This document discusses sterilization methods. It defines sterilization as killing all microorganisms including bacterial spores. Various physical, chemical, and mechanical sterilization methods are described, including dry heat, moist heat using an autoclave, radiation, ethylene oxide gas, and heating with bactericides. Key differences between sterilization and disinfection are provided. Aseptic technique and sterility testing are also summarized.

1. Sterilization
Mr. G.A.Shete

2.  Sterillization
 The process of killing or removing bacteria & all other forms of living micro-organisms &
their spores from preparation.
 Methods of Sterilization
 Physical Method Chemical Method Mechanical Methods
 Dry Heat Sterilization Sterilization by heating with bactericide Ceramic Filters
 Moist Heat Sterilization Gaseous Sterilization Seitz Glass Filters
 Radiation Sterilization Sintered Glass Filters
Sintered Metal Filters
Membrane Filters

3. Difference between Sterilization & Disinfection
Sr.
No
Sterilization Disinfection
1 It is a process by which all viable
forms of micro-oganisms are
removed or destroyed
It is a process that removes infection
potential by destroying micro-
organisms.
2 The bacterial spores are also
destroyed
The bacterial spores are not destroyed
3 Sterilization is done by any of the
physical, chemical & mechanical
methods
Disinfection is done by using any one
of the disinfectants.
4 Ex : Ethylene dioxide Ex : Phenol cresol

4.  Physical Methods
 Dry Heat Sterilization
 Hot Air Oven
Fig: Hot Air Oven

5.  Principle:
All the microorganism including spores are destroyed. Principle of killing is by
dehydration and oxidation of essential metabolites. In hot air oven heating is done
at 160 C for 2 hours.
 Construction:
It consists of double walled chamber made of steel. Insulation is given of
asbestos or other material for preventing heat loss. The door is also double
walled having insulation. Two perforated shells provided to keep the material.
An electric fan is provided for uniform circulation of hot air. A heater is fitted at
the bottom for heating. A thermometer for maintaining the temperature.
 Working:
Wrap the material with paper. Keep the Wrapped material in perforated shelves.
Material should not be kept at floor of the oven. Close the door. Switch on the
oven and set the temperature and time as required. After time is over. Switch off
the oven. Allow to cool. Take out the material.

6.  Precautions while placing the material in hot air oven
It should be filled to its capacity only should not be overloaded.
Glass apparatus and equipment should be wrapped individually.
Articles should be placed in such a way that they should not interfere with air
flow.
Articles should not be placed at the floor of the oven.
Once in operation oven should not be open.
Proper biological indicators should be used.
Thermolabile substance should not be sterilized in hot air oven.

7.  Moist Heat Sterilization
It is more effective than dry heat sterilization.
Following temperature & time are normally employed for sterilization
Sr No Temperature Time (minutes)
1 115-118°C 30
2 121-124°C 15
3 126-129°C 10
4 134-138°C 5

8.  Moist Heat Sterilization (Autoclave)
 Principle:
 The steam has more penetration power than dry heat and thermal capacity of steam is
more than thermal capacity of dry heat. The method is useful for killing of bacterial
spores. The moist steam penetrate the spores and capsules of bacteria, rupture it and
escaping protoplasm it coagulated.
 Construction:
 It consists of a strong metallic chamber usually made of stainless steel. It has cover
fitted with steam vent, pressure gauze, and a safety valve. Rubber gasket is fitted in
inner wall of lid in order to make it air tight. The cover is closed with wing nut and
bolts. An electric heater is fitted at the bottom to heat the liquid. A perforated basket is
provided to keep the material in the autoclave.
 Working:
 A sufficient quantity of water is poured into the chamber after removing the
perforated basket. The level of water adjusted in such a way that it should not touch
the bottom of perforated basket.

9.  The material is placed in the basket and it placed in the autoclave. Close the lid with
wing nuts and bolts. Switch on the heater. Vent is opened and safety valve is set to
required pressure. When steam comes out for 5 min, then close the vent, the steam
pressure stats rising and it should be maintained to required level. After the stated time,
switch off the autoclave. Allow to cool to about 40C . Open the vent and allow the
complete steam to pass from autoclave. Lid is opened and sterilized material is taken out
 Advantages:
It destroy microorganism more efficiently than dry heat.
It is used for sterilization of s large number of official injection.
Rubber, plastic can be sterilized.
A large quantity of material can be sterilized in one batch.
 Disadvantages:
It not suitable for powder or oils.
It is not suitable for sterilization of plastic which melt at 115C.

10. Fig: Autoclave

11.  Others Methods of Moist Heat Sterilization
 Tyndallization, also called fractional sterilization and discontinuous heating, is a
form of sterilization. This method is relatively simple but somewhat time-consuming.
 Process: This is a fractional sterilization method. This method is used for sterilization
of medicaments unstable at 1150C but able to withstand low temperature heating. This
method consist of heating the material at 800C for 1 hour on three successive days
presuming that on the first day all vegetative bacterial cells will be destroyed and the
spores may germinate in the days to follow and will be killed subsequently.
 Pasteurization It is a partial sterilization method used to make milk safe and to
improve its keeping properties.
 Different methods are as follows:
 Holder method: Here the milk is heated at 62.80C for 30 minutes in a steam jacketed
stainless steel tank.
 Flash method: The milk is heated to 71.60C for 15 sec. and then cooled quickly.

