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STERILIZATION METHODS
BY- GOPAL M KUMBHANI
M.SC BIOTECHNOLOGY1/18/2019 1

 Contents:-
 what is sterilization?
 History of sterilization
 Methods of sterilization
 Physical method
 Chemical method
 Filtration (mechanical method) method
 Refrences
1/18/2019 2

WHAT IS STERILIZATION?
Sterilization can be defined as a process by which all viable
forms of micro-organisms(such as fungi, bacteria, viruses) are
removed or destroyed.
Micro organisms are present at everywhere. Since they cause
contamination, infection, and decay, it becomes necessary to
remove or destroy them from materials or from areas.
Sterilization procedures used is should be simple but effective
and of relatively short duration.
1/18/2019 3

HISTORY OF STERILIZATION
LOUIS PASTEUR of France was among the first to use
sterilization techniques, he developed the steam sterilization,
hot air oven and the autoclave. Pasteurization was a method
developed by him to rid of bacilli from milk.
JOSEPH LISTER, applied Pasteur’s work and introduced
antiseptic technique in surgery(1867). He is the father of
antiseptic surgery.
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METHODS OF STERILIZATION
(1)Physical
(2)Chemical
(3)Filtration (Mechanical)
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• Physical methods
1) Sunlight
2) Drying
3) Heat:- i) Moist heat
ii) Dry heat
4) Radiation
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1) Sun light - 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 tanks and lakes.
2) Drying - Moisture is essential for growth of bacteria.
Drying in air has dangerous effect on many bacteria.
However, spores are unaffected.
Therefore, it is not satisfactory method for sterilization.
1/18/2019 7

3) Heat sterilization- it has also two type
A)Dry heat sterilization- In dry heat sterilization, dry heat is used for
sterilizing different materials. Heated air or fire is used in this process.
As compared to the moist heat sterilization, the temperature is higher.
The temp. is maintained for almost an hour to kill the most difficult of the
resistant spores.
Dry heat sterilization also have 4 type:- 1) Hot air oven
2) Red hot sterilization
3) Flaming
4) incineration 1/18/2019 8

1) Hot air oven:- Hot air ovens are electrical devices which use dry
heat to sterilize. They were originally developed by pasteur.
 Generally, they can be operated from
50 to 300 °C, using a thermostat to control
the temperature. ... An air circulating fan
helps in uniform distribution of the heat.
2) Red hot sterilization:- Sterilization by holding them in
Bunsen flame till they become red hot. It use for
bacteriological loops, straight wires, tips of
forceps & spatulas
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3) Flaming:- This is a method of passing article
over a flame, but not heating it to redness.
Use- scalpels, mouth of test tubes,
flask, glass slide & coverslips
4) Incineration:- Incineration is a waste treatment process that
involves of organic substances contained in waste materials.
 This method also burns any organism to ash.
It is used to sterilize medical and other
biohazardous waste before it is discarded
with non-hazardous waste
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B) Moist heat sterilization :- Moist heat method is used for heat
sensitive materials and materials through which steam is permeable.
culture media is also sterilized through moist heat sterilization.
It has also 3 types:- 1) Below 100°C 2) Above 100°C 2) At 100°C
1) Below 100°C:- It has also 2 types
i) Pasteurization- Its purpose is to reduce the bacterial
population of a liquid such as a milk & destroy
Organism that ma cause spoilage.
Spores are not affected by this method.
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ii) inspissation:- Heating at 80-85°C for half an
hour daily on three consecutive days.
Serum or egg media are sterilized.
2) At 100°c:- It has also 2 types.
i) Boiling:-Boiling is a very simple method of water disinfection.
Heating water to a high temperature, 100°C, kills most of the
pathogenic organisms, particularly viruses and bacteria causing
waterborne diseases. In order for boiling to
be most effective, the water must boil for at
least 20 minutes. 1/18/2019 12

