Transcript Sterilization_and_Disinfection_F

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Killing or destroying All forms of living
microorganisms from objects. (bacteria and
their spores).
Sterilization is an absolute term means germ free objects.
Disinfection:
◦ Is removing or reducing the number of the
pathogenic microorganisms from objects.
(Not absolute)
◦ Unable to destroy spores and some could
not kill non enveloped viruses.
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Physical methods.
◦ Chemical methods.
◦ Physical methods.
◦ Are more commonly used, simple,
economical and reliable.
prevention of microbial growth:
important in:
• Food industry.
• Health care: nosocomial infections.
•Drinking water distribution systems
• other industrial processes: e.g., biofouling of oil
pipeliness
•The objective is to render the articles non infectious.
Sterilization
by
Physical
methods
Heat
Radiation
Filtration
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Heat:
◦ Exposure of the objects to heat will kills microbes
by coagulation of protein, denaturation of
enzymes and oxidation.
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Filtration:
◦ Sterilization through removing of microbes from
fluids by exposing them to small size filter. This
is used for heat sensitive fluids like serum,
sugars and urea.
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Radiation:
◦ Exposure to radiation: This causes denaturation
of proteins and enzymes.
Heat
Dry heat
Moist heat
Dry heat
Red hot
Flaming
Hot air
oven
Incineration
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Exposure of wires and forceps to the Bunsen
flame until it becomes red hot, then cooled
down and used.
Used for wire loop, straight wire, forceps,
scalpels and metal rods.
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Slowly passing of objects to the Bunsen flame
to reduce the number of microorganisms.
The Bunsen flame give partially sterile area
around it. This is used for sterilization of the
mouth of bottle, flasks, containers and test
tubes.
it is also used for the preparation of smears,
cultivation of bacteria ands. subculture
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This is a metal chamber (instruments) consists
of a heater, thermometer, thermostat,
perforated shelves, door and timer.
It kills microbes by oxidation-reduction.
There are certain thermal death points and
Thermal death times for the articles to be
sterilized:
◦ 160C for 60 min.
◦ 170C for 40 min.
◦ 180C for 30 min.
oven for dry heat sterilization
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Bowei Dick test (Adhesive tabe).
Browne’s tube No 3. (Red ----- green).
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Is treating of an objects to heating over 250
until they become black.
Done for used equipment.
Moist heat
Less than 100C
At 100C
Above 100C
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Pasteurization of milk:
◦ Holding method (65C for 30 min)
◦ Flash method (72C for 15 - 20 sec)
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Inspeciation:
◦ Heating at 80C for 30 - 1hrs until coagulation of
protein.
◦ Used for preparation of Dorset egg medium and L.J
medium for TB.
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Preparation of vaccine:
◦ By heating at 56C for 30-60 min.
pasteurization:
• 71ºC for 15 seconds (high
temperature short time, HTST,
method)
• significantly reduces microbial
population size
• does not sterilize
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Steaming (Koch steamer):
◦ Single exposure of the microbe to steam at 100C
for 90 min (Shelves over the boiler).
◦ Tyndalization:
 Steaming at 100C for 30 min for 3 successive days.
 1st day kill vegetative bacteria and germinate sporulated
one ---- Put on the bench.
 2nd day kills all vegetative bacteria ---- Put on the bench.
 3rd day insure complete sterilization.
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Boiling (Inside the boiler,no shelves)
◦ At 100C for 30 min.
It is a double jacketed steal cylinder or
chamber supplied with:
1. Heater, water and pressure.
2. Has a thermometer and pressure supplier.
3. Control valves for steam, water and
pressure
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flow of steam through an autoclave:
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Depends on steam and pressure.
◦ Steam is a hot sticky air able to penetrate
through things.
◦ Pressure will rise the temperature from 100C to
121C
Kills microbes and their spores by
coagulation of protein and denaturation of
enzymes.
Make complete killing of bacteria, their
spores, fungi and their spores, parasites
and viruses including Envelop and non
Envelop virus.
Thermal death point and thermal death
time:
◦ 121C (15 bound or 1.1 bar) for 15 min.
◦ Flash autoclaving at 134C for 4-5min.
an autoclave:
• steam under pressure
enables temperature to reach
121ºC
• sufficient to destroy
endospores as well as
vegetative cells
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Sterilization by mechanical removal of
pathogenic microbes by passing through
filter.
Used for sterilization of heat sensitive fluids
like serum, glucose, urea, and Amino acids.
Different filtration units is used according
to the amount of filtrate and pores of the
filter:
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Seitz filter ---- use asbestos
Chamberland filter ------ use ceramic
Sintered filter ----- use glass filter.