12.  Sterilization using UV rays
 Principle: Direct sunlight can destroy the microorganism on account of its ultra-
violet rays of longer wave length.UV light of shorter wavelength kills or inactivates
microorganisms by destroying nucleic acids and disrupting their DNA, leaving them
unable to perform vital cellular functions. UV light of 265 nm wave length is lethal to
microorganism.
 Method: UV rays for sterilization are produced by passing a low current at high
voltage through mercury vapour in an evacuated glass tube.
 Application:
Sterilization of air.
Sterilization of aseptic area.
Sterilization of thermolabile material.
Sterilization of surface of working table.

13.  Gaseous Sterilisation
 Ethylene oxide:
It is colourless gas at room temperature. It can be liquefied easily and boil at 10.80C.
It is highly inflammable so it us used in mixture form:
Ethylene oxide 1 part + carbon dioxide 9 part.
Ethylene oxide 11% w/v part + Trichlofluromethane 79% w/w +
dichlorodiflurometane 10%w/w
Ethylene oxide 12%w/w + dichlorodifluromethane 88%w/w.
Sterilization is done in a chamber which can be heated to the desired degree of
temperature. The material is to be sterilized is packed in chamber and treated with
Ethylene oxide gas.
Sterilization in absence if air:
It is carried out in a evacuated sterilizer at sub atmospheric pressure with Ethylene
oxide 90% + carbon dioxide 10%.

14.  Advantages:
High penetration.
Can maintained high conc.
Very reactive.
Non Irritant to respiratory tract.
Used for heat sensitive material.
Used for sterilization of moist –sensitive material.
 Disadvantages:
Method very slow.
Cost is high.
Apparatus very expensive.
It is highly inflammable.
Certain toxic substance produced such as ethylene chlorohydrins.

15.  Disadvantages of formaldehyde gas when used for sterilization.
Weak penetration power.
Difficult to maintain high conc.
Require high humidity for effectiveness.
Readily inactivated.
Irritant to respiratory tract.
Difficult to remove adsorbed gas.

16.  Sterilisation by heating with Bactericide
 Significance of sterilization using bactericidal solution
The lethal effect of bactericide increases with the rise of temperature.
This method is used for sterilizing aqueous preparation, which is unstable at the
higher temperature, hence moist heat sterilization is not applicable method for
sterilization.
It is official in British Pharmacopoeia and Indian Pharmacopoeia.
 Method
 In this process the medicament is dissolved or suspended in a suitable solution of
following bactericidal, as given in table, then preparation is sealed in final container
and heated at 98°-100°C for 30 minutes in boiling water.
IP 1985 permitted the use of following bactericides-

17. Name of Bactericides Concentration of
Bactericide % w/v
For Injection:
1) Chlorocresol
0.2
2) Phenyl mercuric acetate
or Phenyl mercuric nitrate
0.002
For Eye drops:
1) Chlorohexidine acetate
0.01
2) Benzalkonium chloride 0.01
3) Thiomersal 0.01
4) Phenyl mercuric acetate
or Phenyl mercuric nitrate
0.002

18.  Stages involved in sterilization of surgical dressing.
Pack or wrap the unsterilized surgical dressing into a suitable device/perforated
container or any packaging material i.e parchment paper.
Load this container into sterilizer. Loading ang packaging should be done properly
to ensure the uniform steam penetration and movement.
Close the sterilizer and expose the surgical dressing at 121°C for 30-45 min.
Switch off the sterilizer and condense the steam in it, allow to cool and unload the
sterilizer.
Containers are labelled with date of sterilization to prevent overload storage.

19.  Aseptic technique:
 The method which is used to prevent the access of microorganism during the
preparation of parenteral product and their testing are called “Aseptic Technique”.
 Sources of contamination:
1) Atmosphere, which is contaminated with dust, droplet and droplet nuclei becomes
the breeding ground of microorganism.
2) The hands are a major means of transmitting infection.
3) Coughing, sneezing and spitting can cause contamination considerable distance.
4) The clothes which absorb dust particles are also a source of contamination. A
handkerchief is the richest source of contamination.
5) The hair.
6) Unsterile equipment.
7) Working surface.

20.  Test for Sterility:
 Principle: These tests are based on the principle that if bacteria or fungi are placed in
medium provided favourable conditions like nutritive material, moisture temperature,
the organism will grow and their presence can be indicated by the turbidity in clear
solution. These test should be carried out in strictly aseptic condition.
 Method of testing : Test of sterility may be carried out by
1) Direct inoculation method
2) Membrane filtration method
 1) Direct inoculation method: The substance to be tested is aseptically drawn from
the container by a suitable device and transferred to the final culture medium in the test
tube. The inoculated medium (test tubes) are incubated at 20-25°C for fungi and 30-
37°C for bacteria for the period of seven days. Observe the growth of micro-organism
in the medium.

21.  2) Membrane filtration method : This method is preferred in the following cases-
An oil or oily preparations, ointment, A non-bacteriostatic solid, soluble powder or a
liquid that possesses bacteriostatic and fungistatic properties, liquid products where
volume in a container is 100 ml or more. Carry out filtration of sample under test
through membrane filter having pore size of 0.45 μ and diameter of about 47 mm.
 After the filtration, the membrane is removed aseptically from the metallic holder and
divided into two halves. The first half is transferred into 100 ml of culture media
meant for fungi and incubated at 20 to 25°C for not less than 7 days. The other half is
transferred into 100 ml of fluid thioglycolate medium meant for bacteria and
incubated at 30 to 35°C for not less than 7 days. Observe the growth of the media.
 Results :If no growth of micro-organism is found in any of the tubes, the sample is
declared to have pass the test and same test is repeated for two times.

22. Thank You