Sterilization

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.
METHODS OF STERILIZATION

Physical Methods

1.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.

2.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

a) 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.

B)Moist Heat Sterilization: Procedure

1.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.
2.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.

3.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.
 . 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
 Bacterial spores of Bacillus stearothermophilus used at test organisms.
 Autoclave tips.
4.Ozone
Ozone sterilizer uses oxygen, water, and electricity to produce ozone within the sterilizer and
provide sterilization without producing toxic chemicals. It runs at 25-35 0C temperature. .

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
1.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.

2.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
1.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.

2.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.
3.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
1.Cresols
An example of cresol is Lysol which is mostly used for sterilization of infected glasswares,
floors, etc.
2.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.
3.Chloroxylenol
Dettol is commercially available as a chloroxylenol solution. It is less toxic and less irritant.
4.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.

BACTERICIDAL
Bactericidal refers to substances or agents that kill bacteria. Bactericidal agents cause
irreversible damage to bacterial cells, leading to their death
Eg: Antibiotics like penicillins, cephalosporins, aminoglycosides, fluoroquinolones, and
vancomycin

BACTERIOSTATIC

Bacteriostatic refers to substances or agents that inhibit the growth and reproduction of
bacteria but do not directly kill them.

DISINFECTANTS
Disinfectants are chemical agents used to destroy or inhibit the growth of harmful
microorganisms (bacteria, viruses, fungi) on inanimate objects and surfaces. Disinfectants
are typically too harsh for use on living tissues and are designed for use on non-living
materials, such as floors, countertops, surgical instruments, and other surfaces that may
harbor pathogens.
DISINFECTION
Disinfection is the process of eliminating or reducing harmful microorganisms (pathogenic
bacteria, viruses, fungi) on inanimate objects and surfaces to a level that is not harmful to
human health. Unlike sterilization, which completely destroys all forms of microbial life,
disinfection primarily targets pathogenic microorganisms, though it may not eliminate all
bacterial spores.
ANTISEPTIC
Antiseptics are substances that inhibit the growth of or kill microorganisms on living
tissues, such as skin, wounds, and mucous membranes. Unlike disinfectants, which are used
on inanimate objects and surfaces, antiseptics are specifically designed to be safe for use on
the body and are commonly used to prevent infections.
Common Types of Antiseptics:
1. Alcohols (e.g., ethanol, isopropanol):
o Rapidly kill many bacteria, viruses, and fungi.
o Commonly used in hand sanitizers and skin preparation before injections.
2. Iodine and Iodophors (e.g., povidone-iodine):
o Effective against a broad range of microorganisms.
o Frequently used to disinfect skin before surgical procedures.
3. Hydrogen Peroxide:
o Used as a wound cleaning agent due to its ability to release oxygen and
create a foaming action that mechanically cleans wounds.
4. Chlorhexidine:
o Commonly used as a surgical scrub and in mouthwashes to reduce bacteria.
5. Phenolic Compounds:
o Found in some mouthwashes and skin cleansers.

ANTISEPSIS
Antisepsis is the process of applying antiseptics to living tissue to reduce or eliminate the
presence of pathogenic microorganisms, thereby preventing infection. It focuses on
disinfecting and protecting skin, wounds, and other body surfaces from harmful bacteria,
viruses, and fungi.