam at temperatures in the range
121–134°C.
 High pressure increases the boiling point of water and thus helps achieve a higher
temperature for sterilization.
 High pressure also facilitates the rapid penetration of heat into deeper parts of material
and moisture present in the steam causes the coagulation of proteins causing an
irreversible loss of function and activity of microbes.
 The high temperature-short time cycles not only often result in lower fractional
degradation, but they also provide the advantage of achieving higher levels of sterility
assurance due to more significant inactivation factors.
 The most commonly used standard temperature-time cycles for clinical porous
specimens (e.g. surgical dressings) and bottled fluids are 134°C for 3 minutes and
121°C for 15 minutes, respectively.
 An autoclave is a device that works on the principle of moist heat sterilization through
the generation of steam under pressure.
  In this method, the microorganisms are killed by coagulating their proteins, and this
method is much more effective than dry heat sterilization where microbes are killed
through oxidation.
 In the pharmaceutical and medical sectors, it is used in the sterilization of dressings,
sheets, surgical and diagnostic equipment, containers, and aqueous injections,
ophthalmic preparations, and irrigation fluids, in addition to the processing of soiled
and contaminated items.
 Moist heat can be used in sterilization at different temperatures:

AT TEMPERATURE BELOW 100°C

 The sterilization technique employed at a temperature below 100°C involves
pasteurization.
 In this process, all non-spore forming microbes are killed in milk by subjecting the milk
to a temperature of 63°C for 30 minutes (the holder method) or 73°C for 20 seconds
(the flash method).
 In pasteurization, however, not all the pathogenic organisms are killed. The principle
of pasteurization is the logarithmic reduction in the number of viable microbes so that
they can no longer cause diseases.
 All mesophilic non-sporing bacteria can be killed by exposure to a moist heat at 60°C
for half an hour with the exception of some organisms which require different
temperature-time cycles.
 The milk is not heated above its boiling point as the milk might curdle, and its
nutritional value might be destroyed.
 Besides milk, other fluids and equipment like vaccines of non-sporing bacteria are also
pasteurized at 60°C for 1 hour in special water baths.
 Similarly, serum and body fluids with congealable proteins are also sterilized at 56°C
for 1 hour in water baths.

AT A TEMPERATURE OF 100°C
 Boiling at 100°C is a moist heat sterilization technique that doesn’t ensure complete
sterility, but is enough for the removal of pathogenic vegetative microbes and some
spores.
 In this case, the items to be sterilized are immersed in boiling distilled water for 30-40
minutes.
 Distilled water is preferred because hard water might result in the formation of a film
of calcium salts on the instruments.
 Tyndallization is a method that is used for sterilization of media with sugar and gelatin
at 100°C for 30 minutes on three successive days so as to preserve sugar which might
be decomposed at a higher temperature.
 Moist heat at 100°C is applicable for contaminated dishes, beddings, pipettes, and other
instruments that are not soiled or contaminated as well as for objects that are
temperature sensitive.

AT TEMPERATURES ABOVE 100°C

 Moist heat sterilization above 100°C involves sterilization by steam under pressure.
 Water usually boils at 100°C under normal atmospheric pressure (760 mm of Hg);
however, the boiling point of water increases if the pressure is to be increased.
 This principle is employed in an autoclave where the water boils at 121°C at the
pressure of 15 psi or 775 mm of Hg.
 As a result, the steam under pressure has a higher penetrating power. When this steam
comes in contact on the surface, it kills the microbes by giving off latent heat.
 The condensed liquid ensures the moist killing of the microbes.
 Autoclaves are used for the sterilization of contaminated instruments along with
different culture media as it ensures complete sterility.

Fig.2:(AUTOCLAVE)

Table 2:(Pressure temperature relationship in autoclave)

Pressure temperature relations in

Autoclave
Pressure in Temperature Temperature
psi in °C in °F
5 109 228
10 115 240
15 121 250
20 126 259
25 130 267
30 135 275
 Table 3:(Heat sterilization method, its mechanism, merits, demerits & applications)

Sl Method Mechanis Merits Demerits Applications

no m
1 Heat Destroys Most widely Can be Dry heat is applicable for
sterilizatio bacterial used and applied only sterilizing glasswares and
n endotoxins reliable to the metal surgical instruments
method of thermastabl and moist heat is the most
sterilization e products dependable method for
involving decontamination of
destruction of laboratory waste and the
enzymes and sterilization of laboratory
other glassware, media and
essential cell reagents.
constituents

IRRADIATION
 Irradiation is the process of exposing surfaces and objects to different kinds of radiation
for sterilization.
 Mainly electromagnetic radiation is used for sterilization.
 The major target for these radiations is considered to be microbial DNA, where damage
occurs as a result of ionization and free radical production (gamma-rays and electrons)
or excitation (UV light).

