Sterilization is the process that eliminates (removes) or kills all forms of life, including

transmissible agents (such as fungi, bacteria, viruses, spore forms, etc.) present on a surface,
contained in a fluid, in medication, or in a compound such as biological culture media.

Different methods of sterilization are:

(1) Heat method of sterilization

(2) Chemical method of sterilization
(3) Irradiation method of sterilization
(4) Filtration method of sterilization

Heat Method of sterilization: there are two methods, they are:

(a) Moist heat method of sterilization

(b) Dry heat method of sterilization

(a) Moist heat method of sterilization: Simply boiling a sample for 30 minutes or more will kill
virtually all vegetative cells present, but will not kill spores. Therefore, boiling is not sufficient to
achieve sterilization.

A more commonly used method is to use an autoclave or pressure cooker.

Construction of an autoclave:

Autoclave is an instrument used for moist heat sterilization in which materials are sterilized by
saturated steam under pressure.

The autoclave is basically closed vessel made up of stainless steel or aluminum. The thickness of
the metal should withstand the operating pressures. It consists of electric coil for heating, a base
on which the container with the load to be sterilized is placed and a lid. Lid is closed with the
clamps. A gasket made up of asbestos is used along the lining of the lid, so that steam does not
leak out. Release valve, safety valve and pressure recording dial meter is fixed on the lid. The
temperature at which the autoclave is operated is 121° C at 15 lb/ inch2 pressure for about 15-30
mins.

Different types of autoclave: (i) Pressure monitored autoclave (ii) Temperature control
autoclave (iii) Front loaded autoclave (iv) Top loaded autoclave (v) portable autoclaves etc.
Operation of an autoclave:

The samples are placed into the autoclave, and is closed and heated so that steam forces air out
of the vents. Pressure is then applied so that the interior temperature reaches 121°C, and this
temperature is maintained for between 15 and 30 minutes. This elevated temperature and
pressure is sufficient to sterilize samples of any commonly encountered microbes or spores.
Additional sterilizing time is usually required for liquids and instruments packed in layers of
cloth, as they may take longer to reach the required temperature. Following sterilization, liquids
in a pressurized autoclave must be cooled slowly to avoid boiling over when the pressure is
released.
Mechanism of action: Moist heat causes destruction of micro-organisms by denaturation of
macromolecules, primarily proteins. Destruction of cells by lysis may also play a role.

For effective sterilization, steam needs to penetrate the autoclave load uniformly, so an autoclave
must not be overcrowded, and the lids of bottles and containers must be left ajar. During the
initial heating of the chamber, residual air must be removed.

Application of moist heat sterilization:

 Surgical dressings and instruments.

 Water, normal saline, aqueous fluids and thermostable solutions.
 Microbiological media
 Rubber gloves and rubber material, glass apparatus, containers and medical waste etc.
Note: this method is not used for oils, anhydrous liquids, ointments and dry powders etc.

Other Moist heat method:

(i) Tyndallization: Tyndallization essentially consists of heating the substance to boiling point
for 15 minutes, three days in succession. After each heating, the resting period will allow spores
that have survived to germinate into bacterial cells; these cells will be killed by the next day's
heating. During the resting periods the substance being sterilized is kept in a moist environment
at a warm room temperature, conducive to germination of the spores. When the environment is
favorable for bacteria, it is conducive to the germination of cells from spores, and spores do not
form from cells in this environment.
The Tyndallization process is usually effective in practice. But it is not considered totally
reliable—some spores may survive and later germinate and multiply. It is not often used today,
but is used for sterilizing some things that cannot withstand pressurized heating, such as plant
seeds.
(ii) Pasteurization is a process that kills bacteria in liquid food.
It was invented by French scientist Louis Pasteur during the nineteenth century. Pasteur
discovered that heating beer and wine was enough to kill most of the bacteria that caused
spoilage, preventing these beverages from turning sour. The process achieves this by eliminating
pathogenic microbes and lowering microbial numbers to prolong the quality of the beverage.
Today, pasteurization is used widely in the dairy and food industries for microbial control and
preservation of food.
The two main types of pasteurization used today are:

 HTST (high-temperature, short-time) milk is forced between metal plates or through pipes
heated on the outside by hot water, and the milk is heated to 72 °C for 15 seconds. Milk
simply labeled "pasteurized" is usually treated with the HTST method.
 A less conventional, but US FDA-legal, alternative (typically for home pasteurization) is to
heat milk at 63 °C for 30 minutes.

