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Sterilization:
The use of chemical or physical procedure that completely destroy all
forms of microbial life including their spores
Disinfectant:
The chemical substance that kills infectious microbes but not their spores
Antiseptic:
Chemical agent usually applied to surface of body that prevent microbes
from multiplying
It may do this either by killing the microorganisms or by inhibiting their
growth & metabolic activity
Sanitizer:
The process by which microbial contaminant is brought to safe level (the
process is refers to cleaning of inanimate objects)
Biostatic:
The substance that prevents the growth of microorganisms but does not
necessarily kill them
Biocidal:
The substance that kills all living microorganisms including spores
Sterilization methods are of two types
A. Physical methods
B. Chemical methods
A. Physical methods are of many types such as
1. Heat (moist or dry)
2. Radiation (ionizing & nonionizing)
3. Filtration
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B. Chemical methods
1. Agent acting on cell membrane
2. Agent that denatures protein
3. Heavy metals
4. Oxidizing agent
5. Dyes
6. alkylating agents
Autoclaving (Moist heat method)
• Autoclave is a device that uses steam to sterilize
equipment and other objects. This means that all
bacteria, viruses, fungi, and spores are inactivated.
• Sterilization done at 121⁰C at 15 psi (pounds per
square inch) for 15 min
• Autoclave consist of a steel chamber capable of with
standing more than 1 atm pressure
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At the high temperature and high pressure denaturation
of cell protein occurs as well as cell membrane become
more fluid and leakage of cell internal material take
place leads to killing of microbes
Autoclave is work on the principle of steem under
pressure and it works just like our pressure cooker
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Culture media, instruments, dressings, intravenous
equipment, solutions, syringes, glassware, numerous
other items that can withstand high temperature &
pressure
Surgical and clinical specimens and waste can be
disposed after autoclaving
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Heat sensitive items such as vaccines, antibiotics, etc
cannot sterilized by autoclave
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Heating at 100⁰C in boiling water for 15 min is
sufficient to kill vegetative forms of contaminating
microorganisms and pathogens
Boiling disinfects material at home for sanitizing
certain material like baby bottles & rubber nipples
Shortcoming of this method is that it does not destroy
many of bacterial spores and some of viruses
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Generally employed for milk, wine, fruit juices
Greater significance of pasteurization is to prevent
milk borne disease such as tuberculosis, brucellosis, Q
fever, etc
It is done at 62.9⁰C for 30 min or 71.6 ⁰C for 15
seconds
Milk can be pasteurized in two ways. In the older
method the milk is held at 63°C for 30 minutes.
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Large quantities of milk are now usually subjected to
flash pasteurization or high-temperature short-term
(HTST) pasteurization, which consists of quick
heating to about 72°C for 15 seconds, then rapid
cooling.
The dairy industry also sometimes uses ultrahightemperature (UHT) sterilization. Milk and milk
products are heated at 140 to 150°C for 1 to 3
seconds. UHT-processed milk does not require
refrigeration and can be stored at room temperature
for about 2 months without flavor changes.
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The Direct Flame:
One of the simplest methods of dry heat sterilization is direct flaming.
Mode of Action :
Dry heat probably does most of its damage by oxidizing molecules. A simple
analogy is the slow charring of paper in a heated oven, even when the
temperature remains below the ignition point of paper.
Procedure:
To effectively sterilize the inoculating loop, one must heat the wire to a red
glow. The flame of the Bunsen burner is employed for a few seconds to
sterilize the bacteriology loop .
Applications:
Most rapid sterilization method, for dispose of contaminated paper cups,
bags, and dressings, is the use of a direct flame in the process of incineration.
It is still common practice to incinerate the carcasses of cattle that have died
of anthrax and to put the contaminated field to the torch because anthrax
spores cannot adequately be destroyed by other means.
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Mode of Action:
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.
Procedure:
Items to be sterilized by this procedure are placed in an
oven. Generally, a temperature of about 170oC
maintained for nearly 2 hours ensures sterilization.
Hot air oven
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Disadvantages:
The hot-air oven utilizes radiating dry heat for
sterilization. This type of energy does not penetrate
materials easily, and therefore, long period of exposure to
high temperatures are necessary. For example, at a
temperature of 160oC, a period of 2 hours is required for
the destruction of bacterial spores.