ii) Tyndallization:- Exposure at 100°C for 20-45 minutes
for 3 successive days. Used for sterilizing
sugars, gelatin, & serum containing media.
3) Above 100°C:- Autoclave
 Autoclaving is the most reliable method for sterilization.
Autoclave use pressurized steam to destroy microorganism, and are
the most dependable system available for the decontamination of
laboratory waste & the sterilization of glass wares, media, & reagents.
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For efficient heat transfer, steam must flush the air out of the
autoclave chamber.
Generally the conditions employed are temperature up to 121-134°
for 15-20 minutes under 15 lbs pressure.
The condition based on the type of the
material used for sterilization.
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4) Radiation sterilization:- It has 2 types.
A) Non-ionizing(Hot sterilization):-
i) Infrared rays- used for rapid mass sterilization of prepacked items
such as syringe, catheters( a thin tube that is put into the body in
order to remove liquids)
ii) U.v. rays- used for disinfecting enclosed area such as entry ways,
operation theatres & labs.
1/18/2019 15

B) Ionizing(Cold sterilization):-
i) Gamma rays & X-rays:- Used for sterilizing plastics, syringes,
swabs, animal feeds, oils, greases, fabric & metal foils
 Advantages of radiation sterilization:- clean & dry process,
ensure full exposure of object from all direction.
Disadvantages:- posses threat to human,
lengthy process, requires very qualified person
1/18/2019 16

Chemical methods
1) Gaseous sterilization:- i) Ethylene oxide
ii) Formaldehyde gas
2) Liquid sterilization:- i) Alcohol
ii) Phenol
1/18/2019 17

1) Gaseous sterilization
i. Ethylene oxide:- EtO sterilization is mainly use to sterilize medical
& pharmaceutical products that can not support conventional high
temperature steam sterilization- such as devices that incorporate
electronic components, plastic packaging or plastic container.
 This method uses automatic device filled with ethylene oxide gas
at temperature below 100°C to sterilize complex & delicate material.
 EtO destroys microorganism by chemically reacting with nucleic
acid.
1/18/2019 18

 Advantages :- Fully automatic, high efficiency, 100% result
Disadvantages:- Complex and time consuming process, carcinogenic
safety concern
ii. Formaldehyde gas:- Another low temperature method for sterilizing
heat sensitive items is formaldehyde sterilization. Formaldehyde is an
organic chemical compound which is a by-product of the metabolism of
many organisms and is commonly found in fresh air, rainwater, foods,
industrial products and fabrics.
It is considered even more dangerous than EtO and is therefore less
commonly used for sterilization. 1/18/2019 19

Formaldehyde sterilization is used where sterilization by steam or
high temperature is not possible
Formaldehyde is soluble in water and its inactivation power is greatly
improved by the presence of humidity. It is most commonly used as a
disinfectant, but sometimes formaldehyde is used as a sterilizing
agent. The process is known as low temperature steam and
formaldehyde (LTSF)
In countries such as united kingdom, germany, sweden, denmark and
norway sterilization by LSTF is accepted, but not common. On the
other hand in several countries formaldehyde as a sterilizing agent is
discouraged. LTSF has not been FDA cleared for use in healthcare
facilities in the USA.
1/18/2019 20

Advantages:- Very reactive molecule, Faster cycle time compared
to EtO, cost per cycle is lower than EtO, after sterilization most
loads are available for immediate use
Disadvantages:- The vapour is extremely irritating to the eyes,
weak penetrating power compared to EtO, operates on a higher
temperature than EtO, formaldehyde residue can remain on the
sterilized goods if the rinsing phase is not 100% efficient. This
can be harmful for the patients
1/18/2019 21

