STERILIZATION

Definitions
• Sterilization: it is a process by which an article , surface or medium is freed
from all microorganisms either in vegetative or spore state.
• Disinfection: destruction of all pathogenic organisms capable of giving rise
to infection. It is applied to inanimate objects.
• Sanitization: it is the process of disinfection including cleansing action. It is
applied to inanimate objects.
• Antiseptic: any substance which would prevent sepsis either by killing m.o
or by inhibiting their growth. It can be applied to b ody tissues.
• Germicide: an agent that kills vegetative cells. Bactericide, fungicide,
virucide etc
• Microbiostasis: it is the process of preventing the growth, reproduction and
multiplication of m.o.
• Preservative: a substance that prevents the growth of m.o. These can be
added to food and pharmaceuticals to prevent microbial growth.
 STERILIZATION METHODS

Physical Method Chemical Method Mechanical Method

Dry Heat Moist Heat Gaseous

Sterilization Strerilization Radiation Sterilization Filtration

Hot Air
Oven Autoclave UV Rays Asbestos filter
Disinfectant

Flaming Seitz filters

Tyndallization Ethylene
Gamma Rays oxide
Infra red Membrane
radiation filters
Pasteurization Formaldehyde

Sun light Sintered glass

filters
Heating with β-Propiolactone
Bactericide
 I. PHYSICAL METHODS
1. DRY HEAT STERILIZATION
Mechanism: protein denaturation, oxidative damage and toxic
effect of elevated levels of electrolytes.
Principle:
• The killing of microorganisms by heat is a function of the time-
temperature combination used. If the temperature is
increased then the time required for killing all the bacteria will
be decreased.
a) Hot air oven
• A hot air oven is a double walled chamber made up of mild
steel or aluminium. The door is insulated with asbestos gasket
to minimize the heat loss.
• The inner surface is made reflecting to reduce heat loss by
radiation.
• It consists of one or more shelves.
• It is thermostatically controlled. Heaters are fitted to heat the
air inside. The heat is circulated by normal convection. To
spread the heat uniformly forced convection is arranged by
fitting fans in strategic places.
• Care should be taken so that once in operation the oven is not
opened in the middle of the cycle.
TEMP (oC) TIME (hrs)
170 1
160 1-2
150 2 1/2
140 3
b) Flaming
• This is an emergency method, the forceps-tips, the surfaces of
the scalpels and the needles may be sterilized by holding the items
directly in the flame of a Bunsen burner. This method is generally
done in microbiology.

c) Infrared radiation
• Infrared radiation (IR) is a thermal radiation, often known as
radiant energy.
• The instruments and glass wares are kept in trays are passed
through tunnel having an IR source keeping on the conveyor belt, at
a controlled speed exposing them to 1800C for 17 minutes, thereby
achieving the sterility. This is a continuous process and is used in
hospitals for regular supply of sterile syringes and other apparatus.
• Heating at or above 2000C by IR in vacuum is employed as a means
of sterilizing surgical instruments.

d) Sunlight and drying:

• germicidal activity. Natural method for sterilization of water in
tanks, rivers and lakes.
Application of dry heat sterilization:
Dry heat is used to sterilize
• Glass ware e.g. test tubes, Pasteur-pippettes, petridishes, flasks, glass
syringes etc. The glass wares should be prewashed with apyrogenic water.
• Porcelain and metal equipment such as forceps, scalpels, scissors etc.
• Dry materials in sealed container like powders.
• Fats, oils and greasy materials (like petroleum jelly) those are
impermeable to moisture.
Limitations:
It is not suitable for rubber, plastics and heat labile substances.
2. MOIST HEAT STERILIZATION
Mechanism of killing microrganisms:
Bacterial death by moist heat is due to denaturation and coagulation of
essential protein molecules (enzymes) and cell constituents.
• Principles of sterilization by steam under pressure:
• Pressure itself has no sterilizing power. Steam is used under pressure
as a means of achieving an elevated temperature.
Steam production: This may be achieved in two ways:
• Penetration of steam:
• Steam flows quickly into every article in the load (and into porous
articles). This is due to its condensation creating a low-pressure region into
which more steam flows.
• Rapid heating: The saturated steam heats the load rapidly due to the
release of its considerable amount of latent heat.
• Moist heat: The condensate produced on cooling hydrates the
microorganisms and thus helps in coagulating microbial proteins.
• No residual toxicity: The product is free from toxic contamination
It is of three forms:
a) Temperature below 100oC/Pasteurization
b) Temperature at 100oC/Tyndallisation
c) Temperature above 100oC/Autoclave
d) Heating with Bactericide

