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Sterilization- Physical and chemical methods

August 3, 2023 by Somak Banerjee

Edited By: Sagar Aryal

Microorganisms play an important role in causing infection and contamination.

Therefore, Sterilization is an important technique in microbiology which helps to
remove or destroy microorganisms from materials or surfaces.

Table of Contents
Common Definitions
Sterilization
Disinfection
Antiseptics
Asepsis
Methods of Sterilization
Physical Methods
Sunlight
Heat
Dry Heat: Procedures
Red Heat
Flaming
Incineration
Hot Air Oven
Sterilization control
Moist Heat Sterilization: Procedure
Temperature below 1000C
Pasteurization
Inspissation
Vaccine bath
Low-temperature steam formaldehyde sterilization (LTSF)
At a temperature of 1000C
 Boiling
Tyndallisation
Steam sterilizer
Temperature above 1000C (under pressure)
Autoclave
Sterilization control
Ozone
Filtration
Radiation
Ionizing radiation
Non-ionizing radiation
Chemical Method
Alcohols
Aldehydes
Formaldehyde
Glutaraldehyde
Orthophathalaldehyde
Phenols
Cresols
Chlorhexidine
Chloroxylenol
Hexachlorophene
Halogens
Oxidizing agents
Hydrogen peroxide
Peracetic acid
Salts
Dyes
Vapor phase Disinfectants
Ethylene Oxide (ETO)
Betapropilolactone (BPO)
Reference

Interesting Science Videos

Common Definitions

Sterilization
It is the process by which an article, surface or medium is made free of all
microorganisms either in vegetative or spore form.

Disinfection

It refers to the destruction of all pathogens or organisms which can cause infection
but not necessarily spores. All organisms may not be killed but the number is
reduced to a level that no longer harmful to health.

Antiseptics

These are chemical disinfectants that are safe to apply on living tissues and used to
prevent infection by arresting the growth of the microorganism.

Asepsis

This refers to the technique which helps to prevent the occurrence of infection into
an uninfected tissue.

Image Source: https://doi.org/10.1016/B978-0-12-814421-3.00014-2

Methods of Sterilization

Physical Methods

Sunlight
Sunlight has an active germicidal effect due to the presence of ultraviolet rays. It is a
natural procedure of sterilization which reduces the number of microorganisms in
water tanks, lakes, etc.

Heat

Heat is a mostly used method of sterilization. Moreover, it is a highly effective and

most reliable process. There are two major methods of using heat in sterilization
which are dry heat and moist heat. The principle behind both of these methods is
similar. Dry heat induces the denaturation of protein, oxidative damage and toxic
effect due to the high level of electrolytes. Moreover, the dry heat can also damage
the DNA of the microorganism. As a result, the microorganism got killed. Moist Heat
kills the microorganisms by denaturation and coagulation of proteins. There are
several factors that can influence the heat killing procedure. Such as

Temperature and Duration: The duration and temperature are inversely connected
to each other. Therefore, in the case of the long duration of heat provided for
sterilization, the temperature will be reduced while in case of high temperature the
duration will be reduced.
Characteristic of the Microorganism: Microorganisms can be present in both
vegetative and spore form. Spore forms are generally heat resistant. Therefore, the
sterilization process will vary on the basis of the characteristic of the
microorganism.
Type of Material: Organic substances often provide protection to the vegetative
and spore form of microorganisms which reduces the lethal property of heat.
Apart from that, the materials containing the substances are also needed to be
heat stable for proper sterilization.

Dry Heat: Procedures

Red Heat

Inoculation loops, wires, forceps tips, needles are needed to be sterilized to inhibit
microbial contamination. These instruments are held in the flame of a Bunsen burner
until they become red hot.

Flaming
Glass slides, scalpels, and mouths of culture tubes or conical flasks are passed
through Bunsen flame without allowing them to become red hot.

Incineration

This procedure is used to reduce the infective material into ashes by burning. The
incinerator is used for the process. Soiled dressings, animal carcasses, bedding, and
pathological materials are dealt with this method.

