Transcript CONTROL OF MICROORGANISMs- NEW

Control of Microorganisms
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The process of sterilization is important in microbiology
to prevent contamination from extraneous organisms
Sterilization is essential :
in hospitals for maintaining asepsis,
in laboratories for ensuring safety from contaminating
organisms.
in food and drug manufacture to prevent microbial
growth and also
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Sterilization is also defined as a process by which an
article, surface or medicine is made free of all
microorganisms either in the vegetative or the sporing
forms.
The sterile object is totally free of viable microorganisms,
spores and other infectious agents.
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Definitions of commonly used Terms:
Disinfection: It is killing, inhibiting or removal of
microorganisms that may cause disease.
Disinfectants :These are agents, usually chemical, used to
carry out disinfection.
Sanitation: It is the reduction in the microbial population
to the level that is considered to be safe by public health
standards.
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Antisepsis: It is the prevention of infection or sepsis by
killing or inhibiting the pathogen growth.
Antiseptics: These are chemical agents applied to tissue.
These are not as toxic as disinfectants.
Germicide: A chemical agent which kills germs; most of
pathogens but not endospores.
Bactericide, Fungicide, Viricide and Algicide: Chemicals
which kills bacteria, fungi, viruses and algae
respectively.
Bacteriostatic and Fungistatic: Chemicals which do not
kill but prevent the growth of bacteria and fungi
respectively.
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Conditions That Affect the Efficiency of Antimicrobial
agents
Destruction and inhibition of microorganisms depend on
the factors:
1.Population Size.
2.Population Composition.
3.Concentration and intensity of an antimicrobial agent
4.Duration of exposure
5.Temperature
6.Local environment
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Methods of Sterilization
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The different physical agents are:
1.Sunlight
2.Drying
3.Heat
4.Filtration
5.Radiation
6.Vibration
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i ) Dry Heat :
a) Flaming: Different materials used in microbiological
laboratories such as forceps, scalpels are passed over the
Bunsen burner for bringing about sterilization before use.
b) Red heat: Inoculation loop or wires are usually made
hot before it can be used for inoculation.
c) Incineration: It is an effective method for rapidly
destroying materials such as surgical dressings, animal
carcasses and any other material used in hospital. It is
usually done in a special device called incinerator.
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d) Hot- Air Sterilization: This is used where it is either
undesirable or unlikely that steam under pressure will
make direct contact with materials to be sterilized. Only
dry articles such as glass wares, bandages, instruments ,
mineral oils, talcum powder may be sterilizes by this
method. The apparatus used for this type of sterilization
may be a special electric or gas oven. Normally for
laboratory glass wares, a 2 hour exposure to a
temperature of 160°C is employed.
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ii) Moist Heat:
a)Temperature below 100°C : This is mainly used in
pasteurization and temperature applied is either 63°C for
30 minutes ( holder’s method) or 72°C for 15 seconds (
flash Method). By this process all non sporing pathogenic
organisms present in milk are destroyed.
b) Temperature at 100°C:
Boiling: The vegetative forms are killed at 90-100°C but
spore forming bacteria require considerable periods of
boiling. Sterilization may be promoted by the addition of
2% sodium carbonate to the water. In cases where boiling
is considered to be adequate , the material should be
immersed in water and boiled for a period of 10-30
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minutes.
c) Steam at atmospheric Pressure ( 100°C ) : An
atmosphere of free steam is used to sterilize culture
media which may decomposed if subjected to high
temperatures. Special devices such as Koch’s steam
sterilizer or Arnold steam sterilizer can be used for
sterilization. One exposure of 9 minutes usually ensures
complete sterilization but for media containing sugars
and gelatin, an exposure of 100°C for 20 minutes on
three successive days is used. This is known as Fraction
Sterilization or Tyndallization. The principle is that the
first exposure kills all vegetative bacteria and spores
present will germinate and be killed on subsequent
exposures.
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Arnold steam sterilizer
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d) Temperature above 100°C: This is commonly
obtained by holding the steam under pressure in an
instrument called Autoclave. Steam under pressure
provides temperatures above those obtainable by
boiling
Temperatures of steam under Pressure
Steam Pressure,
Lb/in2
0
5
10
15
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Temperature in °C
100
109
115
121.5
126.5
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In routine use of autoclave, the temperature of 121.5°C at
15 lb / in2 for 15 minutes is employed. This period is referred
to as holding Period during which effective sterilization
takes place. Many media, solutions, discarded cultures,
and contaminated materials are regularly sterilizes with
autoclave.
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Sterility checks
Sterility checks are done by using certain indicators.
1). Chemical indicator-this includes Bowie _Dick tape or
Browne’s tubes and
2) Biological indicators- this contains spores strip of
Bacillus stearothermophilus.