A) NON IONOZING
INFRARED RADIATION
 Infrared radiation (IR) is a method of thermal sterilization in which the radiation is
absorbed and then converted into heat energy.
 For this purpose, a tunnel containing an IR source is used. The instruments and
glassware to be sterilized are kept in a tray are then passed through the tunnel on a
conveyer belt, moving at a controlled speed.
 During this movement, the instruments will be exposed to the radiation, which will
result in a temperature of about 180°C for about 17 minutes.
 IR is applicable for mass sterilization of packaged items like syringes and catheters.

ULTRAVIOLET RADIATION
 Ultraviolet radiation includes light rays from 150-3900 Å, of which 2600 Å has the
highest bactericidal effect.
 Non-ionizing waves have a very little penetration power, so microorganisms only on
the surface are killed.
 Upon exposure, these waves are absorbed by many materials, particularly nucleic
acids.
 The waves, as a result, cause the formation of pyrimidine dimers which bring error in
DNA replication and cause the death of microbes by mutation.
 UV radiation owing to its poor penetrability of conventional packaging materials is
unsuitable for sterilization of pharmaceutical dosage forms.
  It is, however, applied in the sterilization of air, for the surface sterilization of aseptic
work areas, and the treatment of manufacturing-grade water.

B) IONIZING RADIATION
 X-ray and gamma rays are the commonly used ionizing radiation for sterilization.
 These are high energy radiation which causes ionization of various substances along
with water.
 The ionization results in the formation of a large number of toxic O2 metabolites like
hydroxyl radical, superoxide ion, and H2O2 through ionization of water.
 These metabolites are highly oxidizing agents and kill microorganisms by oxidizing
various cellular components.
 With ionizing radiation, microbial resistance decreases with the presence of moisture
or dissolved oxygen (as a result of increased free radical production) and also with
elevated temperatures.
 Radiation sterilization is generally exposed to items in the dried state which include
surgical instruments, sutures, prostheses, unit-dose ointments, plastic syringes, and dry
pharmaceutical products.

FILTRATION
 The process of filtration is unique among sterilization techniques in that it removes,
rather than destroys, microorganisms.
 Further, it is capable of preventing the passage of both viable and nonviable particles
and can thus be used for both the clarification and sterilization of liquids and gases.
 The primary mechanisms involved in filtration are sieving, adsorption, and trapping
within the matrix of the filter material.
 Filtration uses membranous filters that have tiny pores that let the liquid pass through
but prevent bigger particles such as bacteria from passing through the filter. Therefore,
the smaller the pore, the more likely the filter is to stop more things from going through
it.
 Certain types of filter (membrane filters) also have an essential role in sterility testing,
where they can be employed to trap and concentrate contaminating organisms from
solutions under test.
 These filters are then placed in a liquid nutrient medium and incubated to encourage
growth and turbidity.
 The principal application of sterilizing-grade filters is the treatment of heat-sensitive
injections and ophthalmic solutions, biological products, air, and other gases for supply
to aseptic areas.
 They may also be required in industrial applications where they become part of venting
systems on fermenters, centrifuges, autoclaves, and freeze dryers.

FILTRATION STERILIZATION OF LIQUIDS

 Membrane filters, in the form of discs, can be assembled into pressure-operated filter
holders for syringe mounting and in-line use or vacuum filtration tower devices for
filtration of liquid.
 Filtration under pressure is generally considered most suitable, as filling at high flow
rates directly into the final containers is possible without problems of foaming, solvent
evaporation, or air leaks.
 Membrane filters are often used in combination with a coarse-grade fiberglass depth
profiler to improve their dirt-handling capacity.
 FILTRATION STERILIZATION OF GASES
 Filters employed for this generally consist of pleated sheets of glass microfibres
separated and supported by corrugated sheets of Kraft paper or aluminum which are
employed in ducts, wall or ceiling panels, or laminar air flow cabinets.
 These high-efficiency particulate air (HEPA) filters can remove up to 99.997% of
particles >0.3mm in diameter and thus are acting as depth filters.
 In practice, their microorganism removal efficiency is rather better as the majority of
bacteria are found associated with dust particles.
 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 fiber filters); treatment of
exhausting air from microbiological safety cabinets (HEPA filters); and the clarification
and sterilization of medical gases (glass wool depth filters and hydrophobic membrane
filters).[6]