Unlike sterilization, pasteurization is not intended to kill all microorganisms in the food. Instead,
it aims to reduce the number of viable pathogens so they are unlikely to cause disease (assuming
the pasteurized product is stored as indicated and is consumed before its expiration date).
(iii) Sterilization of vaccines: Vaccines are antigen containing preparations which when
introduces into the host confers immunity to the particular disease. There are different types of
vaccines and one of them is killed bacterial vaccines. Here the bacterial cells are killed by mild
heat treatment, because higher temperature may destroy the antigenicity. Examples are 15 min at
55oC for plague and 1 h at 56oC for cholera and typhoid. But these temperatures do not kill other
bacteria, particularly spores and hence, strict aseptic conditions are maintained for preparation of
vaccines.
(iv) Heating with a bactericidal agent: bactericides are more effective at high than low
temperatures. Based on this fact, a method was developed to achieve sterilization by heating with
a bactericidal agent. Heating at 80oC or boiling water with brilliant green (1 in 3000),
formaldehyde solution B.P. (1 in 500), mercuric chloride (1 in 10000), chlorocresol (0.2 %),
phenyl mercuric chloride (0.002 %). But care must be taken so that the preparation is free from
toxicity, compatible and stable.

(b) Dry heat method of sterilization: Dry heat, as the name indicates, utilizes hot air that is
either free from water vapour, or has very little of it, and where this moisture plays a minimal or
no role in the process of sterilization

Process:

The Dry-Heat sterilization process is accomplished by conduction; that is where heat is absorbed
by the exterior surface of an item and then passed inward to the next layer. Eventually, the entire
item reaches the proper temperature needed to achieve sterilization. The proper time and
temperature for Dry-Heat sterilization is 160 °C for 2 hours or 170 °C for 1 hour.

There are two types of Hot-Air convection (Convection refers to the circulation of heated air
within the chamber of the oven) sterilizers:

 Gravity convection
 Mechanical convection

Gravity convection process:

As air is heated, it expands and possesses less density (weight per unit volume) than cooler air.
Therefore, the heated air rises and displaces the cooler air (the cooler air descends). The method
of Dry-heat gravity convection produces inconsistent temperatures within the chamber and has a
very slow turn over.
Mechanical convection process:
A mechanical convection oven contains a blower that actively forces heated air throughout all
areas of the chamber. The flow created by the blower ensures uniform temperatures and the
equal transfer of heat throughout the load. For this reason, the mechanical convection oven is the
more efficient of the two processes.

Instruments used for dry heat sterilization:

Instruments used for dry heat sterilization include hot air oven, incineration or
burning, flaming, Radiation, Microwave, bunsen burner, and glass bead sterilizer.

Effect on microorganisms:

Dry heat coagulates the proteins in any organism, causes oxidative free radical damage, causes
drying of cells, and can even burn them to ashes, as in incineration.

Hot air oven:

Construction of Hot-air oven:

Hot air oven is an apparatus used for dry heat sterilization in which substances are sterilized by a
hot air at 150°-160° C for one hour. Here heat is transferred from source to articles by
conduction, convection and radiation. The microorganisms are killed due to coagulation of
proteins (by oxidation).
Hot air oven is closed chamber, made up of heat resistant metal. The walls are double layered,
thick, well insulated in the middle. Inner surface is a reflecting surface, no heat is given out.
Door is also double walled, with insulation. Insulting gaskets are placed between door and
chamber to avoid heat losses. Electric heating coils are placed at the bottom, shelves are
provided through out the chamber. A vent is provided at the top for placing thermometer to
record the temperature within the oven. Temperature setting knobs are provided outside to set the
required temperatures.

Operation of Hot-air oven: all the materials to be sterilized are uniformly loaded on the shelves
of hot-air oven. The material should not touch the walls of hot-air oven and there is sufficient
space between the loads. The material is placed in such a way that there more surface area
exposed for proper heat transfer. If the load is taken in packets, then the size of the pack must be
small and uniform. Care must be taken that the material of packing should not reflect the heat.
And a single type of material must be sterilized at a given time.

One the hot-air oven is loaded, and then it is heated to the required temperature. That
temperature is maintained for the specified time depending on the type of material to be
sterilized. Then it is switched off and sufficiently cooled before opening it.

Uses: used for sterilization

 Of substances which come in contact with mixture ointment base pharmaceutical

lubricants.
 Sterilization of glassware such as flask, pipettes, scissors, spatula, knife and blade etc.
 Of injectables like syringes and needles made off glass.
 Of porcelain materials such as motor and pestle bacteria proof, filter candles.
 Of thermostable powders and oils.

