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STM 312 (Microbial Technique I) : Sterilization Techniques

This document discusses sterilization techniques. It defines sterilization as eliminating all forms of life, including spores, from surfaces or materials. There are physical and chemical methods of sterilization. Physical methods include heat sterilization using moist heat (autoclaving, fractional sterilization) or dry heat (flaming, hot air ovens), radiation (UV, X-rays), and filtration. Heat sterilization, especially autoclaving, is the most common and reliable method for materials that can withstand heat. Choice of sterilization method depends on the material's properties.

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489 views

STM 312 (Microbial Technique I) : Sterilization Techniques

This document discusses sterilization techniques. It defines sterilization as eliminating all forms of life, including spores, from surfaces or materials. There are physical and chemical methods of sterilization. Physical methods include heat sterilization using moist heat (autoclaving, fractional sterilization) or dry heat (flaming, hot air ovens), radiation (UV, X-rays), and filtration. Heat sterilization, especially autoclaving, is the most common and reliable method for materials that can withstand heat. Choice of sterilization method depends on the material's properties.

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Iruafemi victoria
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© © All Rights Reserved
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Download as DOCX, PDF, TXT or read online on Scribd
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STM 312 (MICROBIAL TECHNIQUE I)

LECTURE II

STERILIZATION TECHNIQUES

Sterilization refers to any process that eliminates, removes, kills, or deactivates all
forms of life present in or on an object. Sterilization is a broad term that refers to
any process that removes or kills all forms of microorganisms. This includes
bacteria, fungi, viruses and protozoans including their spore forms which are
usually very resistant. This refers to life forms that are on the surface, within a
fluid, medications, or compounds such as buffers and culture media. Sterilization is
an absolute term, i.e a material can either be sterile or not. Disinfection on the
other hand is the process of elimination of most pathogenic microorganisms
(excluding bacterial spores) on inanimate objects. However the word
“sterilization” is loosely use atimes when sterilization is not achieved such as when
the bench surface is wiped with cotton wool soaked in 70% alcohol, this process is
disinfection because spores of microorganisms will not be destroyed by this
process if it is present on the bench surface. Therefore sterilization and disinfection
are not synonymous.

There are several methods used to bring about sterilization, the choice depend on
the properties of the materials such as the physical nature of the material to be
sterilized i.e. filter paper which is a solid material cannot be sterilized by filtration.
The methods are classified as Physical and Chemical methods.

Physical Methods of Sterilization:


 Heat method of sterilization
 Radiation
 Filtration
Heat Method of Sterilization
This is the most common method of sterilization. It is considered to be the most
reliable method of sterilization of materials that can withstand the heat treatment.
The heat used kills the microbes in the substance. The temperature of the heat and
duration of heating are the factors that affect the extent of sterilization.

In heat sterilization process, the longer the exposure to heat the better is the
sterilization at a given temperature. As the temperature of heat rises the timespan
required for sterilization decreases and vice-versa. But one needs to maintain
minimum sterilization time or minimum contact time for the heat to be in touch
with the microorganisms and thereby kill them.

The heat method of sterilization is again of two types based on the type of heat
used. They are;

A) Moist heat methods


B) Dry heat methods
a) Moist heat method of sterilization:
Here heat is applied in the form of steam or just boiling. It acts by coagulation and
denaturation of proteins. This method includes techniques like:

 The use of steam (Autoclave)


 Fractional Sterilization

● Using Steam (autoclaving): here the substances are subjected to sterilization in


autoclave steam sterilization equipment. The process is carried out at a temperature
of 115 degrees for 60 min or 121 degrees for 20 min at 15psi pressure. Bacterial
spores are the forms of bacteria which are inert. They form a rigid cover over
the cell wall during harsh climate. This cover prevents any damage to cell and
drying of the cell. By steam sterilization, these forms of bacteria are also killed as
steam destroys the cell wall. This method is used for sterilization of; Flammable
substances, Culture media, Solutions, Glass wares etc.

A simple autoclave has vertical or horizontal cylindrical body with a heating


element, a perforated tray to keep the articles, a lid that can be fastened by screw
clamps, a pressure gauge, a safety valve and a discharge tap. The lid is closed but
the discharge tap is kept open and the water is heated.

As the water starts boiling the steam drives air out of the discharge tap, when all
the air is displaced and steam starts appearing through the discharge tap, the tap is
closed. The pressure inside is allowed to rise up to 15 lbs. per square inch. At this
pressure the articles are heated for 15 minutes, after which the heating is stopped
and the autoclave is allowed to cool.

Once the pressure gauge shows the pressure equal to atmospheric pressure, the
discharged tap is opened to let the air in. The lid is opened and articles are
removed. Culture media, dressing, certain equipment can be sterilized by
autoclave.

● Fractional Sterilization is another steaming method which is by Passing the


steam at 100°C over the materials, it kills bacteria. The method was called
fractional sterilization because a fraction was accomplished on each day. It was
also called tyndallization after its developer, John Tyndall and intermittent
sterilization because it was a stop-and-start operation. Sterilization by the fractional
method is achieved by an interesting series of events. During the first day’s
exposure, steam kills virtually all organisms except bacterial spores, and it
stimulates spores to germinate to vegetative cells. During overnight incubation the
cells multiply and are killed on the second day. Again, the material is cooled and
the few remaining spores germinate, only to be killed on the third day. Although
the method usually results in sterilization, occasions arise when several spores fail
to germinate. The method also requires that spores be in a suitable medium for
germination, such as a broth.
Fractional sterilization has assumed importance in modern microbiology with the
development of high-technology instrumentation and new chemical substances.
Often, these materials cannot be sterilized at autoclave temperatures, or by long
periods of boiling or baking, or with chemicals. An instrument that generates free-
flowing steam, such as the Arnold sterilizer, is used in these instances.