a) Temperature below 100oC: heat labile fluids

• Temp is either 630C for 30 min (holder method) or 720C for 20 sec (flash
method) followed by rapid cooling to 130C or lower – Pasteurization.
• Widely used in dairy product like milk, butter etc.
• Serum may be disinfected by heating at 560C for one hour.
• Vaccines prepared from nonsporing bacteria may be inactivated in a water
bath at 600C for one hour.
b) Temperature at 100oC/Tyndallization:
• Boiling at 1000C for 10 to 30 min kills all vegetative bacteria and some
spores. Arnold steam sterilizer is used.
• Single exposure to steam for 90 min ensures complete sterln but media
containing sugar and gelatin, gets decomposed. Such materials may be
exposed at 1000C for 20 min on three successive days –
Tyndallization/Fractional Sterilization.
• First exposure to steam kills all veg bacteria and at sec all spores and killed
on subsequent.

c) Temperature Above 100oC/Autoclave:

• Heat in form of saturated steam under pressure
a) it provides greater lethal action of moist heat
b) it is quicker in heating up the exposed articles
• It can penetrate easily porous material such as cotton, wool stoppers,
paper or cloth wrappers.
Design and operation of autoclave:
• A portable autoclave is an elaborate pressure cooker. It is a hollow cylindrical
vessel fitted with a lid that can be tightly secured in a position by nut-bolts.
The body is made up of aluminium or steel or gun metal.
• The lid is provided with a steam vent, a safety valve and a pressure gauge.
The electrical element is fitted at the bottom of the autoclave. First water is
added so that the heating element is fully immersed in water. Then the
materials to be sterilized are placed over a perforated platform.
• Heater is switched on. Initially the steam-vent (outlet) is opened. The
displaced air is first removed, then the vent is closed. The pressure will then
rise to 15 lb/sq inch (psi), temperature to 1210C and kept for 15 minutes,
then the heater is switched off. When the pressure inside and outside the
autoclave equals, the steam-vent is opened and the lid is removed to take out
the sterilized articles.
Temp (oC) Steam pressure (lb/sq. inch) Holding time (min)

115-118 10 30
121-124 15 15
126-129 20 10
135-138 30 3
 Vertical autoclave Horizantal autoclave

Applications:
• Aqueous parenteral solutions e.g. distilled water, saline solutions.
• Aqueous liquid media e.g. liquid media with or without carbohydrate and gelatin.
• Surgical dressings and fabrics.
• Plastic and rubber closures.
• Metal instruments.
• Glass apparatus and containers.
• Autoclaving is recommended in I.P for many official injections.
• It is unsuitable for anhydrous materials.
d) Heating with a Bactericide:
• This method is used for sterilizing aqueous solutions that are
thermolabile to withstand normal autoclaving temperature.
Condition:
Temperature : 98 to 1000C boiling water
Time :30 minutes
Bactericides : Bactericide that is compatible with the product,
container and closure:
e.g. for injection – Chlorocresol, Phenylmercuric nitrate, Phenylmercuric
acetate )
e.g. for eye drop – Thiomersal , Chlorhexidine acetate, PMA and PMN,
Benzalkonium chloride
Applications:
• Injection fluids in the final container
• Eye drops.
Advantages of moist heat sterilization:
• High heat content plus rapid heat transfer.
• Destroys micro-organism more efficiently than dry heat.
• It can be used for a large number of injections, ophthalmic solutions,
irrigants, dialysis fluids etc.
• It rapidly penetrates porous materials and is therefore very suitable for
sterilizing surgical dressings and materials.
• The process is adaptable for plastic containers and some other special
dosage forms.
• It is more suitable than dry heat for sharp instruments.
• Accurate control and monitoring of the process is possible.
• No toxic contaminants are left in the materials sterilized.
Disadvantages of moist heat sterilization:
• It is not suitable for anhydrous materials such as powders and oils.
• It cannot be used for thermolabile substances.
• It does not destroy pyrogens.
3. RADIATION:
Energy transmitted through space in a variety of forms is generally called
Radiation/cold sterilization because ionizing radiations produce
relatively little heat in the material being irradiated.
Heat sensitive substances in food and pharmaceuticals.
• Ionizing radiation – high energy and ionize the target molecule. Ionizing
radiation can cause excitations, ionization and where water is present free
radical formation. Free radicals are powerful oxidizing ( OH, HO2) and
reducing (H) agents, which are capable of damaging essential molecules
(enzymes and DNAs) in living cells. This results in cell death.
• Non ionizing radiations – less energy and do not disturb the atomic
configuration of target molecule.
a) Non Ionizing - UV Rays:
Source: Low current of high voltage is passed through mercury vapor in an evacuated
tube made of borosilicate glass.
Dose of sterilizing radiation: 10 to 60 microwatts / cm2 reduce the populations of
vegetative cells by 90% in a short period.
Mode of action: Only a narrow range of wavelength (220 to 280 nm) is effective in
killing micro-organisms, and wavelengths close to 253.7 nm are the most effective.
UV light is absorbed by the nucleic acid – thymine dimers-interfere in DNA
replication.
Use:
• (i) Surface sterilization and (ii) hospital operating rooms, aseptic filling rooms in
the pharm industry, food and dairy indusrty for treatment of contaminated
surfaces.
• UV rays have also been employed in sterilizing biological fluids such as blood
plasma and vaccines.