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Browne’s tubes
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Filtration
This is a method adopted to remove organisms from eat
labile liquids. This is used for antibiotic solutions, serum
preparations and carbohydrate solutions used in culture
media. By this technique, one can obtain bacteria free
filtrates. This method is also useful where separation of
microorganisms is required which less in specimens.
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Types of Filters
a) Earthenware candle filter. Ex: Berkefeld and
Chamberland filter Candle Type Filters: Berkefeld are
made up of diatomatious earth or Kieselgarh. These
filters are available in different pore sizes. The filters are
in the form of hollow candle open at one end.
b) Asbestos disc filter. Ex: Seitz filter: Thse are made of
asbestos and chemically composed of magnesium
silicate. These filters are usually in the forms of discs and
are available in different pore sizes.
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Berkefeld
Sinter glass filter
Seitz filter
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c) Sinter glass filter: These are made of finely ground glass
which are then made to fuse. They are also available in
different pore sizes.
d) Collodion or Membrane filter: These are usually made
of nitrocellulose and have precise pore sizes from 25μm
0.025 μm. Advantages of these filters are, they relatively
inexpensive ,do not clog easily and can filter large
volumes of fluid rapidly. They can be autoclaved.
Membrane filters are used to sterilize materials likely to
be damaged by heat sterilization. Materials like special
nutrients that are added to culture media, sera, drugs,
and vitamins are regularly filtered through membrane
filters.
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Membrane filters
The Isopore Filter
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e) High-efficiency particulate air filters ( HEPA ): These
are used in the ventilation systems of areas where
microbial control is especially important ,such as
operating rooms, burn units, and laminar flow transfer
units in laboratories. These filters remove all organisms
larger than 0.3 μm in diameter.
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High-efficiency particulate air filters ( HEPA )
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Radiation
Four types of radiation-ultraviolet light, ionizing
radiation, microwave radiation and in some case strong
visible light can be used to control microorganisms.
i) Ultraviolet ( UV ) light: UV light consists of wavelength
between 40 and 390nm,but wave length in the 200 nm
range are most effective in killing microorganisms by
damaging DNA and proteins.
UV light does not penetrate glass, cloth, paper or most
other materials and it does not reach corners and
crevices of laboratory surfaces and materials thus
limiting its use.
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Electromagnetic Spectrum
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ii) Ionizing Radiation: X rays which have wave length
of 0.1 to 40nm and gamma rays which have even
shorter wavelengths are forms of ionizing radiation.
These cause ionization of molecules when they hit them
resulting in death of microorganisms and viruses.
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X-RAY DAMAGE OF DNA
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UV RADIATION DAMAGE OF DNA
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iii) Microwave Radiation: This radiation fall at the
longer wavelength end of electromagnetic spectrum
with the wave length of approximately 1mm to 1 m.
Microwave oven frequencies are turned to match
energy level in water molecules. In the liquid state,
water molecules quickly absorb the microwave energy
and then release it to surrounding materials as heat.
Specialized microwave oven is available now that can
be used to sterilize the medium in just 10 minutes.
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iv) Strong visible light: It has bactericidal property
because of UV rays in sunlight. Strong visible light which
has wave length from 400 to 700 nm can oxidize light
sensitive molecules
such as riboflavin
and
porphyrins.The fluorescent dyes eosin and methylene
blue can be denature proteins on the presence strong
light. Therefore the combination of a dye and strong
light can be used to rid materials of both bacteria and
viruses.
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Vibrations: Sonic or sound waves in the audible range
can destroy bacteria if waves are of sufficient intensity.
Ultrasonic waves or waves with frequencies above
15,000 cycles per second can cause bacteria to cavitate
( formation of partial vacuum in a liquid). Bacteria so
treated disintegrate and their proteins are denatured.
The disruption of cells by sound waves is called
sonication. Neither sonic nor ultrasonic waves are a
practical means of sterilization.
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Refrigeration, Freezing and Freeze –Drying: Cold
temperatures retard the growth of microorganisms by
slowing the rate of enzyme –controlled reactions but do
not kill microbes.
Refrigeration of foods at 5°C prevents spoilage from
microorganisms. However, the storage should be
limited to a few days as some bacteria and fungi
continue to grow at this temperature.
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Osmotic pressure: High concentration of salt, sugar or
other substances creates a hyper osmotic medium which
draws water from microorganism by osmosis. Plasmolysis
or loss of water severely interfere with cell function and
eventually leads to cell death. The use of sugar in jellies ,
jams and syrups or salt solution in curing meat and
making pickles plasmolyzes most organisms present and
prevents growth of new organisms. A few halophilic
organisms grow even in such conditions and spoil the
food stuff.
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