Specific

Objectives
Ø Explain the importance of sterilization.

Ø Describe the various techniques employed in sterilization.

Ø Outline the validation and in-process monitoring of sterilization

procedures.
 Sterilization and Its Importance
Sterilization
§ Sterilization is a process by which we make an object (glassware,
surgical instruments, operation theatre etc.) free from all types of
microbial life, including vegetative cells, spores and viruses.

§ The principal reasons for controlling microorganisms are:

Ø To prevent transmission of disease and infection.

Ø To prevent contamination by undesirable microorganisms.

Ø To prevent deterioration and spoilage of materials by microorganisms.

 Sterilization Methods
1. PHYSICAL METHODS :
A. Heat
B. Radiation
C. Filtration
A. Heat Methods
ü This is most widely used and reliable method of sterilization.

ü It involves in destruction of enzymes and other essential cell

constituents.

ü Applicable only on THERMO STABLE PRODUCTS.

 I-Dry Heat Sterilization
ü Dry heat destroys bacterial endotoxins which are difficult to eliminate by
other means.

ü Protein denaturation.

ü Oxidative damage.

ü Toxic effect of elevated electrolyte (in absence of water).

ü It utilizes temperatures between 160 and 180 ° C.

ü Exposure time up to 2 hours, depending upon the temperature employed.

ü Used for sterilizing glass-wares, metal surgical instruments, non-aqueous

thermo-stable liquids and thermostable powders.
 q Incineration
ü It involves the combustion of organic substances contained in waste
materials.

ü This method also burns any organism to ash.

ü It is used to sterilize medical and other biohazardous waste before it is

discarded with non-hazardous waste.

ü Bacteria incinerators are mini furnaces that incinerate and kill off any
microorganisms that may be on an inoculating loop or wire.
 q Flaming (Red Heat)
ü Item to sterilized are held almost vertically in a Bunsen flame until red
hot along their whole length, almost up to the tip of their metal holder.

ü E.g.: Inoculating wire loops, Points of forceps.

ü Only exposed to the flame for a few seconds & not heating to red hot.
ü E.g.: Scalpel blades, mouth of culture tubes.
 q Hot Air Oven
ü Hot air ovens are electrical devices which are used dry heat to sterilize.

ü Generally, they can be operated from 50 to 300 °C, using a thermostat

to control the temperature.

ü Glassware and metal surgical instruments, non-aqueous thermostable

liquids and thermostable powders, glass bottles.
Temperature Time

140 °C 3 hrs.

160 °C 1 hr.

180 °C 30 minutes
 II. Moist Heat Sterilization

ü Moist heat acts by denaturation and coagulation of protein, breakage

of DNA strands, and loss of functional integrity of cell membrane.
 q Sterilization below 100 °C: Pasteurization
ü Pasteurization is a process in which certain packaged and non-
packaged foods (such as milk and fruit juice) are treated with mild heat,
usually less than 100 °C to eliminate pathogens and extend shelf life.

ü Mycobacterium bovis, Salmonella spp., Escherichia coli and Brucella spp.

may be present in milk.

ü It does not sterilize the milk as many living organisms including spores
are not destroyed.
v Heating is done at 72°C for 15 seconds.
v Heating is done between 63-66°C for 30 minutes.
 Sterilization at 100 °C: Boiling
ü Boiling is done in a water bath at 100°C for 30 minutes.

ü Syringes, rubber goods and surgical instruments may be sterilized by

this method.

ü All bacteria and certain spores are killed. It leads to disinfection.

Sterilization above 100 °C: Autoclaving

ü In this method sterilization is done by steam under pressure.

ü Steaming at temperature higher than 100 °C is used in autoclaving.

ü Autoclave is an instrument in which pressurized steam is used to
destroy microorganisms.
ü Used for decontamination of laboratory waste and the sterilization of
laboratory glassware, culture media, and reagents.
ü Most recommended conditions employed in autoclave are :
Ø Temperature : 121oC
Ø Pressure : 15 psi
Ø Time : 15-20 minutes
 B. Radiation Sterilization
ü Electromagnetic radiations are used for sterilization. E.g.:- Gamma rays,
UV rays, Accelerated electrons.