Sugars and gelatin medium may get decomposed by autoclaving. So, these can be
sterilized by exposing them to free steaming for 20 minutes for three successive
days.

b) Dry heat methods: It acts by protein denaturation and oxidative


damage. Materials such as bacteriological loops, straight wires, tips of forceps and
searing spatulas are sterilized by holding them in a Bunsen flame, till they become
hot red. Following methods are subjected for dry heat sterilization:
 Flaming
 Incineration/Red heat
 Hot air oven.
 Flaming  is the process of exposing metallic device like the needle, scalpels, and
scissors to flame for few minutes. The fire burns the microbes and other dust on
the instrument directly.
 Incineration/Red heat is done especially for inoculating loops used in microbe
cultures. The metallic end of the loop is heated to red hot on the flame. This
exposure kills all the microorganisms.
● Hot air oven is suitable for the sterilization of dry material like metal
devices, glassware, and other such laboratory stocks. Dry heat destroys
microorganisms by dehydration and oxidation or even incineration. Materials are
exposed to high temperature (160°C) for duration of one hour in an electrically
heated oven. If the temperature goes above 180°C., there will be danger of the
cotton stoppers charring. Therefore, the thermometer must be watched closely at
first until the sterilizer is regulated to the desired temperature. The necessity of
watching the sterilizer may be avoided by having the oven equipped with a
temperature regulator.

The effect of dry heat on microorganisms is equivalent to that of baking. The heat
changes microbial proteins by oxidation reactions and creates an arid internal
environment, thereby burning microorganisms slowly. It is essential that organic
matter such as oil or grease films be removed from the materials, because organic
matter insulates against dry heat. Moreover, the time required for heat to reach
sterilizing temperatures varies among materials. Thus this factor must be
considered in determining the total exposure time.

Radiation
This method involves exposing the packed materials to radiation for sterilization.
There are two types of radiations available for sterilization i.e.
a) non-ionic and

b) ionic radiation.

a) Non-ionic radiations: These are low-energy rays with poor penetrative power,
e.g., U.V. rays (wavelength 200-280 nms, effective 260 nm). They are safe to the
operator of sterilization, and they can be used even at the door entrances to prevent
entry of live microbes through the air. When microorganisms are subjected to
ultraviolet light, cellular DNA absorbs the energy, and adjacent thymine molecules
link together. Linked thymine molecules are unable to position adenine on
messenger RNA molecules during the process of protein synthesis. Moreover,
replication of the chromosome in binary fission is impaired. The damaged
organism can no longer produce critical proteins or reproduce, and it dies quickly.
This method is used to sterilized the air inside the inoculating chamber.

b) Ionizing radiation sterilization: These are high-energy rays with good


penetration power. They are powerful radiation and very useful for sterilization.
The operator needs to protect himself from exposure from these radiations by use
of special clothing because they cause any molecule on their path to undergo
ionization e,g X-rays, γ-rays, etc. This method is used to sterilized disposable petri
dished and syringes. In some parts of Europe, fruits and vegetable are irradiated to
increase their shelf life up to 500 percent. Since radiation does not generate heat, it
is called Cold sterilization.
Filtration
Heat is valuable physical agent for controlling microorganisms but sometimes it is
important to use other methods. For example, no one would suggest removing the
microbial population from a tabletop by using a Bunsen burner, nor can heat-
sensitive solutions be subjected to an autoclave. In instances such as these and
numerous others, a heat-free method must be used. Both liquids and air can be
sterilized by filteration. The filter act to retain microorganisms partly by the small
size of its pore and partly by adsorption on the pore walls as the liquid flows
through it. Several types of filters are available for use in the microbiology
laboratory. They include; i. Porcelain or Chamberland Filters. ii. Berkefeld Filters.
iii. Mandler Filters. iv. Fritted-Glass Filters. v. Asbestos Filters. vi.Jenkins Filter.
vii. Ultrafilter. viii. Membrane Filter. ix. Cleaning Filters etc.

Chemical Methods of Sterilization


Heat sterilization is mostly unstable for thermolabile solid medicament and
thermolabile equipment including materials of plastics, delicate rubber items.
Because of high capital cost and use of elaborate precautionary measures, the
radiation method which is one of the methods of sterilization has become
unpopular.

Thus the sterilization of such materials with a chemical in gaseous state finds a
greater application. Previously formaldehyde was widely used, but at present
ethylene oxide is the only compound of outstanding importance in pharmaceutical
and medical fields. The materials are subjected to sterilization by using toxic
gasses. The gas penetrates quickly into the material like steam so, the sterilization
is effective. But the chances of explosion and cost factors are to be considered.
Gasses used for sterilization are very poisonous. The commonly used gas
is ethylene oxide with a combination of carbon-dioxide. Carbon dioxide is added to
minimize the chances of an explosion.

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