Physical and

Chemical Methods
of Sterilization
Definition of Terms
1. Sterilization– the process of killing or removing all microbial forms, including spores.
2. Disinfection– the process by which most microbial forms on inanimate objects are
killed without necessarily destroying saprophytes and bacterial endospores which
leads to a reduction in the number of organisms to a level that they cannot produce
infection.
3. Antisepsis– use of chemical agents on living tissue (e.g., skin) to prevent the spread
of microorganisms by inhibiting their growth or destroying them.
4. Bactericidal or germicidal agent– agent, physical or chemical, that kills bacteria.
5. Bacteriostatic agent– agent, physical or chemical, capable of inhibiting the growth
of bacteria without necessarily killing them.
6. Sporicidal, fungicidal, viricidal– agents capable of destroying spores, fungi, and
viruses, respectively
Physical Methods of Sterilization
Heating
• Heating is the most common physical method of
sterilization. The rate of killing is expressed in thermal
death time,
• i.e., the minimum time required to kill a suspension of
an organism at a predetermined temperature and
environment.
The mechanisms of action of heating include:
(1)Formation of single strand breaks in the bacterial DNA;
(2) Coagulation and denaturation of proteins;
(3) Accumulation of toxic levels of electrolytes; and
(4) alteration of cell membrane structure.
Several factors can affect the process of
sterilization through heating. These include:
1. Nature of the heat– moist heat has greater killing action than dry heat.
2. Temperature and time– as temperature increases, the time taken to sterilize
decreases.
In other words, there is an inverse relationship between time and temperature.
3. Number of microorganisms– the more microorganisms there are, the higher the
Temperature and the longer the duration of the process required to destroy all of them.
4. Nature of microorganisms– spore forming microorganisms are more difficult to destroy
than non spore forming ones.
5. Type of material– the temperature required to sterilize materials depend on the
sensitivity of
the material to heat. Heat sensitive materials will require lower temperature than heat
resistant
materials.
6. Presenceof organic material– the presence of organic materials such as fats, proteins,
and sugars may necessitate higher temperatures.
• Types of Heat
1. Moist heat– preferred over dry heat because of its more
rapid killing action. Its main mechanism of action is to cause
coagulation and denaturation of proteins. The various
methods of moist heat maybe classified according to the
temperature used.
• These include:
a. Temperature below 100 °C
• Pasteurization
This is the method of destroying disease producing organisms
in milk and milk products as well as other beverages. There
are several variations of this method based on the
temperature utilized. One method is called the conventional
method where the milk is heated at 60 °C–65°C followed by
rapid cooling
The flash method involves heating at 72 °C for 15
seconds followed by quick cooling to 13 °C. A newer
pasteurization method developed is what they call ultra
high temperature (UHT) method where heating is done
at 140 °C for a period of 15 seconds and 149 °C for 0.5
seconds.
• Vaccine bath
This is used to destroy contaminating bacteria in vaccine
preparations. The vaccine preparation is heated in a
water bath at 60 °C for one hour. This procedure is not
sporicidal. Only the vegetative forms of the bacteria are
destroyed
• Serum bath
This is used to inactivate bacteria contaminating serum
preparations and is done by heating at 56 °C for several
successive days. Like vaccine bath, only the vegetative forms
are destroyed since higher temperatures will cause
coagulation of proteins present in the serum.
• Inspissation
This technique is used to solidify and disinfect egg containing
and serum
Containing media. The culture medium is placed in the slopes
of a device called an inspissator and is heated at 80 °C–85 °C
for 30 minutes for three successive days. The basis for the
method is that on the first day, vegetative forms will die and
the spores that will germinate the following day will also die
 b. Temperatureof100 °C
• Boiling
This method involves utilizing water at boiling temperature of 100 °C. It is
not sporicidal and will destroy only the vegetative forms. The killing action
can be enhanced by the addition of 2% sodium bicarbonate. Certain metal
articles and glass wares can be disinfected using this method for 10–20
minutes without
opening the lid of the boiler.
• Fractional sterilization(Tyndallization)
This method is also known as intermittent sterilization and involves exposing
the material to be sterilized to live steam at 100 °C for 30–90 minutes for
three
consecutive days, depending on the material to be sterilized. This
sterilization method can be used to sterilize culture media such as TCBS and
selenite broth.
The vegetative forms are killed on the first day and the spores that will
germinate will be destroyed on the next successive days. Only vegetative
forms of the bacteria are destroyed with this method
c. Temperatureabove100 °C
• Autoclave(Steam under pressure)
This is the most efficient method of sterilization because
it can destroy all microbial forms. The temperature for
sterilizing is dependent on the pressure of the steam.