Hot Air Oven

It is a widely used method of sterilization by dry heat. The heat inside the oven is
maintained by electricity and a fan fitted inside it provides the adequate distribution
of hot air inside the chamber. A thermostat is also connected which maintains the
temperature inside the chamber. 1600C for two hours is required for sterilization.
There are also some alternative temperatures and holding time which include 1700C
for 1 hour and 1800C for 30 minutes.

Uses: Sterilization of

Glasswares like glass syringes, Petri dishes, flasks, pipettes, and test tubes.
Surgical instruments like scalpels, scissors, forceps, etc.
Chemicals such as liquid, paraffin, fats, sulphonamides powders etc.

Sterilization control

The spores of Bacillus subtilis subsp. Niger (NCTC 10075 or ATCC 9372) are kept
inside the oven. These spores should be destroyed if the sterilization is proper.
Thermocouples may also be used.
Browne’s tube with a green spot is available. After proper sterilization, a green
color is produced (after two years at 1600C).

Moist Heat Sterilization: Procedure

Temperature below 1000C

Pasteurization

There are two different types of pasteurization methods that are used for sterilization
of milk, Holder method (63oC for 30 minutes) and flash method (720C for 20 seconds
followed by cooling quickly to 130C). This method is effective against all non-sporing
pathogens such as mycobacteria, Salmonella, etc. except Coxiella burnetii which
survives the holder method due to heat resistant characteristics.

Inspissation

Media like Lowenstein- Jensen’s and Loeffler’s serum are required to sterile at 80-
850C for 30 minutes daily on three consecutive days. This process is known as
inspissation and the instrument used is called inspissator.

Vaccine bath

It is used for sterilization of bacterial vaccines at 600C for one hour. Serum or other
body fluids can be sterilized by heating in a water bath at 560C for several successive
days.

Low-temperature steam formaldehyde sterilization (LTSF)

This method is applicable for materials that cannot withstand 1000C temperature. In
this method, steam at subatmospheric pressure at 750C with formaldehyde vapor is
used. Bacillus stearothermophilus plays an important role as a biological control to
test the efficacy of the test.

At a temperature of 1000C

Boiling

It is an effective method that can kill vegetative cells. Boiling for 10-30 minutes can
kill most of the vegetative cells; however, many spores can withstand this
temperature. Boiling can be employed when adequate methods are not available to
sterilize glass syringes, rubber stopper, etc.

Tyndallisation

In this case, steam at 1000C for successive 3 days is used. It is also known as
intermittent sterilization. In this case, the first exposure kills the vegetative forms,
and in the intervals between the heating and remaining spores germinates into
vegetative forms which are killed on subsequent heating. This process is applied for
sterilization of egg, serum or sugar-containing media which can be damaged due to
exposure in high temperature for a longer period.
Steam sterilizer

Koch’s and Arnold’s steam sterilization is usually used for media which can easily
decompose due to the high temperature in the autoclave. Those media are kept on a
perforated tray and steam at 1000C and at atmospheric pressure passes through the
media for 90 minutes. It is an effective method to kill vegetative cells.

Temperature above 1000C (under pressure)

Autoclave

Steam above 1000C or saturated steam has a better killing capacity than dry heat.
Bacterial proteins coagulate rapidly at moist heat. Saturate steam has the ability to
penetrate any porous material. When steam comes into contact with the cooler
surface it condenses into water and releases its latent heat to the surface. The large
reduction in volume sucks in more steam to the same site and the process continues
until the temperature of the substance raised to that of steam. The condensed water
produced moist conditions for killing the microbes present.

The autoclave is a modified pressure cooker which contains a vertical or horizontal

cylinder. The cylinder is made up of stainless still. A lid on the cylinder is placed and
fastened by screw clams to make it airtight. The lid contains a steam discharge unit,
a pressure gauge, and a safety valve. Moreover, a thermostat is present to monitor
the temperature. Heat is produced by electricity. At the time of sterilization, the
cylinder is filled with an adequate amount of water and it is kept for some time for
preheating. After that, the materials which are needed to be sterilized are inserted
into the cylinder and the lid is then closed tightly. The temperature will increase
eventually along with the pressure. When the temperature reaches 121.10C and the
pressure at 15 psi the sterilization is performed for 15 minutes.

