This document discusses various sterilization methods including physical (heat, radiation, filtration), chemical (gaseous), and their mechanisms and applications. Heat sterilization is the most widely used method and can be dry heat or moist heat. Radiation uses gamma rays or electrons to damage DNA. Filtration removes microbes physically. Gaseous methods like ethylene oxide act as alkylating agents. Selection depends on material properties and desired sterility level. In-process controls monitor manufacturing to ensure quality. Membrane filtration and direct inoculation are used in sterility testing.

1. Sterilization
Prof. Devender Sharma
Assistant Professor
Goenka College of Pharmacy, Sikar

2. 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.

3. Sterilization
 A process that completely eliminates or kills all
microorganisms

4. METHODS OF STERILIZATION
 he various methods of sterilization are:
 1. Physical Method
 a. Thermal (Heat) methods
 b. Radiation method
 c. Filtration method
 2. Chemical Method
 a. Gaseous method

5. PHYSICAL METHODS:
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
THERMO STABLE PRODUCTS, but it can be used for
MOISTURE-SENSITIVE MATERIALS.
 i) Dry Heat (160-1800˚C) Sterilization for thermo
stable products
 ii) moist heat (121-1340 ˚C) sterilization is used for
moisture- resistant materials.

6.  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.

7.  THERMAL (HEAT) METHODS
 Thermal methods includes:
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

8.  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.

9.  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.
 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.

10.  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
on type of metiral used.

11. Autoclave
 Steam must come into direct contact with the surface
 Air must be completely removed
 Downward displacement
 Pre-vaccuum

14.  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 is
microbial DNA.
 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.

15.  Radiation sterilization is generally applied to articles
in the dry state; including surgical instruments,
sutures, prostheses, unit dose ointments, plastic
syringes and dry pharmaceutical products.
 UV light, with its much lower energy, and poor
penetrability finds uses in the sterilization of air, for
surface sterilization of aseptic work areas, for
treatment of manufacturing grade water, but is not
suitable for sterilization of pharmaceutical dosage
forms.

16. Ethylene oxide
 Extremely penetrative
 Non-corrosive
 Toxic, irritant, and explosive when mixed with air at
conc. >3%
 Odorless

18.  Filtration Sterilization
 Filtration process does not destroy but removes the
microorganisms. It is used for both the clarification
and sterilization of liquids and gases as it is capable of
preventing the passage of both viable and non viable
particles.
 The major mechanisms of filtration are sieving,
adsorption and trapping within the matrix of the filter
material.
 Ex:HEPA FILTERS

19.  Sterilizing grade filters are used in the treatment of
heat sensitive injections and ophthalmic solutions,
biological products and air and other gases for supply
to aseptic areas.
 They are also used in industry as part of the venting
systems on fermentors, centrifuges, autoclaves and
freeze driers.
 Membrane filters are used for sterility testing

20.  CHEMICAL STERILIZATION METHOD GASEOUS
METHOD
 The chemically reactive gases such as formaldehyde,
(methanol, H.CHO) 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.

21.  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

22.  MERITS, DEMERITS AND APPLICATIONS OF
DIFFERENT METHODS OF STERILIZATION

23.  METHOD MECHANISM MERITS DEMERITS
APPLICATIONS
1 Heat sterilization
Destroys bacterial endotoxins Most widely used and
reliable method of sterilization, involving destruction
of enzymes and other essential cell constituents Can
be applied only to the thermo stable products
2 Dry heat is applicable for sterilizing glass wares and
metal surgical instruments and moist heat is the most
dependable method for decontamination of laboratory
waste and the sterilization of laboratory glassware,
media, and reagents.