Disadvantages:
• It has very poor penetration power.
• It is not absolutely reliable because DNA may get repaired in some favorable
condition.
• Bactericidal UV -light causes eye problems and erythema (reddening) of the skin if
those parts are exposed to UV- radiation for a prolonged period of time.
Ionizing radiations (Cold sterilization):
X-rays, gamma rays and cathode rays are highly lethal to DNAand other vital cell
constituents.
a) X-RAYS:
• It have considerable energy and penetration ability that is used to produce lethal
effect to m.o.
• Expensive and difficult to utilize. Widely employed to produce microbial mutants.
b) GAMMA RAYS
Source: Radiation from the radioactive isotope of Cobalt 60Co.
Dose: 1.25 MeV, Some users take the adequate dose as 2.5 Mrad (Mega radiation unit)
Procedure: Articles are packed in boxes of standard size, which are suspended from a
monorail and sterilized by slow-passage around the gamma-ray source.
Uses:
• Articles regularly sterilized on a commercial scale include plastic syringes, catheters,
hypodermic needles and scalpel blades, adhesive dressings, single-application
capsules of eye-ointment and catgut
• Containers made of polyethylene and packaging materials using aluminum foil and
plastic films.
Disadvantages:
• The damage of the cells is mediated through radiation-induced free radicals in water.
• Expensive.
 II. MECHANICAL METHODS
FILTRATION
This method is used for sterilizing thermolabile solutions, which will otherwise be
degraded by other conventional heating methods.
Principle:
The drug solutions are passed through the sterile bacteria proof filter unit and
subsequently transferring the product aseptically into the sterile containers which
are then sealed. The process involves considerable hazards. Hence IP and BP require
that the tests for sterility be carried out on the filtered product.
Procedure:
The solutions 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 side.
Mode of action:
The filters are thought to function by one or usually a combination of the following:
• Sieving or screening,
• Entrapment,
• 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. 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. Electrostatic attraction causes
particles, opposite in charge to that of the surface of the filter pore, to be held or
adsorbed onto the surface.
a) Sintered glass filters:
• Borosilicate glass is finely powdered in a ball mill and packed n to disc
moulds heated until adhesion takes place between the granules.
• Filters are available with different porosities a no. or grade 5 or 5 on 3
must be used.
• Low adsorptive property and
• cleaned easily.
• Brittle and expensive.
b) Filter candles:
• Purification of water for industrial and drinking purpose.
• Made up of either porous porcelein or kiesulghur.
• The filter is fixed to filter assembly and placed in a mantle. The liquid to be
filtered is poured into the mantle where vacuum forces it to filter.
• Inexpensive and available in different sizes.
c) Membrane filter:
• Membrane filters are made of cellulose-derivative. They are very fine.
• Nominal pore size is 0. 22  0. 02 mm or less is required.
• The membranes are brittle when dry. They become very tough when dipped in
water.
• They are sterilized by autoclaving or by ethylene oxide gas.
• They are suitable for sterilizing aqueous and oily solutions but not for organic
solvents such as alcohol, chloroform etc.
Examples of membrane filters:
• MF-Millipore – it is a mixture of cellulose esters
• Sartorius Regular – it is made of cellulose nitrate
• Gelmen Triacetate Metricel – cellulose triacetate
d) Asbestos filters:
• Disposable, single use discs
• Pore size of filters is 0.01 to 5 microns.
• Supported on metal disc.