Other dry-heat methods include flaming, incineration, and using dry heat.

Flaming is done to loops and straight-wires in microbiology labs. Leaving the loop in the flame
of a flame until it glows red ensures that any infectious agent gets inactivated. This is commonly
used for small metal or glass objects, but not for large objects.

Incineration will also burn any organism to ash. It is used to sanitize medical and other
biohazardous waste before it is discarded with non-hazardous waste.
Chemical method of sterilization:

Chemicals are also used for sterilization. Heat method is not always appropriate, because it will
damage heat-sensitive materials such as biological materials, fiber optics, electronics, and many
plastics.

Low temperature gas sterilizers function by exposing the articles to be sterilized to high
concentrations (typically 5 - 10% v/v) of very reactive gases like alkylating agents such as
ethylene oxide, and oxidizing agents such as hydrogen peroxide and ozone. Liquid sterilants and
high disinfectants typically include oxidizing agents such as hydrogen peroxide and peracetic
acid and aldehydes such as glutaraldehyde and more recently o-phthalaldehyde.

While the use of gas and liquid chemical sterilants/high level disinfectants avoids the problem of
heat damage, users must ensure that article to be sterilized is chemically compatible with the
sterilant being used.

Ethylene Oxide: EtO is a liquid at temperatures below 10.8OC. vapours of EtO are highly
inflammable. So EtO is mixed with inert gases like CO2 and Freon. EtO is an efficient sterilent
and has good penetration power. The variety of materials on which EtO can be used are: spices,
biological preparations, soil, plastics, certain medical preparations and contaminated medical
equipment.

The mode of action of EtO is believed to be alkylation reactions with organic compounds such as
enzymes and other proteins. Alkylation consists in the replacement of an active hydrogen atom
in an organic compound e.g. the hydrogen atom in a free carboxyl, amino or sulfhydryl group,
with an alkyl group.

EtO gas is commonly used to sterilize objects sensitive to temperatures greater than 60°C and /
or radiation such as plastics, optics and electrics. Ethylene oxide treatment is generally carried
out between 30°C and 60°C with relative humidity above 30% and a gas concentration between
200 and 800 mg/l, and typically lasts for at least three hours. Ethylene oxide penetrates well,
moving through paper, cloth, and some plastic films and is highly effective. EtO can kill all
known viruses, bacteria and fungi, including bacterial spores and is compatible with most
materials (e.g. of medical devices), even when repeatedly applied. However, it is highly
flammable, toxic and carcinogenic.

A typical process consists of a preconditioning phase, the actual sterilization run and a period of
post-sterilization aeration to remove toxic residues, such as ethylene oxide residues and by-
products such ethylene glycol and ethylene chlorohydrine.

The two most important ethylene oxide sterilization methods are: (1) the gas chamber method
and (2) the micro-dose method.

Ethylene oxide is still widely used by medical device manufacturers for larger scale sterilization
(e.g. by the pallet), but while still used, EtO is becoming less popular in hospitals. Since EtO is
explosive from its lower explosive limit of 3% all the way to 100%.
In hospitals, most EtO sterilizers use single use cartridges because of the convenience and ease
of use compared to the former plumbed gas cylinders of EtO blends. Another 100% method is
the so-called micro-dose sterilization method, using a specially designed bag to eliminate the
need to flood a larger chamber with EtO. This method is also known as gas diffusion
sterilization, or bag sterilization. This method minimizes the use of gas.

Another reason for the decrease in use of EtO are the well known health effects. In addition to
being a primary irritant, EtO is now known as human carcinogen.

Beta-propioolactone: this is a liquid at room temperature with high boiling point (155OC). It is
not flammable like EtO but is a vesicant and lachrymator and must be handles with care. It is less
penetrating and is more active against microorganisms. It is sporicidal, fungicidal, virucidal. Just
2% of liquid is sufficient to achive the required effect. It has carcinogenic effect.

Ozone: Ozone is used in industrial settings to sterilize water and air, as well as a disinfectant for
surfaces. It has the benefit of being able to oxidize most organic matter. On the other hand, it is a
toxic and unstable gas that must be produced on-site, so it is not practical to use in many settings.

Ozone is a very efficient sterilant because of its strong oxidizing properties capable of destroying
a wide range of pathogens, including prions without the need for handling hazardous chemicals
since the ozone is generated within the sterilizer from medical grade oxygen.