When the pressure reaches 15 pounds per square inch
(psi), the temperature inside the vessel reaches 121 °C.
Because of the high temperature and pressure, it would
take only 15–20 minutes to sterilize the material. This
method is used to sterilize instruments, surgical
bandages, culture media, and other contaminated
materials that can withstand high temperature and high
pressure.
2. Dry heat– the effectiveness of dry heat depends on
the penetration of heat through the material to be
sterilized. It is used to sterilize materials in enclosed
tubes, oils, jellies, powders, and glass wares such as test
tubes and Petri dishes.
a. Red flame
This method is used to sterilize articles like
bacteriological wire loops, straight wires, tips of forceps,
and searing spatulas. The materials are held over the
flame of a Bunsen burner until they become red hot. It is
limited only to articles that can be heated to redness in
flame.
b. Open flame (Flaming)
This method also makes use of the Bunsen burner or alcohol lamp. The material
to be sterilized is passed over the flame several times but is not heated to
redness.
It is aimed at burning the organism into ashes and is used to sterilize such articles
as mouth softest tubes, scalpels, glass slides, and cover slips. Only vegetative
forms are destroyed. In addition, cracking of the glassware may occur.
c. Incineration
This method is aimed at burning the organism into ashes. The contaminated
material is burned using an incinerator. Articles that must be incinerated
include soiled dressings and beddings, animal carcasses, and pathological
material.
This will result in loss of the article and hence must be used only for articles that
must be disposed. Some materials such aspolystyrene emit dense smoke and
must
not be incinerated.
d. Hot air oven
The use of the hot air oven was first introduced by Louis
Pasteur. Articles to be sterilized are placed in the oven
with a temperature of 160 °C for a period of one hour.
This can be used to sterilize metallic instruments such as
forceps, scalpels, and scissors. It can also be used to
sterilize certain glass wares (e.g. Petri dishes, pipettes,
flasks)and it is the only method used to sterilize powders
and ointments. The disadvantage of using this method is
that because air is a poor conductor of heat, then hot air
will have poor penetration of the materials to be
sterilized. In addition, cotton wool and paper may get
slightly charred and glasses can become smoky.
e. Infrared rays
In this method, the articles to be sterilized are placed in a
conveyor belt and passed through a tunnel that is heated
by infrared radiators. The temperature to which the
materials are subjected to is 180 °C for a period of 7.5
minutes. It can be used to sterilize metallic equipment
and glassware.
 Desiccation
• This method is based on the principle of depriving the
microorganism of moisture. It is used mainly for food
preservation, such as in the preparation of dried fish
and fruits. It may destroy vegetative forms. Endospores
are resistant to drying.
Freezing
• Freezing is not a reliable method of sterilization
because most pathogenic organisms are resistant to low
temperatures. Its main use in the laboratory is for the
preservation of microorganisms in a process called
lyophilization or freeze drying where the organism is
rapidly frozen then dehydrated in high vacuum and
stored in a vacuum sealed container.
 Filtration
• This is a form of mechanical sieving that does not kill
microorganisms but merely separates them from the
fluid. A cellulose ester filter with a pore size of 0.22 μm–
0.45 μm is used which can filter all microorganisms
except viruses and the three smallest bacteria-
Mycoplasma, Rickettsia, and Chlamydia. It is used for
liquid solutions that will be destroyed by heat or
freezing such as serum, antibiotic solutions, sugar
solutions, or urea solution. This method can be used to
remove bacteria from culture media or to prepare
suspensions of viruses and phages.
Radiation
1. Ultraviolet Light (UVL)/Nonionizing radiation– the effective
UVL wavelength is in the range of 200 nm–280nm, with 260 nm as the
most effective. This corresponds with the maximum absorption of
bacterial DNA. UVL acts by inducing formation of thymine-thymine
dimers resulting in lethal frame shift mutations. Microorganisms such
as bacteria, viruses, and yeasts can be inactivated within seconds.
However, UVL is not sporicidal and is more frequently used for surface
disinfection. It is used to disinfect hospital wards, operating rooms,
laboratories, and other rooms in the hospital that need to be sterilized.
The disadvantage of UV ray is that it has low penetrance. It is also
limited by the lifespan of the Uv bulb. In addition, there are some
bacteria that have DNA repair systems that can counteract the
damage done by UV rays. Care should also be observed by the handler
because UV rays can be harmful to the skin and eyes.