Uses of Hot Air Ovens:
The hot-air method is useful for sterilizing dry powders
and water-free oily substances, as well as for many types
of glassware, such as pipettes, flasks, and syringes. Dry
heat does not corrode sharp instruments as steam often
does.
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Although our emphasis is on the destruction of microorganisms,
often the most convenient control technique is to inhibit their
growth and reproduction by the use of either freezing or
refrigeration.
This approach is particularly important in food microbiology
Refrigeration:
At low temperature of (0-7⁰C) the metabolic rate of most
microbes is reduced so they can’t reproduce
Freezing:
Very low temperature does not kill microorganisms rather it
inhibits their growth by slowing enzymatic reaction
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Freezing is done at only temperature below 0⁰C so
cellular water starts to freeze & create an environment
that is not conductive to metabolic activities
If cells are frozen rapidly at low temperature of -80⁰C
in viable form in appropriate medium, however they
can be preserved indefinitely
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Electromagnetic radiation classified according to its
wavelength
The energy content of radiation is inversely related
to the wavelength i.e. shorter the wavelength greater
the energy content
Radiation are of two type
Non-ionizing radiation
Ionizing radiation
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Non ionizing radiation has wavelength longer than
that of ionizing radiation, Ex. UV rays
UV radiation produces chemical modification of
nucleotide , creating cross-linking between pairs of
thymine molecules and T-T dimmers forms in cell
This lead misreading of genetic code, resulting in
mutation that impair vital function of the organism
& death occurs
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Used in hospital operating room, aseptic filling room,
in pharmaceutical industries where sterile products are
being dispensed in to vials or ampoules, food & dairy
industry for treatment of contaminated surfaces
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low penetration power, so organisms to be killed
directly gets exposed
UV rays can damage retina of eyes, burns skin & cause
cancer too
Effectiveness decreases as the distance from radiation
sources increases
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The wavelength is shorter than nonionizing
radiation
It is called ionizing means , it has sufficient energy
to cause ionization of molecules
Ex is Gamma & X rays
Dislodge electrons from atoms and form ions.
Cause mutations in DNA and produce peroxides.
Used to sterilize pharmaceuticals and disposable
medical supplies. Food industry is interested in
using ionizing radiation.
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High penetrative power
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No appreciable increase in the temperature –
COLD STERILISATION
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Sterilise plastics Syringes, catheters, grease
fabrics metal foils
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Disadvantages: Penetrates human
tissues. May cause genetic mutations
in humans.
Microbial Control
Methods
Physical Agents
Chemical Agents
Mechanical Removal
Methods
Mechanical Removal
Methods
Filtration
Air
Liquids
Disinfection
Sterilization
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Filtration involves the passage of liquid or gas through
screen like material that has pores small enough to
retain microorganisms of certain size
Filtration is used for sterilizing substances that are
sensitive to heat, like enzyme solution, intravenous
solution, bacterial toxins, cell extracts etc
For liquid filtration different filters are used, sintered
glass filter, asbestos (seitz) filter, membrane filter etc
Filtration helps to remove bacteria from heat
labile liquids such as sera and solutions of
sugar, Antibiotics.
The following filters are used
Candle filters
Asbestos filters
Sintered glass filter
Membrane filters
CANDLE FILTER
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Widely used for purification of water
Two types
(a) Unglazed ceramic filter – Chamberland
filter
(b) Diatomaceous earth filters – Berkefeld
filter
SEITZ FILTER
ASBESTOS DISCS
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Disposable single use discs
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High adsorbing tendency
Carcinogenic
Eg: Seitz filter
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SINTERED GLASS FILTER
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Prepared by heat fusing powdered glass
particles of graded size
Cleaned easily, brittle, expensive.