The disadvantage of using ozone is that the gas is very reactive and very hazardous. It is
immediately dangerous to life and health.

Bleach: Chlorine bleach is another accepted liquid sterilizing agent. Household bleach consists
of 5.25% sodium hypochlorite. Bleach will kill many organisms immediately, but for full
sterilization it should be allowed to react for 20 minutes. Bleach will kill many, but not all
spores. It is also highly corrosive. Bleach decomposes over time when exposed to air, so fresh
solutions should be made daily.

Glutaraldehyde and Formaldehyde: Glutaraldehyde and formaldehyde solutions are accepted

liquid sterilizing agents, provided that the immersion time is sufficiently long. To kill all spores
in a clear liquid can take up to 22 hours with glutaraldehyde and even longer with formaldehyde.
The presence of solid particles may lengthen the required period or render the treatment
ineffective.

Glutaraldehyde and formaldehyde are volatile, and toxic by both skin contact and inhalation.
Glutaraldehyde has a short shelf life (<2 weeks), and is expensive. Formaldehyde is less
expensive and has a much longer shelf life. Formaldehyde is also used as a gaseous sterilizing
agent; in this case, it is prepared on-site by depolymerization of solid paraformaldehyde. Many
vaccines, such as the original salk polio vaccine, are sterilized with formaldehyde.
Phthalaldehyde: Ortho phthalaldehyde (OPA) is a chemical sterilizing agent that received Food
and Drug Administration (FDA) clearance in late 1999. Typically used in a 0.55% solution, OPA
shows better myco-bactericidal activity than glutaraldehyde. It also is effective against
glutaraldehyde-resistant spores. OPA has superior stability, is less volatile, and does not irritate
skin or eyes, and it acts more quickly than glutaraldehyde. On the other hand, it is more
expensive, and will stain proteins gray in color. Some side effects from equipment sterilized
using this reagent have been reported.

Hydrogen Peroxide: Hydrogen peroxide is another chemical sterilizing agent. It is relatively

non-toxic when diluted to low concentrations, such as the familiar 3% retail solutions although
hydrogen peroxide is a dangerous oxidizer at high concentrations (> 10% w/w). Hydrogen
peroxide is strong oxidant and these oxidizing properties allow it to destroy a wide range of
pathogens and it is used to sterilize heat or temperature sensitive articles such as rigid
endoscopes. In medical sterilization hydrogen peroxide is used at higher concentrations, ranging
from around 35% up to 90%. The biggest advantage of hydrogen peroxide as a sterilant is the
short cycle time. Whereas the cycle time for ethylene oxide (discussed above) may be 10 to 15
hours, the use of very high concentrations of hydrogen peroxide allows much shorter cycle times.
Some hydrogen peroxide modern sterilizers, have a cycle time as short as 28 minutes.

Hydrogen peroxide sterilizers have their drawbacks. Since hydrogen peroxide is a strong oxidant,
there are material compatibility issues and users should consult the manufacturer of the article to
be sterilized to ensure that it is compatible with this method of sterilization. The hydrogen
peroxide would be completely absorbed by the paper product. The penetrating ability of
hydrogen peroxide is not as good as ethylene oxide and so there are limitations on the length and
diameter of lumens that can be effectively sterilized and guidance is available from the sterilizer
manufacturers.

Hydrogen peroxide can also be mixed with formic acid or per-acidic acid for sterilization of
endoscopes.

Dry sterilization process

Dry sterilization process (DSP) uses hydrogen peroxide at a concentration of 30-35% under low
pressure conditions. This process achieves bacterial reduction of 10 −6...10−8. The complete
process cycle time is just 6 seconds, and the surface temperature is increased only 10-15 °C (18
to 27 °F). Originally designed for the sterilization of plastic bottles in the beverage industry,
because of the high germ reduction and the slight temperature increase the dry sterilization
process is also useful for medical and pharmaceutical applications.

Peracetic acid

Peracetic acid (0.2%) is used to sterilize instruments.

Radiation sterilization:

Methods of sterilization exist using radiation such as electron beams, X rays, gamma rays, or
subatomic particles.