Table 4:(Filtration method, its mechanism, merits, demerits & applications)

Sl Method Mechanism Merits Demerits Applications

no.
1 Filtration Does not It is used for Does not In this method
destroy but both the differentiate sterilizing grade filters
remove the clarification and between are used in the
microorgani sterilization of viable and treatment of heat
sms liquids and non viable sensitive injections
gases as it is particles and opthalmic
capable of solutions, biological
preventing the products and air and
passage of both other for supply to
viable and non asceptic areas
viable particles

PHYSICAL STERILISATION

HEAT IRRDIATION FILTRATION

DRY HEAT MOIST HEAT NON IONISING RADIATION IONISING

Classification of physical sterilization

CHEMICAL STERILIZATION

 Chemical Sterilization is the process of removal of microorganisms by the use of

chemical bactericidal agents.
 Even if physical methods of sterilization are more appropriate for effective
sterilization, it is not always appropriate to use for heat-sensitive materials like
plastics, fiber optics, and biological specimens.
 Under such conditions, chemical either in liquid or gaseous state can be used for
sterilization. However, it is crucial to ensure that the materials undergoing
sterilization are compatible with the chemical being used.
 Besides, it is important to adopt safety rules in the workplace safety during the use of
chemical agents.
 The chemical method of sterilization can be categorized as liquid and gaseous
sterilization.

GASEOUS STERILIZATION
 Gaseous sterilization involves the process of exposing equipment or devices to
different gases in a closed heated or pressurized chamber.
 Gaseous sterilization is a more effective technique as gases can pass through a tiny
orifice and provide more effective results.
 Besides, gases are commonly used along with heat treatment which also facilitates the
functioning of the gases.
 However, there is an issue of release of some toxic gases during the process which
needs to be removed regularly from the system.
 The mechanism of action is different for different types of gases.
Some of the common gases used for gaseous sterilization are explained below

ETHYLENE OXIDE
 Ethylene sterilize, pasteurize, or disinfect different types of equipment and surfaces
because of its wide range of compatibility with different materials.
 EO treatment often replaces other sterilization techniques like heat, radiation, and even
chemicals in cases where the objects are sensitive to these techniques.
 This method is a widespread method used for almost 70% of all sterilizations and
around 50% for disposable medical devices.
 The mechanism of antimicrobial action of this gas is assumed to be through the
alkylation of sulphydryl, amino, hydroxyl, and carboxyl groups on proteins and imino
groups of nucleic acids.
 EO treatment is usually conducted at the temperature range of 30-60°C for several hours
which aids in the activity of the gas.
 The efficacy of the gas depends on the concentration of gas available for each article
which is greatly assisted by the good penetrating nature of the gas, which diffuses
readily into many packaging materials including rubber, plastics, fabric, and paper.
 Ethylene oxide kills all known microorganisms, such as bacteria (including spores),
viruses, and fungi (including yeasts and molds), and is compatible with almost all
materials even when repeatedly applied.
 This process, however, is not without drawbacks as the level of gas in the sterilizer goes
on decreasing due to absorption, and the treated articles need to undergo a process of
desorption to remove the toxic residual wastes
  Organisms are more resistant to ethylene oxide treatment in a dried state, as are those
protected from the gas by inclusion in crystalline or dried organic deposits.

Table 5: (Gaseous & Radiation sterilization methods, mechanism, merits,

demerits & applications)

Sl no. Method Mechanism Merits Demerits Applications

1 Gaseous Alkylation Penetrating Gases being Ethylene oxide
sterilization ability of alkylating agents gas has been used
gases and potentially widely to process
mutagenic and heat sensitive
carcinogenic devices
2 Radiation Ionization It is a Undesirable Radiation
sterilization of nucleic useful changes occur in Sterilization is
acids method for irradiated products, generally applied
the an to articles in the
industrial example is aqueous dry state;
sterilization solution where including surgical
of heat radiolysis of water instruments,
sensitive occurs. sutures,
products Prosthesis,
unit doses.