Uses: Sterilization of

Culture media, rubber material, dressing gloves.

Materials that are unable to withstand dry heat in a hot air oven.

Sterilization control

Thermocouple
 Bacterial spores of Bacillus stearothermophilus used at test organisms.
Browne’s tubes contain red solution which turns into green when exposed to the
specific temperature for 15 minutes in an autoclave.
Autoclave tips.

Ozone
Ozone sterilizer uses oxygen, water, and electricity to produce ozone within the
sterilizer and provide sterilization without producing toxic chemicals. It runs at 25-
350C temperature. Inside this device, the oxygen is converted into atomic oxygen
due to the intense electrical field. The atomic oxygen is then combined with the
oxygen molecule to produce ozone. The ozone provides a sterility assurance of 10-6
in approximately 4 hours.

Filtration
This process is useful for sterilizing those materials which are unable to withstand
heat. There are several types of filters such as

Candle filter: Used for purification of water. These filters consist of hollow candles

and water passes through the candles for purification.

Asbestos disc filters: These are made up of magnesium silicate.
Sintered glass filters: These are prepared by fusing finely powdered glass powders.

Membrane filters: These are made up of cellulose esters and are used for water
analysis, sterility testing and for the preparation of the solutions. Membrane filters
are available in pore size 0.015 to 12 micron. The .22 micron filter is most
commonly used as it is smaller than bacteria.
Air filters: These filters are used in laminar airflow chambers to give bacteria-free

air supply. These are also known as High-efficiency particulate air (HEPA) filters.
These filters can separate particles of 0. Micron or larger.
Syringe filters: Syringes fitted with the membrane of different diameters are
available.

A limitation of using the filtration process is that the pores are not small enough for
viruses.

Radiation
Ionizing radiation
Ionizing radiations such as gamma rays, X rays, and cosmic rays are used for
sterilization process. Due to the high penetrating power, these radiations are lethal
for cells. The bacterial cells are killed by damage in the DNA. Gamma radiations from
a cobalt 60 source are commercially used for sterilization of disposable items. This
procedure is also known as cold sterilization.

Non-ionizing radiation
Infrared radiation and UV radiation comes under this of radiation. Infrared radiation is
used for mass sterilization of syringes and catheters. UV radiation with a wavelength
of 240nm to 280nm has bactericidal capacity. The UV radiation causes protein
denaturation and interferes with DNA replication of bacteria. UV radiations are used
for sterilization of close areas, surfaces, operation theaters, laminar airflow, etc.

Chemical Method
Several chemical agents are used as antiseptic and disinfectants. The properties of a
chemical antiseptic or disinfectant are following

The chemical disinfectants need to have a broad spectrum of activity against all
microorganisms such as bacteria, viruses, protozoa and fungi.
The chemical agents should act in the presence of organic matter.
High penetration power is an important property of the chemical agents
The chemical agent needs to be chemically stable under both acidic and basic
environments.
The chemical substances should not have any corrosion activity in metals.
The disinfectants are needed to be non-toxic if absorbed into circulation.
Finally, the chemical agents are needed to be easily available and less expensive.

Alcohols

Ethyl alcohol and isopropyl alcohol are frequently used as chemical agents for
disinfection. Both of the chemicals facilitate the protein denaturation of bacterial
proteins. 70% ethyl alcohol is the standard concentration which is used for
disinfection. These are used as skin antiseptics. Apart from this methyl alcohol has
activity against fungal spores and used to disinfection of inoculation cabinets.

Aldehydes
Formaldehyde
It is known for its bactericidal, sporicidal and virucidal activities. It can be used in
both aqueous and gaseous form. A 10% formalin solution is a standard chemical
disinfectant. It is used for

Prevention of tissues for histological examinations.

Sterilization of bacterial vaccines
Preparation of toxoids from toxins.