Appln:
• Used to sterilize water purification and analysis, sterility testing and for
preparation of parentral use.
• Also for identification and enumeration of m.o. in water and other
materials .
 III. CHEMICAL METHODS
1.by using disinfectant:
• Chemical agents most commonly used as disinfectant and antiseptics
• Eg: phenol,alcohols, halogens, dyes, aldehydes etc.
• MOA: pptn of cell protein, inactivation of enzymes and leakage of
aminoacids.
• Bacteriocidal or bacteriostatic.
• Appln: Disinfect sputum, clinical oral thermometers etc.
2. Gaseous Sterilization
Destruction of all living organisms with a chemical in gaseuos or vapour state.
This process involves exposure of materials to sterilize gasses such as
ethylene oxide, formaldehyde, glutaraldehyde, propylene oxide.
a) Ethylene oxide:
• Colourless liquid inflammable – explosive when mixed with air.
• When mixed with CO2 or fluorinated hydrocarbons makes non-
inflammmable.
Conc (mg/lit) exposure time (hrs)
44 24
88 10
442 4
884 2
MOA: Action of ethylene oxide is due to is power of alkylating the amino,
carboxyl, hydroxyl and sulphydryl groups in the enzymes and protein
molecules. it reacts with DNA and RNA.
Appln:
• It is powerful sterilizing agent for heat and moisture sensitive materials.
• Used for sterilization of medical and biological prep, catgut, plastic
equipments, antibiotics, plaster bandages, culture media, disposable
syringes, hypodermic needles, prepackaged materials hospital bedding,
food stuff, heavy euipment, books, clothing and soil etc. are sterilized by
this method.
Advantages:
• It is suitable for thermolabile substances.
• It does not damage moisture-sensitive substances and equipment
because only a low humidity is required.
• Used for prepackaged articles, because of the great penetrating power of
ethylene oxide.
• Itis highly reactive compound .
Disadvantages:
• It is slow. Long exposures and desorption periods are necessary and
therefore cannot be used in emergency.
• The running costs are high.
• Ethylene oxide is inflammable.
b) β-propiolactone:
This heterocyclic ring compound is a colourless liq at room temp with a high
B.P (155oC).
It is capable of killing all m.o and is very active against viruses.
Bactericidal – used in conc. of 2 to 5 mg/litre.
Low power of penetration – irritation – carcinogenic properties – not
recommended for use in pharm industry.
c) Formaldehyde
• Like ethylene oxide this is an alkylating agent but it is generally inferior for use as a
sterilizing agent. Because formaldehyde has poor penetrating power and is readily
inactivated by organic matter.
• HCHO in aqueous solution is known as formalin or paraformaldehyde, used to
sterilize enclosed area.
• High concentrations are difficult to maintain in the atmosphere because it tends to
deposit in the form of solid polymers on contact with cool surfaces.
• MOA: It combines with vital organic nitrogen compounds such as proteins and
nucleic acids.
• Condition:
• Input concentration : 2 g/L of HCHO in subatmospheric steam
• Temperature : 900C
• Exposure time : 3 hours
• Appln: used for disinfection and steriln of enclosed areas such as Operation
theatres, hospital rooms, aseptic area and microbiology lab.
Disadvantages:
• Low penetrability. It cannot penetrate polymeric packaging.
• Polymerize to inactive forms on the surface of low exposure temperature.
• Can fall in the active concentration
• Cannot sterilize narrow lumen.
Solution of the above problems:
• Reducing the size of the load.
• Increasing exposure temperature.