FORMALDEHYDE
 Formaldehyde is another important highly reactive gas which is used for sterilization.
 This gas is obtained by heating formalin (37%w/v) to a temperature of 70-80°C.
 It possesses broad-spectrum biocidal activity and has found application in the
sterilization of reusable surgical instruments, specific medical, diagnostic and electrical
equipment, and the surface sterilization of powders.
 Formaldehyde doesn’t have the same penetrating power of ethylene oxide but works on
the same principle of modification of protein and nucleic acid.
 As a result of the low penetrating power, its use is often limited to paper and cotton
fabrics.
 Formaldehyde can generally be detected by smell at concentrations lower than those
permitted in the atmosphere and thus can be detected during leakage or other such
accidents.

NITROGEN DIOXIDE
 Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of
common bacteria, fungi, and even spores.
 NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard
temperature.
 This property of NO2 enables the use of the gas at standard temperature and pressure.
 The biocidal action of this gas involves the degradation of DNA by the nitration of
phosphate backbone, which results in lethal effects on the exposed organism as it
absorbs NO2.
  An advantage of this gas is that no condensation of the gas occurs on the surface of the
devices because of the low level of gas used and the high vapor pressure. This avoids
the need for direct aeration after the process of sterilization.
 Nitrogen dioxide is a rapid and effective sterilant that can be used for the removal of
common bacteria, fungi, and even spores.
 NO2 has a low boiling point (20°C) which allows a high vapor pressure at standard
temperature.
 This property of NO2 enables the use of the gas at standard temperature and pressure.
 The biocidal action of this gas involves the degradation of DNA by the nitration of
phosphate backbone, which results in lethal effects on the exposed organism as it
absorbs NO2.
 An advantage of this gas is that no condensation of the gas occurs on the surface of the
devices because of the low level of gas used and the high vapor pressure. This avoids
the need for direct aeration after the process of sterilization.

OZONE
 Ozone is a highly reactive industrial gas that is commonly used to sterilize air and water
and as a disinfectant for surfaces.
 Ozone is a potent oxidizing property that is capable of destroying a wide range of
organisms including prions, without the use of hazardous chemicals as ozone is usually
generated from medical-grade oxygen.
 Similarly, the high reactivity of ozone allows the removal of waste ozone by converting
the ozone into oxygen by passing it through a simple catalyst.
 However, because ozone is an unstable and reactive gas, it has to be produced on-site,
which limits the use of ozone in different settings.
 It is also very hazardous and thus only be used at a concentration of 5ppm, which is 160
times less than that of ethylene oxide.

LIQUID STERILIZATION
 Liquid sterilization is the process of sterilization which involves the submerging of
equipment in the liquid sterilant to kill all viable microorganisms and their spores.
 Although liquid sterilization is not as effective as gaseous sterilization, it is
appropriate in conditions where a low level of contamination is present.
Different liquid chemicals used for liquid sterilization includes the following

HYDROGEN PEROXIDE
 Hydrogen peroxide is a liquid chemical sterilizing agent which is a strong oxidant and
can destroy a wide range of microorganisms.
 It is useful in the sterilization of heat or temperature-sensitive equipment like
endoscopes. In medical applications, a higher concentration (35-90%) is used.
 H2O2 has a short sterilization cycle time as these cycles are as short as 28 minutes
where ethylene oxide has cycles that as long as 10-12 hours.
 However, hydrogen peroxide has drawbacks like low material compatibility, lower
capacity of penetration, and associated health risks.
 Vaporized hydrogen peroxide (VHP) is used to sterilize largely enclosed and sealed
areas, such as entire rooms and aircraft interiors.
 GLUTERALDEHYDE
 Glutaraldehyde is an accepted liquid sterilizing agent which requires comparatively
long immersion time. For the removal of all spores, it requires as long as 22 hours of
immersion time.
 The presence of solid particles further increases the immersion time.
 The penetration power is also meager as it takes hours to penetrate a block of tissues.
 The use of glutaraldehyde is thus limited to certain surfaces with less contamination

