Mrs. Tole S.B.

Asst. Prof.
K.T.Patil College Of Pharmacy, Osmanabad.
Sterilization is a process by which an article, surface or
medium is freed of all microorganism either in the
vegetative or spore state.
It is an essential stage in the processing of any product
used for parenteral administration, broken skin,
mucosal surfaces or internal organs.
For to minimize the health hazards it is very necessary
to sterilize surgical dressings & equipments.
Why sterilization ??
 1. To prevent contamination in sterile products.
 2. To prevent transmission of pathogenic
microorganism which are responsible for causing
diseases in plants , animals and human beings.
 3. To prevent decomposition and spoilage of food and
food products.
 3. To prevent contamination of unwanted microbes in
pure culture & other microbiological experiments
performed for research studies.
 4. To prevent contamination in aseptic areas,in
antibiotic, enzymes, vitamin fermentation and other
industrial processs.
Methods of sterilization
 TWO METHODS-
 Physical method
1. Dry heat sterilization:
 Eg: Incineration, Direct flame, Red heat, Hot air.
2. Moist heat sterilization:
 Eg: Pasteurization, Tyndallisation, Autoclave
3. Sterilization by radiation:
 Eg: Use of Ultra-violet rays: UV light(Non-
ionising), Ionising radiations: X-rays,
 Gamma rays, beta rays
4. Filtration/mechanical method:
 Eg: Abestos filter(seitz), sintered
glass filter(morton), filter
candles(ceramic), membrane filter(millipore)
Chemical method

1. Gaseous sterilization
 Eg: Ethylene oxide gas, Formaldehyde, Beta
propiolactone
2. Sterilization by disinfectant
 Eg: Alcohols and Aldehydes, Phenols and
Halogens, Oxidizing agents and Salts
Physical Method
Physical Methods
 Involves processes by the use of physical means
Utilization of heat in the presence or in the
absence of heat, moisture, radiation or
membrane filtration methods.
Dry heat Sterilization.
 Heat is the most reliable and rapid method of
Sterilization.
 Mechanism: Protein denaturation, oxidative
damage and toxic effect of elevated levels of
Electrolytes
 Time required for sterilization is inversely
proportional to the temperature of exposure.This
can be expressed as thermal death time,which is
the minimum time required to kill a suspension of
microorganisms at a temperature and specific
conditions.
1. Sunlight and drying:
 Action primarily due to UV rays however, effects
vary due to places
Eg: Natural method for sterilization of water in
tanks, reservoir, lakes, etc
2. Heat:
 Most reliable method of sterilization and should
be the method of choice.
Eg: Inoculating wire, needles, forceps, etc
3. Flaming:
Passed over flame without allowing it to become
red hot
Eg: Culture tube, glass slides, scalpels, needles,
cover slips, etc.
4. Incineration:
Excellent method for rapid destroying materials
Eg: Pathological material, contaminated cloth,
animals carcasses
HOT AIR OVEN
 Hot air ovens are electrical devices used in sterilization.
 The oven uses dry heat to sterilize articles.
 Generally, they can be operated from 50 to 300 C (122 to 572 F) .
 There is a thermostat controlling the temperature.
 This is the most widely used method of sterilization by dry heat.
 Items: glassware, forceps, scissors, scalpels, all-glass syringes,
swabs, liquid paraffin, dusting powder, fats, grease
 (Materials should be properly arranged to allow free circulation of
 air)
 IT IS NOT SUITABLE FOR SURGICAL DRESSING, RUBBER,
PLASTIC,
 VOLATILE AND HEAT LIABLE SUBSTANCES
Hot air oven
Precautions
 Glass wares should be dry.
 Oven should not be over loaded.
 Glass materials after drying are allow to cold
down before use
 Articles are to be arranged in a manner to
allow free circular of air.
 Door of the Oven should be opened after it
cools down (2Hours)
 Temperature Time (in
Nono (C) Minutes )

 170 60

160 120

150 150

140 180

Normally the spores

as well as the
vegetative forms of all
microorganism are
killed in two hours at
a Temp of 160 C
Advantages
 Advantages:
& Disadvantages
 It is suitable method for sterilization of
substances destroyed by moisture.
 They do not require water and there is not much
pressure build up
 within the oven, unlike an autoclave, making
them safer to work with.
 Suitable and easy to be use in a laboratory
environment.
 They are much smaller than autoclaves but can
still be as effective.
 Disadvantages:
 long heating time, high temperature.
 As they use dry heat instead of moist heat, some
organisms like prions, may not be killed by them
every time
MOIST HEAT STERILIZATION
 Killing of microorganisms with hot water or steam
 Mechanism: Denaturation and coagulation of
proteins
 Divided into three forms in terms of temperature:
 1. Temperature below 100°C (Pasteurization
method)
2. Temperature at 100°C (Tyndallization method)
3. Temperature above 100°C (Autoclaving method)
Temperature below
 I : Pasteurization: below100°C
100°C
 Used for milk, ice cream, yogurt, and fruit juices
 Heat-tolerant microbes survive
 Batch method
 Temperature below 100° Pasteurization of milk
 Developed by Louis Pasteur to prevent the spoilage of
beverages.
 Used to reduce microbes responsible for spoilage of
beer, milk, wine, juices, etc.
 Milk was exposed to 65°C for 30 minutes
 Inspissation is the process used when heating
high-protein containing media; for
example to enable recovery of bacteria for testing.( by
making THICKINING /DENSE)
 High Temperature Short Time Pasteurization
(HTST): Used today. Milk is exposed to 72°C for 15
seconds
 Target: all non-sporing pathogens
 Eg: Mycobacteria, Brucellae, Salmonella,
relatively heat resistant, may survive
the holder method.
Principle of Pasteurization
II: A temperature at 100°C
 1. Boiling : Boiling for 10 – 30 minutes may kill most
of vegetative forms but spores with stand boiling.

