METHODS OF STERILIZATON

By: Miss Lumingu

 INTRODUCTION:

Microbes are ubiquitous and many microorganisms are

associated with undesirable consequences, such as food
spoilage and disease. Therefore, it is essential to kill a wide
variety of microorganisms or inhibit their growth to minimize
their destructive effects.
The goal is twofold:
(a) To destroy pathogens and prevent their transmission
(b) To reduce or eliminate microorganisms responsible for
the contamination of water, food, and other substances
Sterilization is defined as a process by which an
article, surface, or medium is freed of all living
microorganisms either in the vegetative or in the spore
state.
Any material that has been subjected to this process is
said to be sterile.
An object cannot be slightly sterile or almost sterile; it
is either sterile or not sterile.
Sterilization is performed with a physical agent, such as
heat, a few chemicals called sterilants can be classified
as sterilizing agents because of their ability to destroy
spores.
A germicide, also called a microbicide, is any chemical
agent that kills pathogenic microorganisms.
A physical or chemical agent that kills “germs” is said to
have germicidal properties.
 DEFINITIONS

Bactericidal
kills bacteria
• Bacteriostatic
• Inhibits growth of bacteria
Sterilization
Removal or killing of all living microorganisms
including bacteria and their spores
• Disinfection
• Removal or killing of disease-causing
microorganisms (not necessarily all microorganisms)
PHYSICAL METHODS OF STERILIZATION
Methods of sterilization can be broadly classified as:
Physical methods of sterilization include:
•Sterilization by sunlight
•Sterilization by Heat
•Sterilization by Filtration
•Sterilization by Irradiation
•Sterilization by Sound (sonic) waves
 SUNLIGHT
Direct sunlight is a natural method of sterilization of water in
tanks, rivers, and lakes.
Direct sunlight has an active germicidal effect due to its
content of ultraviolet and heat rays.
Bacteria present in natural water sources are rapidly destroyed
by exposure to sunlight
 STERILIZATION BY DRY HEAT
Sterilization by dry heat makes use of air with a low moisture content
that has been heated by a flame or electric heating coil.
In practice, the temperature of dry heat ranges from 160°C to several
thousand degrees Celsius.
The dry heat kills microorganisms by protein denaturation, oxidative
damage, and the toxic effect of increased level of electrolytes.
Dry heat is not as versatile or as widely used as moist heat, but it has
several important sterilization applications.
Sterilization by dry heat includes sterilization by (a) flaming, (b)
incineration and (c) hot air oven:
 STERILIZATION BY HEAT
Heat is the most dependable method of
sterilization and is usually the method of choice
unless contraindicated.
As a rule, higher temperatures (exceeding the
maximum) are microbicidal, whereas lower
temperatures (below the minimum) tend to have
inhibitory or microbistatic effects.
Two types of physical heat are used in
sterilization are:
Moist and Dry heat.
 FLAMING
Principal: Passing the object
through the flame of Bunsen
burner without heating to
redness.
Used for: Sterilization of:
glass slides
Mouth of culture tubes.
 Red Heat
Principal: Holding object in
Bunsen flame till they become
red hot.
Used for: Sterilization of:
Bacteriological loops
Tips of forceps
 INCINERATION:
Incineration is an excellent method for safely destroying
infective materials by burning them to ashes. It has many uses:
Incinerators are used to carry out this process and are
regularly employed in hospitals and research labs to destroy
hospital and laboratory wastes.
The method is used for complete destruction and disposal of
infectious material, such as syringes, needles, culture material,
dressings, bandages, bedding, animal carcasses, and pathology
samples.
This method is fast and effective for most hospital wastes, but
not for metals and heat-resistant glass materials.
Incineration
Principal: Infective materials are
converted to sterile ash by
burning in incinerator