HYPOCHLORITE SOLUTION
 Hypochlorite solution, which is also called liquid bleach, is another liquid chemical that
can be used as a disinfectant, even though sterilization is difficult to obtain with this
chemical.
 Submerging devices for a short period in liquid bleach might kill some pathogenic
organisms but to reach sterilization submersion for 20-24 hours is required.
 It is an oxidizing agent and thus acts by oxidizing organic compounds which results in
the modification of proteins in microbes which might ultimately lead to death.
 Appropriate concentrations of hypochlorite can be used for the disinfection of
workstations and even surfaces to clean blood spills and other liquids .[7]

CHEMICAL STERILIZATION

GASEOUS STERILIZATION LIQUID STERILIZATION

Classification of chemical sterilization

EVALUATION OF EFFICIENCY OF STERILIZATION METHODS

Evaluation of the efficiency of sterilization methods: The term 'sterile' in a microbiological
context, means no surviving organisms, whatsoever. Thus, there are no degrees of sterility; an
item is either sterile or it is not, and so there are no level of contamination which may be
considered negligible or insignificant and therefore acceptable. True sterility, represented by
zero survivors, can only be achieved after an infinite exposure period or radiation dose. Clearly
then, it is illogical to claim, or expect, that a sterilization procedure will guarantee sterility.
Thus, the likelihood of a product being produced free of microorganisms is best expressed in
terms of the probability of an organism surviving the treatment process, a possibility not
entertained in the absolute term 'sterile'. From this approach has arisen the concept of 'sterility
assurance' or a microbial safety index which gives a numerical value to the probability of a
single surviving organism remaining to contaminate a processed product. For pharmaceutical
products, the most frequently applied standard is that the probability, post sterilization, of a
non-sterile unit is 1 in 1 million units processed (i.e., <=10 -6). The sterilization protocol
necessary to achieve this with any given organism of known D-value (decimal reduction time)
can be established from the inactivation factor (IF) which may be defined as
IF = 10 × t/D
where 't' is the contact time (for a heat or gaseous sterilization process) or dose (for ionizing
radiation; and 'D' is D-value (time taken at a fixed temperature or the radiation dose required
to achieve a 90% reduction in viable cells. (D-value is one the functions to indicate the
efficiency of sterilization process)
IF for selected sterilization protocols and their corresponding biological
indicator organisms-
Moist heat (121℃ for 15 min) - B. stearothermophilus, D-value - 1.5 min, Log IF -
10; Dry heat (160℃ for 120 min) - B. subtilis var niger, D-value - max. 3 min,
Log IF - Min. 40;
Irradiation (25 kGy) - B. pumilus, D-value - 1.9 kGy,
Log IF - 13.2
This is the simplest method of calculating the probability of achieving sterility for any given
initial survival level.
From the above-mentioned D-values and Log IF (or IF) values, it is clear that moist heat. [8]

STERILITY INDICATORS
PHYSICAL INDICATORS
 Monitoring physical indicators involves observing the gauges or displays on the
sterilizer and recording the time, temperature, and pressure associated with each
sterilization cycle for each load.
 Some sterilizers have recording devices that print out these parameters.
 Correct readings do not guaranty sterilization, but incorrect readings can be the first
indication of a problem with the sterilization cycle and suggest the load may not be
sterile. [9]
CHEMICAL INDICATORS
 Chemical indicators use sensitive chemicals to assess critical variables (e.g., time,
temperature, or steam saturation) during a sterilization cycle.
 They are applied either to the outside or placed on the inside of each instrument unit
(e.g., packs, peel pouches, containers, etc…).
 They do not prove that sterilization has been achieved, but they can provide an early
indication of a problem and where in the sterilization process the problem might
exist.[10]
BIOLOGICAL INDICATORS
 Biological indicators (BIs), or spore tests, assess directly the killing of known highly
resistant, non pathogenic bacterial spores.
 Geobacillus stearothermophilus (G. stearothermophilus) spores test steam and
unsaturated chemical vapor sterilizers.
 Bacillus atrophaeus (B. atrophaeus) spores test dry heat sterilizers.
 Bacterial spores in the test products are more resistant and are present in greater
numbers than common microbial contaminants found on patient-care items..[11]
 EQUIPMENTS EMPLOYED IN LARGE SCALE
STERILIZATION ARE:
 Steam sterilizer
 Dry heat sterilizer
 ETO Sterilizer
 Sterilizing tunnel
 CIP System
 SIP System[12]

(A) (B)

(C) (D)

Fig. 3: (A) Steam sterilizer; (B) Dry heat

sterilizer; (C) ETO Sterilizer; and (D) CIP System