2) Liquid sterilization
i) Alcohol:- Alcohols are effective disinfectants for many reasons.
They evaporate quickly, without leaving a residue. They are capable
of dissolving lipids, which makes them effective against lipid-wrapped
viral cells such as HIV and hepatitis A. They are inexpensive and
relatively easy to handle, although their vapors are flammable.
Ethanol and isopropyl alcohol are both members of the alcohol
family and have similar disinfectant properties. Ethanol is the type of
alcohol present in alcoholic beverages. Isopropyl alcohol is also
known as isopropanol, 2-propanol or rubbing alcohol. When used as
disinfectants, both are typically at a concentration of 70 percent in
water.
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ii) Phenols:- Phenol is one of the oldest antiseptic agents.
 phenols acts by damaging cell membrane thus releasing cell contents
& causing lysis.
 phenols is commonly found in mouth washes, scrub soaps, & surface
disinfectants.
 phenols are used for decontamination of the hospital environment,
including laboratory surfaces, & non critical medical items.
Examples:-
1/18/2019 23

Filtration (Mechanical) methods
Filtration sterilization used for heat sensitive materials to sterilize.
Filtration process does not destroy but removes the microorganisms.
Filtration allows for the exclusion of organisms based upon size.
 Procedure:- The solution to be sterilized is passed through the filter
and collected in the sterile receiver by the application of positive
pressure to the nonsterile compartment or negative pressure to the
sterile slide.
1/18/2019 24

Mode of action
The filters are thought to function by one or usually a combination
of the following:
1. Sieving or screening
2. Entrapment
3. Electrostatic attraction
 When a particle is larger than the pore size of the filter the particle
is retained on the filter- this known as sieving or screening
1/18/2019 25

Entrapment occurs when a particle smaller than the size of the pore
enters into the pore channel and lodges onto the curves of the
channel while passing through it.
In Electrostatic attraction Particles are attracted & absorbed at the
surface of the filter bed which is oppositely charged.
There are 4 types of filters:-
1. Membrane filters
2. Sintered or Fritted glass filters
3. Seitz filters
4. Ceramic filters 1/18/2019 26

1) Membrane filters:- They are made of cellulose-derivative (acetate
or nitrate). They are very fine. They are fixed in some suitable
holders.
 Nominal pore size is 0.22 ± 0.02mm or less is required.
 The membranes are brittle when dry. In this condition they can be
stored for years together. They become very tough when dipped in
water.
 They are suitable for sterilizing aqueous and oily solutions but not
for organic solvents such as alcohol, chloroform etc. 1/18/2019 27

 They are sterilized by autoclaving or by ethylene oxide gas. They
can not be sterilized by dry heat as they decompose above 130°C.
 Membrane filters are generally blocked by dirt particles and
organisms. Pre-filtration (through glass-fibre paper prefilter) reduces
the risks of membrane filter.
Examples:- i) MF-Milipore - it is a mixture of cellulose esters
ii) Sartorius regular – it is made of cellulose nitrate
1/18/2019 28

2) Sintered (or Fritted) glass filters:-
 Borosilicate glass is finely powdered in a ball-mill and the particles
of required size are separated.
 This is packed into disc mounted and heated till the particles get
fused. The disc thus made have pore size of 2 mm and are used
for filtration.
 They are cleaned with the help of sulfuric acid.
1/18/2019 30

3) Seitz filters:-
 It consists of two parts.
 Lower part filled with a perforated plate over which compressed
asbestos pad is placed.
 Upper part has a valve through which pressure can be applied.
 Both parts joined together by winged nuts.
 The main advantage of this filter is that no risk of
contamination & easy to use.
 For viscous solution they are more suitable.
1/18/2019 31

4) Ceramic filters:-
 Ceramic water filters are an inexpensive and effective type of water
filter, that rely on the small pore size of ceramic material to filter dirt,
debris, and bacteria out.
 However, filters are typically not effective against viruses since they
are small enough to pass through to the "clean" side of the filter.
 Ceramic filtration does not remove chemical contaminants.
 However, some manufacturers (especially of ceramic candle filters)
incorporate a high-performance activated carbon core inside the
ceramic filter that reduces organic and metallic contaminants.1/18/2019 32

 The two most common types of ceramic water filter are pot-type
and candle-type filters.
1/18/2019 33

 REFRENCES:-
 Principle and practice of disinfection and sterilization by
A P Fraise
 Wikipedia
 www.study.com
1/18/2019 34

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