Glutaraldehyde
It has its activity against bacteria (Mycobacterium tuberculosis), fungi and viruses
(including HIV, hepatitis B, etc). It can also kill spores and is known for its less toxic
nature. It is used as a 2% buffered solution. Glutaraldehyde is used for

Sterilization of cystoscopes, endoscopes, and bronchoscopes

Sterilization of plastic endotracheal tubes, face masks, metal instruments, etc.

Orthophathalaldehyde
Orthophathalaldehyde (OPA) is a high-level disinfectant and is known for its stability
during storage. It has bactericidal effects against mycobacteria. 0.5% OPA is slowly
sporicidal and OPA vapors irritate the respiratory tract and eyes, therefore, it must be
handled with appropriate safety.

Phenols
Lister (father of antiseptic surgery) used phenol for the first time in the sterilization of
surgical instruments. Phenols work as a disinfectant and kill microorganisms by cell
membrane damage. It is toxic for the skin. Different derivatives of phenol are used as
antiseptics which are following

Cresols
An example of cresol is Lysol which is mostly used for sterilization of infected
glasswares, floors, etc.

Chlorhexidine
Savlon is an example of a chlorhexidine solution which is widely used in wounds,
preoperative disinfection of the skin. It is bactericidal at high dilution. Moreover, it
also has fungicidal activity.

Chloroxylenol
Dettol is commercially available as a chloroxylenol solution. It is less toxic and less
irritant.

Hexachlorophene
It is bacteriostatic at very high dilution.

Halogens
Chlorine and iodine are commonly used disinfectants. Chlorine is used in water
supplies, swimming pools, food, and dairy industries. Chlorine compounds in the
form of bleaching powder, sodium hypochlorite, and chloramines. The disinfection
action of all the chlorine compounds is due to the release of free chlorine which
becomes a strong oxidative agent.

Iodine in alcoholic and aqueous solution is used as a skin disinfectant. It is active

against M tuberculosis and slightly active against spores. Compounds with iodine
with surface-active agents known as iodophors are claimed to be more active than
aqueous or alcohol solution.

Oxidizing agents
Hydrogen peroxide
It is effective against most organisms in the concentration of 3-6 %. However, it kills
spores at higher concentrations (10-25%). The mode of action is by the liberation of
free hydroxyl radical on the decomposition of hydrogen peroxide. These free radicals
are active ingredients in the disinfection process.

Peracetic acid
It is an oxidizing agent and is a more potent germicidal agent than hydrogen
peroxide.

Salts
Slats of heavy metals have a toxic effect on bacteria. The salts of copper, silver, and
mercury are used as a disinfectant. They are protein coagulant ant act by combining
with sulphydryl groups of bacterial proteins and other essential intracellular
compounds. Merthiolate (sodium ethyl mercurithiosalicylate) is used in a dilution of
1:10000 for the preservation of sera.

Dyes
Two groups of dyes, aniline and acridine dyes have been used as a skin and wound
antiseptics. Both the dyes have bacteriostatic activity. Aniline dyes include crystal
violet, brilliant green, and malachite green. Acridine dyes include acriflavine, cuflavin,
proflavin, and aminacrine.

Vapor phase Disinfectants

Ethylene Oxide (ETO)
It is a colorless liquid with a boiling point of 10.70C. It is effective against all types of
microorganisms including viruses and spores. It acts by alkylating the amnio
carboxyl, hydroxyl and sulphydryl groups in protein molecules. In addition, it reacts
with DNA and RNA. It is specially used for sterilizing plastic and rubber articles,
respirators, heart-lung machines, dental equipment, etc.

Betapropilolactone (BPO)
This is a condensation product of ketane and formaldehyde. It has rapid action and
used in0.2%. It is more efficient in fumigation than formaldehyde. BPO is used for the
inactivation of vaccines.

Reference

Textbook of Microbiology by C.P. Baveja, 4th Edition, Arya Publications

About Author
 Somak Banerjee

Somak Banerjee has completed his M.Sc. in applied microbiology from the Vellore
Institute of Technology, Vellore, India.

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