Used for: Destruction of

contaminated disposable
materials (waste)
 HOT-AIR OVEN
The hot-air oven provides another means of dry heat sterilization
and is the most widely used method.
The hot-air oven is electrically heated and is fitted with a fan to
ensure adequate and even distribution of hot air in the chamber.
It is also fitted with a thermostat that ensures circulation of hot air
of desired temperature in the chamber.
Heated, circulated air transfers its heat to the materials inside the
chamber.
While sterilizing by hot-air oven, it should be ensured that the oven
is not overloaded.
The materials should be dry and arranged in a manner which allows
free circulation of air inside the chamber.
Hot air oven
Principal
Materials to be sterilized are
exposed to high temperature in an
electrically heated oven.
Even distribution of heat
throughout the chamber is
achieved by a fan.
Holding time:
• 160°C for two hours
• 180°C for one hours
Used for Sterilization of:
All glasses: test tubes, Petri dishes, flasks,
pipettes.
Instruments: as forceps, scalpels, scissors
Dry material in sealed containers such as fat,
oils, powder.
 STERILIZATION BY MOIST HEAT
Moist heat occurs in form of hot water, boiling water, or steam
(vaporized water).
In practice, the temperature of moist heat usually ranges from 60 to
135°C. Adjustment of pressure in a closed container can regulate the
temperature of steam. Moist heat kills microorganisms by
denaturation and coagulation of proteins.
Sterilization by moist heat can be classified as follows:
1. Sterilization at a temperature <100°C
2. Sterilization at a temperature of 100°C
3. Sterilization at a temperature >100°C
 PASTEURIZATION (BELOW 100)
Principal: Pasteurization is
Pasteurization is a commonly used in
process of heating a milk fruit juices,
liquid to a specific beer, and wine
temperature for a
definite length of which are easily
time and then contaminated during
cooling it collection and
immediately. processing.
 BOILING (AT 100 °C)
Principal: Boiling water for fifteen minutes will kill most
vegetative bacteria and inactivate viruses. However
boiling is ineffective against many bacterial and fungal
spores.
 AUTOCLAVING
Principal:
When the pressure is increased inside a
closed container, the temperature at
which water boils exceeds 100°C.
At double atmospheric pressure the
temperature of the steam reaches
121°C.
Autoclaving is the most reliable method
of sterilization that kills all kinds of
bacteria and spores.
Sterilization Temperature: - 121˚C for
20 – 30 minutes.
Used for sterilization of:
Culture media.

Surgical supplies e.g. dressing, and

surgical instruments.
 FILTRATION
Filtration is an excellent way to reduce the
microbial population in solutions of heat-labile
material by use of a variety of filters. Filters are
used to sterilize these heat-labile solutions.
Filters simply remove contaminating
microorganisms from solutions rather than directly
destroying them.
 FILTRATION
It is possible to remove bacteria
from fluids by passing them
through filters with pores so
small that bacteria are arrested.
 FILTRATION
Filtration is used to sterilize
liquids that would be
damaged by heat, liquids
such as (antibiotic solutions
and vaccines).
STERILIZATION BY IRRADIATION“cold
sterilization.”
 ULTRAVIOLET RADIATION
used for:
1.sterilization of operating
theatres.
2. sterilization of the interiors of
biological safety cabinets.
 IONIZING IRRADIATION (GAMMA RAYS)
Used for sterilization of an
article that can not stand
heat,such as rubber catheters,
gloves, plastic syringes.
 RADIATIONS
Ionizing and nonionizing radiations are the two types of radiation used
for sterilization.
Ionizing radiations: Ionizing radiation is an excellent sterilizing agent
with very high penetrating power.
These radiations penetrate deep into objects and destroy bacterial
endospores and vegetative cells, both prokaryotic and eukaryotic.
Ionizing radiations include (a) X-rays, (b) gamma rays, and (c) cosmic
rays. Gamma radiation from a cobalt-60 source is used for sterilization
of antibiotics, hormones, sutures, catheters, animal feeds, metal foils,
and plastic disposables, such as syringes.
This type of radiation is also used to sterilize and “pasteurize” meat
and other food items.
 NON-IONIZING RADIATIONS
Nonionizing radiations include infrared and ultraviolet
radiations.
Infrared radiations are used for rapid and mass sterilization
of disposable syringes and catheters.
Ultraviolet (UV) radiation with a wave length of 240–280
nm is quiet lethal and has a marked bactericidal activity. It
acts by denaturation of bacterial protein and also interferes
with replication of bacterial DNA.
 Sound (sonic) waves
High-frequency sound (sonic) waves beyond the sensitivity of the
human ear are known to disrupt cells.
Sonication transmits vibrations through a water-filled chamber
(sonicator) to induce pressure changes and create intense points of
turbulence that can stress and burst cells in the vicinity.
Sonication also forcefully dislodges foreign matter from objects.
Heat generated by the sonic waves (up to 80°C) also appears to
contribute to the antimicrobial action.
Gram-negative rods are most sensitive to ultrasonic vibrations,
while Gram-positive cocci, fungal spores, and bacterial spores are
resistant to them.
 CHEMICAL METHODS OF
STERILIZATION.
Disinfectants:
Are chemical materials used for sterilization and are toxic
to the human tissues and cells.
Antiseptics:
Are chemicals for sterilization and not toxic to the human
body e.g. "mouth gargles".
Antiseptics and disinfectants are used extensively in hospitals
for a variety of topical and hard surface applications.
They are an essential part of infection control practices and aid
in the prevention of nosocomial infections.
 PROPERTIES OF IDEAL DISINFECTANTS
An ideal disinfectant or antiseptic has the following
characteristics:
Ideally, the disinfectant should have a wide spectrum of
antimicrobial activity.
It must be effective against a wide variety of infectious agents
(Gram-positive and Gram negative bacteria, acid-fast bacteria,
bacterial endospores, fungi, and viruses) at high dilutions.
 ACTION OF DISINFECTANTS
Disinfectants act in many ways as discussed below.
1. They produce damage to the cell wall and alter
permeability of the cell membrane, resulting in exposure,
damage, or loss of the cellular contents.
2. They alter proteins and form protein salts or cause
coagulation of proteins.
3. They inhibit enzyme action and inhibit nucleic acid
synthesis or alter nucleic acid molecules.
 TYPES OF DISINFECTANTS
Disinfectants include the following:
Phenolic compounds
Halogens
Alcohols
Aldehydes
Gases
Surface active agents
Oxidizing agents
Dyes
Heavy metals
Acids and alkalis
 HALOGENS