MEMBRANE FILTER
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Made of cellulose esters or other polymers
Uses
 Water purification & analysis
 Sterilization & sterility testing
 Preparation of solutions for parenteral use
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Filtering air to reduced microbial contaminants has
found significant application in hospital operating room
High efficiency particulate air (HEPA) filter is widely
used which has efficiency of 99.97% for removal of
particles that are 0.3µm or more in diameter
One of the application of HEPA filter in formation of
nonturbulant or laminar air flow (LAF) for maximum
contamination control in given area
Application in pharmaceutical industry, in hospital
specially where patient undergoing bone marrow
transplants , it protects leukemic patient from microbial
contamination by environmental organisms
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Another application is in microbiology laboratory for
media preparation, tissue culture preparation, personal
protection from pathogenic organisms
Chemical agents play important role in controlling
microbes and they classified on the basis of their effect
on microbes
• They may microbiostatic or microbiocidal
• All disinfectant has its mode of action to control or kill
microbes, which may be of as follow
----- Lysis of cell wall
----- Action on cytoplasmic membrane
----- Action on cellular proteins
----- Impairing of function of nucleic acids
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Phenol
Alcohol
Halogens
Heavy metals
detergents
acid
Alkalies
Aldehydes
Gaseous chemical sterilant (ethylene oxide)
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Phenol was the first widely used antiseptic and disinfectant.
In 1867 Joseph Lister employed it to reduce the risk of infection
during operations.
It disrupts cell membrane resulting in leakage of cytoplasmic
contents & also precipitates cellular proteins and finally cell gets
died
0.1-2% concentration is effective
They have some real advantages as disinfectants: phenolics are
tuberculocidal, effective in the presence of organic material, and
remain active on surfaces long after application.
However, they do have a disagreeable odor and can cause skin
irritation.
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Alcohols are among the most widely used disinfectants
and antiseptics.
They are bactericidal and fungicidal but not sporicidal;
some lipid-containing viruses are also destroyed.
The two most popular alcohol germicides are ethanol and
isopropanol, usually used in about 70 to 80%
concentration.
They act by denaturing proteins and possibly by
dissolving membrane lipids.
A 10 to 15 minute soaking is sufficient to disinfect
thermometers and small instruments
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Chlorine, Iodine, Bromine is from halogen family and are strong
oxidizing agents which destruct cell’s vital molecules
Chlorine compounds are widely used for control of microbes in
environment, majority for drinking water treatment & in
swimming pools
It may be applied as chlorine gas, sodium hypochlorite, or
calcium hypochlorite, all of which yield hypochlorous acid
(HClO) and then atomic oxygen.
The result is oxidation of cellular materials and destruction of
vegetative bacteria and fungi, although not spores.
Cl2 + H2O
HCl + HClO
Ca(OCl)2 + 2H2O
Ca(OH)2 + 2HClO
HClO
HCl + O
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Iodine is used as a skin antiseptic and kills by
oxidizing cell constituents and iodinating cell
proteins.
At higher concentrations, it may even kill
some spores.
Iodine often has been applied as tincture of
iodine, 2% or more iodine in a water-ethanol
solution of potassium iodide.
Although it is an effective antiseptic, the skin
may be damaged, a stain is left, and iodine
allergies can result.
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Mercury, lead, zinc, silver, copper are heavy metal
Use of silver or copper utensil for storage of drinking
water by ancient people represent application of inhibitory
action of heavy metals to microbes
Mode of action of heavy metal is to inactivate enzymes by
binding to -SH groups
Widely employed in cosmetics, antiseptic creams, drinking
water tanks etc
1% silver nitrate for prevention of ophthalmia neonatorum
(Neisseria gonorrhoeae)
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Formaldehyde (37%) is known as formalin can
converted to gaseous form
aqueous solution of formaldehyde has application
in preservation of biological specimens, surgical
instruments, to fumigate microbiological laboratory,
hospital clinics etc
Disadvantage of using formaldehyde is that it is
noxious, irritative to human eyes & tissue
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At room temperature ethylene oxide is vaporizes
rapidly in air and it kills vegetative cells as well as
spores of fungi and viruses
It has high penetrative ability and pass through
packages and bundles of cloths and plastic
containers for sterilization purposes
Its practical application has limitation due to slow of
antimicrobial action & overnight treatment is
needed to kill bacteria
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Application of ethylene oxide is in
sterilization of delicate medical instruments
like heart pumps, respirometer, intravenous
catheters, plastic materials, etc