 2. Tyndallisation: Steam at 100°C for 20 minutes on

three successive days. Used for egg , serum and
sugar containing media.

 3. Steam sterilizer : Steam at 100°C for 90 minutes.

Used for media which are decomposed at high
temperature.
III. A temperature
 Autoclave :
above 100°C
 Steam above 100°C has a better killing power than dry
heat.
 Bacteria are more susceptible to moist heat.
 TARGETS BOTH VEGETAIVE AND SPORES
Components of autoclave:
 Consists of vertical or horizontal cylinder of gunmetal
or stainless steel.
 Lid is fastened by screw clamps and rendered air tight
by an asbestos washer.
 Lid bears a discharge tap for air and steam, a pressure
gauge and a safety valve.
REQUIRED TEMPERTURE AND
TIME

Temperature(°C) Duration(min)

121 15

126 10

134 3
Vertical autoclave
Vertical Autoclave
Horizontal autoclave
Transformation in design
Procedure
 Water is added on the bottom of the autoclave and
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
 When the air bubbles stop emitting from the discharge
tap it indicates all the air from inside the autoclave has
been removed
 At this stage, the discharge tap is closed.
 Steam pressure rises inside and when it reaches the
desire set level(15p.s.i) the safety valve opens and
excess steam escapes.
From this point the holding time(15mins) is counted
 When the holding time is over, the heating is stopped
and autoclave is allowed to cool till pressure gauze
indicates that the inside pressure has reached to the
atmospheric pressure
 The discharge tap is opened slowly and air is allowed
to removed from the autoclave.
 The lid is opened and the sterilized articles are
removed.
Uses of Autoclaves
 Useful for materials which can not withstand high
temp.
 To sterilize culture media, heat stable liquids,
saline solutions, heat resistant equipments and
instruments, glassware's, ampoules, syringes, rubber
material, gowns, surgical dressings, gloves etc
 Unsuitable for anhydrous material such as
powders, oils, fats, ointments
More efficient than Dry heat
sterilization
 i) It provides greater lethal action of moist heat
ii) It is quicker in heat up the exposed articles
iii)It can penetrate easily porous material such as
cotton wool stoppers, paper and cloths wrappers
C) RADIATION
 Two types of radiation: Ionizing radiation & Non-
ionising radiation
 1. Non-ionising radiation (HOT STERILIZATION)
Infrared- Used for rapid mass sterilization of pre-
packed items such as Syringe, Cathaters
• UV
• Used for disinfecting enclosed area such as
entryways, operation theatres and labs.
 Ionising radiation (Cold sterilization)
 Gamma rays: X-rays:
 Used for sterilizing plastics, syringes, swabs,
catheters, animal feeds, cardboard, oils, greases,
fabric and metal foils.
MECHANISM
 1. Non-ionizing radiations(UV light)
Induce the production of abnormal nucleotides such as
thymine dimmers in the bacterial cell.
2.Ionizing radiation(X-rays, gammarays,cathode
rays)
 Produces microbial mutant
 Causes ionization resulting in the death of cell
D) Filtration (mechanical) method
 Helps to remove bacteria from heat labile liquids
 Items: sera and solutions of sugars or antibiotics
 Principle: as viruses pass through the ordinary
filters, filtration can be used to obtain bacteria
free filtrates of clinical samples for virus isolation
Types of filters:
 Candle filters

 Asbestos filters

 Sintered glass filters

 Membrane filters
Candle filter
 Manufactured in different grades of porosity
and widely used for purification of water for
industrial and drinking purposes.
Made up of porous procelain or kieselguhr
Inexpensive and available in different sizes
Asbestos filter(Seitz filter)
 Disposable
 Single use disc made up of asbestos(magnesium
trisilicate)
 Tend to alkalinise filtered liquids.
 The pore size of filter ranges from 0.01 to 5 microns.
 Usage is discouraged because of its carcinogenic
property
Sintered glass filter(morton filter)
 Has low absorptive properties
 Borosilicate glass is finely powdered in a ball mill
and packed
 into disc mould and heated until suitable
adhesion take
 place between the granules.
 Brittle and expensive
Sintered glass filter(morton filter)
Membrane filter(millipore/ultra
filter)
 Made of cellulose esters or other polymers
 Usually used for water purification and analysis,
sterilization and sterility testing and preparation
of solutions for parenteral use.
 The are 150μm thick and contain millions of
microscopic
 pores ranging from 0.01 to 10μm in diameter
Membrane filter(millipore/ultra
filter)
Advantages and disadvantages
 Advantages:
All microorganisms are separated by process of sieving
 Membranes have a high and uniform porosity
permitting a rapid rate of filtration
Membranes are disposable. Hence, there is no cross
contamination between filtered products
 Adsorption is very less
 Disadvantages:
 Prefilter is used before the membrane filter to avoid
clogging and breaking
 They have less chemical resistance to certain organic
solvents such as chloroform, ketone and esters