24. 2 Gaseous sterilization
 Radiation sterilization
 Alkylation Ionization of nucleic acids Penetrating
ability of gases. It is a useful method for the industrial
sterilization of heat sensitive products Gases being
alkylating agents are potentially mutagenic and
carcinogenic.
 Undesirable changes occur in irradiated products,an
example is aqueous solution where radiolysis of water
occurs. Ethylene oxide gas has been used widely to
process heat-sensitive devices. Radiation sterilization
is generally applied to articles in the dry state;
including surgical instruments, sutures, prostheses,
unit dose ointments, plastics

25. 1 Filtration sterilization
 Does not destroy but removes the microorganisms It is
used for both the clarification and sterilization of
liquids and gases as it is capable of preventing the
passage of both viable and non viable particles Does
not differentiate between viable and non viable
particles This method is Sterilizing grade filters are
used in the treatment of heat sensitive injections and
ophthalmic solutions, biological products and air and
other gases for supply to aseptic areas

26.  Pharmaceutical Importance of Sterilization
 • Moist heat sterilization is the most efficient biocidal
agent. In the pharmaceutical industry it is used for:
Surgical dressings, Sheets, Surgical and diagnostic
equipment, Containers, Closures, Aqueous injections,
Ophthalmic preparations and Irrigation fluids etc.
 • Dry heat sterilization can only be used for thermo
stable, moisture sensitive or moisture impermeable
pharmaceutical and medicinal. These include
products like; Dry powdered drugs, Suspensions of
drug in non aqueous solvents, Oils, fats waxes, soft
hard paraffin silicone, Oily injections, implants,
ophthalmic ointments and ointment bases etc.

27.  Gaseous sterilization is used for sterilizing
thermolabile substances like; hormones, proteins,
various heat sensitive drugs etc.
 • U.V light is perhaps the most lethal component in
ordinary sunlight used in sanitation of garments or
utensils.
 • Gamma-rays from Cobalt 60 are used to sterilize
antibiotic, hormones, sutures, plastics and catheters
etc.

28.  Filtration sterilizations are used in the treatment of
Heat sensitive injections and ophthalmic solutions,
biological products, air and other gases for supply to
aseptic areas.
 They are also used in industry as part of the venting
systems on fermentors, centrifuges, autoclaves and
freeze driers. Membrane filters are used for sterility
testing.

29.  In-process controls (IPC) are checks that are carried
out before the manufacturing process is completed.
 The function of in-process controls is monitoring and
– if necessary – adaptation of the manufacturing
process in order to comply with the specifications.
 This may include control of equipment and
environment, too.

30.  The In-Process Quality Control system lays emphasis
on the responsibility of manufacturers processors in
ensuring consistency in quality during all stages of
production by adopting quality control drills and
exercising control on raw materials and bought-out
components, manufacturing process, packing and
final testing.

31.  MEMBRANE FILTRATION.
 After transferring the contents of the container or
containers to be tested to the membrane add an inoculum
of a small number of viable micro-organisms (not more
than 100 CFU) to the final portion of sterile diluent used to
rinse the filter.
 After filtration, aseptically remove the membrane(s) from
the holder, transfer the whole membrane or cut it
aseptically into 2 equal parts. Transfer one half to each of
two suitable media.
 Direct Inoculation. After transferring the contents of the
container or containers to be tested to the culture medium
add an inoculum of a small number of viable micro-
organisms (not more than 100 CFU) to the medium

32.  Quantity in each container Minimum quantity to be
used Less than 1 ml Total contents of a container 40 ml
or more but less than 100 ml 20 ml 1 ml or more but
less than 40 ml Half the contents of a container 100 ml
or more 10 per cent of the contents of a container but
not less than 20 ml

33.  There are two types of filters used in filtration
sterilization:
 (a) Depth filters:
 (b) Membrane filters: These are porous membrane
about 0.1 mm thick, made of cellulose acetate,
cellulose nitrate, polycarbonate, and polyvinylidene
fluoride, or some other synthetic material.

34. Cleaning
 Removing all foreign material from objects by using
water and detergents or soaps and washing or
scrubbing the object
 Must be done before any disinfection or sterilization
process

35. Disinfection
 A process that eliminates many or all microorganisms
except spores
 Done with liquid chemicals or by pasteurization
 Proper contact time and dilution of the disinfectant
must be followed

36. Definitions
 High-level disinfection
 All microorganisms except high numbers of bacterial
spores
 Intermediate disinfection
 M. tuberculosis, vegetative bacteria, most viruses, and
most fungi
 Not necessarily kill bacterial spores