Halogens (fluorine, bromine, chlorine, and iodine) are

a group of non-metallic elements that commonly occur in
minerals, sea water, and salts.
Although they can occur either in the ionic (halide) or
nonionic state, most halogens exert their antimicrobial
activity primarily in their nonionic state, but not in the
halide state (e.g., chloride, iodide).
 ALCOHOLS
Alcohols are among the most widely used
disinfectants and antiseptics.
They are bactericidal and fungicidal but not
sporicidal.
They have no action against spores. Ethyl alcohol
and isopropyl alcohol are the two most popular
alcohol germicides.
 ALDEHYDES
• Formaldehyde and glutaraldehyde are the two
most commonly used aldehydes that are used as
disinfectants.
• They are highly reactive molecules that combine
with nucleic and alkylating molecules. They are
sporicidal and can also be used as chemical sterilants
 GASES
• Various gaseous agents are used for sterilization of large
volume of heat-sensitive disposable items and also
instruments.
• Ethylene oxide, formaldehyde gas, and beta
propiolactone are frequently used gaseous agents.
 OXIDIZING AGENTS
This group includes halogens, hydrogen peroxide,
potassium permanganate, and sodium perborate.
They are good disinfectants and antiseptics but are less
effective in the presence of organic matter.
Hydrogen peroxide, used as 3% solution, is a weak
disinfectant.
It is useful for cleaning of the wounds and for mouth wash
or gargle. Potassium permanganate is bactericidal in nature
and active against viruses also.
 DYES
The dyes that have been used extensively as skin and
wound
antiseptics include (a) acridine dyes and (b) aniline dyes.
The acridine dyes include acriflavine, euflavine,
proflavine, and aminacrine. They show more activity
against Gram-positive bacteria than against Gram-negative
organisms.
They act by interfering with the synthesis of nucleic acids
and proteins in bacterial cells.
 SURFACE ACTIVE AGENTS
• Surface active agents, such as detergents are the
substances that alter energy relationship at interfaces
producing a reduction in surface tension.
• Detergents are organic molecules that serve as wetting
agents and emulsifiers because they have both polar
hydrophilic and nonpolar hydrophobic ends.
• Due to their amphipathic nature, detergents solubilize and
are very effective cleansing agents.
• They are different from soaps, which are derived from
fats.
 HEAVY METALS
Soluble salts of mercury, silver, copper, arsenic, and
other heavy metals have antibacterial activity, both
bactericidal and bacteriostatic.
They combine with proteins, often with their sulfhydryl
groups and inactivate them.
 ACIDS AND ALKALIS
Acids (such as sulfuric acid, nitric acid, hydrochloric acid,
and benzoic acid) and alkalis (like potassium and sodium
hydroxide and ammonium hydroxide) are germicidal in
nature.
They kill microorganisms by hydrolysis and altering the pH
of the medium.
They are rarely used as disinfectants.
DIFFERENCES BETWEEN STERILIZATION
AND DISINFECTION