ü The major target : Microbial DNA.

ü Useful method for the industrial sterilization of heat sensitive products.

E.g. :- Surgical instruments, sutures, prostheses, unit dose ointments,
plastic syringes and dry pharmaceutical products etc.

ü UV light : Sterilization of air, for surface sterilization of aseptic work

areas, for treatment of manufacturing grade water.

ü Not suitable for sterilization of pharmaceutical dosage forms.

 ü C. Filtration Sterilization
ü Filtration process does not destroy microbes but removes.
ü It is used for both the clarification and sterilization of liquids and gases.
ü HEPA filters remove at least 99.97% of airborne particles 0.3 micrometers
(µm) in diameter pore size.
ü Millipore Membrane Filters have 0.45/0.22 µm pore size diameter.
ü They are made of biologically inert mixtures of cellulose acetate and
cellulose nitrate which are most widely used membranes in analytical and
research applications.
ü Heat sensitive injections, ophthalmic solutions, biological
solutions/products, air and other gases for supply to aseptic areas.
ü High Efficiency Particulate Air (HEPA) filters are used for air filtration
(Laminar air flow chamber).
ü Millipore Membrane Filters are used for liquid filtration. (Millipore
Filter Assembly).
Membrane Filter Assembly
 2. Chemical Sterilization Method
A. Liquid
ü Disinfectants are those chemicals that destroy pathogenic bacteria from

inanimate surfaces.

ü Some chemicals when used at appropriate concentration for

appropriate duration can be used for sterilization and are called

sterilant liquids.

ü Those chemicals that can be safely applied over skin and mucus

membranes to kill the pathogens are called antiseptics.

ü Denaturation of cellular proteins (E.g.: Alcohol, Phenol).

ü Oxidation of essential sulfhydryl groups of enzymes (E.g.: H2O2,

Halogens).

ü Alkylation of amino, carboxyl and hydroxyl group (E.g.: Formaldehyde).

ü Damage to nucleic acids (E.g. : Formaldehyde).

 B. Gaseous Method
§ Formaldehyde and Ethylene oxide are most common gases possess
biocidal activity (bactericidal & sporicidal, viricidal) and employed to
sterilize the operation theatre as these are cheaper for sterilization of
huge areas like operation theatres.

§ 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.

§ E.g. : Electric components, plastics, and cardboard, Operation theatre

room, Intensive Care Unit room etc.
 Fumigation
ü When Formaldehyde mixed with water and exposed to elevated
temperature, gaseous formaldehyde is generated.
1. First the area to be fumigated is usually covered to create a sealed
environment.
2. The fumigant is released into the space to be fumigated.
3. The space is held for a set period while the fumigant gas percolates
through the space and acts on and kills any infestation in the product.
4. The space is ventilated so that the poisonous gases are allowed to
escape from the space, and render it safe for humans to enter.
Fumigation boiler
 Validation and In-process Monitoring of Sterilization Procedures

ü The validation of sterilization and quality of sterile pharma products must

be checked during each stage of production of drug.

ü E.g.:- Raw material checking, in-process monitoring and finished product

checking must be done.

ü Validation of sterilization procedures can be carried out by 3 ways :

1. Physical indicator
2. Chemical indicator
3. Biological indicator
 A. Physical indicator
ü Physical parameters of sterilizing instruments are observed and
recorded continuously to ensure the sterilization processes.

ü E.g. :- Temperature, pressure etc.

B. Chemical indicator

ü When a chemical substance or any material carrying a chemical

indicator is used to validate the sterilization processes is called
chemical indicator.

ü E.g. :- Various chemical indicator strips.

 C. Biological indicator
§ Biological indicators are the bacterial spores used to validate the
sterilization processes.

§ This is a very effective method to validate the sterilization process.

§ E.g. : Bacillus stearothermophilus , Bacillus pumilus spore ampoules etc.

Organism (BI) Sterilization Process

Bacillus subtilis Dry heat sterilization (Hot air oven)

Bacillus stearothermophilus 1. Moist heat sterilization (Autoclave)

2. Gaseous sterilization
(Ethylene oxide/Fumigation)

Bacillus pumilus Ionizing radiation

Biological Indicator

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