 Gamma rays are very penetrating and are commonly used for sterilization of disposable
medical equipment, such as syringes, needles, cannulas and IV sets, packaged foodsetc.
Gamma radiation requires bulky shielding for the safety of the operators; they also
require storage of a radioisotope (usually cobalt-60), which continuously emits gamma
rays (it cannot be turned off, and therefore always presents a hazard in the area of the
facility). Mechanism by which gamma rays destroy microorganisms include that the
radiant-energy particle makes a direct hit on some essential substances such as DNA
within the bacterial cell, causing ionization which results in the death of the cell. Above
2.5 mega radiation energy is used for sterilization purpose.
 Electron beam (cathode rays) processing is also commonly used for medical device
sterilization. Electron beams use an on-off technology and provide a much higher dosing
rate than gamma or x-rays. Due to the higher dose rate, less exposure time is needed and
thereby any potential degradation to polymers is reduced. A limitation is that electron
beams are less penetrating than either gamma or x-rays.
 X rays, High-energy X-rays are a form of ionizing energy allowing irradiating large
packages and pallet loads of medical devices. Their penetration is sufficient to treat
multiple pallet loads of low-density packages with very good dose uniformity ratios. X-
ray sterilization is an electricity based process not requiring chemical nor radio-active
material. High energy and high power X-rays are generated by an X ray machine that can
be turned off for servicing and when not in use. X rays kill the microorganisms mainly by
targeting phosphodiester bonds of the nucleic acids. Other bio-molecules are also the
targets.
 Ultraviolet irradiation (UV, from a germicidal lamp) is useful only for sterilization of
surfaces and some transparent objects. Many objects that are transparent to visible light
absorb UV. UV irradiation is routinely used to sterilize the interiors of biological safety
cabinets between uses, but is ineffective in shaded areas, including areas under dirt
(which may become polymerized after prolonged irradiation, so that it is very difficult to
remove). It also damages some plastics, such as polystyrene foam if exposed for
prolonged periods of time. UV rays are non-ionizing radiations. They are absorbed by
many cellular materials but most significantly by nucleic acids (268 nanometers). This
leads to the formation of thymidine dimmers in adjacently placed bases. Dna replication
can be inhibited and mutations can result.
 Subatomic particles may be more or less penetrating, and may be generated by a
radioisotope or a device, depending upon the type of particle.
Filtration Sterilization
Fluids that would be damaged by heat, irradiation or chemical sterilization, such as drug
products, can be sterilized by microfiltration using membrane filters. This method is commonly
used for heat labile pharmaceuticals and protein solutions in medicinal drug processing. A
microfilter with pore size 0.2 µm will usually effectively remove microorganisms. In the
processing of biologics, viruses must be removed or inactivated, requiring the use of
nanofilters with a smaller pore size (20 -50 nm) are used. Smaller pore sizes lower the flow rate,
so in order to achieve higher total throughput or to avoid premature blockage, pre-filters might
be used to protect small pore membrane filters.
Membrane filters used in production processes are commonly made from materials such as
mixed cellulose ester or polyethersulfone (PES). The filtration equipment and the filters
themselves may be purchased as pre-sterilized disposable units in sealed packaging, or must be
sterilized by the user, generally by autoclaving at a temperature that does not damage the fragile
filter membranes. Typically, terminal pharmaceutical sterile filtration is performed inside of an
aseptic room to prevent contamination.
Materials used for Filtration:

(a) Sintered Ceramics: Finely ground porcelain or diatomaceous earth material

(b) Fibrous pads: Made up of asbestos and wood cellulose.

(c) Sintered glass: Made from borosilicate

(d) Micro porous plastics: Prepared from cellulosic ester, particularly acetate and nitrate.

Efficiency of the filters must be checked:

(1) To show that the filtration medium retains organisms satisfactorily:

a) Bacteriological Method: A known CFU of Serratia marcesans is filtered across

the filtration medium. Filtrate is collected and incubated with a suitable nutrient
medium at 25oC. Appearance of red color pigment indicates the presence of
organism.

b) Bubble pressure: air is passed across the filtration medium kept in CCl4
environment. The bubbles coming are analyzed for their size to know the porosity
of filtration medium.

(2) To show that the flow rate of aqueous fluids is reasonably fast.

Features of satisfactory medium:

(i) Adequate filtration rate without blockage

(ii) Yield nothing and remove nothing except microorganisms

(iii) Drug properties must not change

(iv) Easy assembled, easily cleanable. Better if disposable unit is used,

(v) No leakage during filtration

Uses:

(i) Membrane filters are used for sterilization of heat sensitive biological fluids.

(ii) Fiber glass filters (HEPA) used for air disinfection to maintain aseptic conditions.

(iii) Various filters are used for filtration of water.

Note: fluid must be relatively free from suspended particles and it is little bit expensive. The process must be carried
out strict aseptic conditions.