Milipore or membrane filter ---- use filter paper.
filter sterilization:
• removal of particles from
liquids
• 0.2 μm pore size usual for
sterilization
5 μm dia pores
• filtration also used to separate
or distinguish organisms
based on size
depth filter:
• fibrous sheet or mat of randomly overlapping fibers of different
substances (paper, glass)
• can use as pre-filter to remove suspended particles
• “trapping action”
conventional membrane filter:
• polymeric compounds such as cellulose acetate or cellulose nitrate
• pore diameter variable during production
• “sieve-like action”
Nucleopore filter:
• thin, polycarbonate films (~10 mm thick)
• pores formed by chemical “etching”
• consistent pore size
• useful for microscopy – filtered material is in a single plane on surface
lab-scale membrane filtration:
disposable, presterilized, assembled membrane filter units
syringe filter: small volumes
system for larger volumes
◦ Sterilization by radiation kills microbes by causing
mutation to the cellular protein and disrupting
cellular elements.
◦ Used for plastic syringes, disposable plastic and
dental e
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2 types:
◦ Ionizing radiation:
 Depends on ionization of water which forms highly reactive
hydroxyl group react with cellular components specially
DNA causing mutation.
 E.g.: X-rays, Gamma radiation and high energy electron
beams.
 Have a wavelength less than 1 nm.
◦ Non ionizing radiation:
 using short wave length rays like Ultraviolet with a
wavelength of 260 nm.
 Causing mutation by making thymine dimers.
 Used for sterilization of plastic and surgical room
ultraviolet (UV) radiation:
Chemical methods of sterilization
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Disinfection:
◦ Is removing of pathogenic microorganism or
reducing their number on the exposed area.
◦ Unable to destroy spores and some could not kill
non envelop viruses.
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Factors affects disinfection action:
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Type of disinfecant used.
Concentration of disinfectant.
Type of microorganism.
Number of microorganism.
Time of exposure.
Temperature.
Presence of organic compound.
examples of modes of action:
• alcohol - lipid solvent, protein denaturant
• hydrogen peroxide (H2O2) – oxidizing agent
• triclosan (a phenolic) – disrupts cell membrane
• chlorine gas – oxidizing agent
• ethylene oxide – alkylating agent
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E.g.: Phenol crystal, Dittol, Lysol, Cresol.
Injuring lipid containing plasma membrane
leads to leakage of cellular contents.
Remain active in the presence of organic
materials, stable and persist for long period
of time.
Suitable for disinfecting pus, blood and
sputum.
Active against G+ve, G-ve, Mycobacterium
& viruses.
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Contains 2 groups of phenol.
Hexaclorophenol used for surgical and
microbial control, Excessive use for infant
could leads to neurologic damage.
Triclosan found in antimicrobial soaps,
inhibits an enzyme needed for synthesis of
fatty acid which affects the integrity of
plasma membrane.
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E.g.: Chlorohexidine.
Broad spectrum activity as disinfectant of skin
and mucus membrane due to it is ability to
bind to mucus membrane.
Cause injury to plasma membrane.
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Iodine have broad spectrum activity to
many bacteria, spores, fungi and some
viruses, it binds to certain amino acids of
enzyme and proteins.
Iodophore (Betadine and Isodine) is non
pigmented iodine comprise from iodine +
organic materials to release the iodine
slowly. Used for skin disinfection and
wounds.
Chlorine in shape of gas or solution
combined with water to give HOCl
(Oxidizing agents). Used for swimming
pools, drinking water and sewage.
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Causes denaturation of proteins, disrupt
membrane and dissolve lipids.
They able to act and Evaporate (Volatile).
Used as skin disinfectant and in vein puncture
in a concentration of 70%.
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Silver, mercury and copper
Causes denaturation of proteins when bind
to it.
Silver nitrate is used as a droper to avoid
opthalmia neontam by N. gonorrhoea.
Silver sulfadiazine (Flamazine) is used in
treating infection associated with burns.
Copper sulfate is used to destroy green
algae (effective in one part/million of
water).
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Gention violet, Crystal violet and Eosine are
very effective antiseptic.
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Soap and other detergent make mechanical
remove of microbe by scrubbing of dead
tissue so reduce their number.
Quaternary ammonium compound are
positively charged molecules kills G+ve
bacteria. Affects plasma membrane and
changes cell permiability. E.g.: Citerimide.
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E.g.: Formaldehyde and Gultraldehyde.
Inactivate proteins by covalent cross-links
with COOH, NH, OH and SH.
Used for disinfect hospital instruments,
benches and room. E.g.: Formaldehyde and
Gultraldehyde.
Inactivate proteins by covalent cross-links
with COOH, NH, OH and SH.
Used for disinfect hospital instruments,
benches and room.
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Used to sterilize plastics in closed chamber
similar to autoclave.
Denaturate proteins.
Bacteriocidal, fungicidal and kills viruses.
Used at high temperature 60C for 4-24 hrs.
Carcinogenic.
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H2O2 is an oxidizing agents kills many
microbes especially who fail to produce
catalase enzymes (Anaerobic bacteria). Used
as disinfectant for gas gangrene.
Ozone (O3) also toxic for microbes and used
instead of chlorine in treating drinking water.
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In-use test:
◦ Used to determine the proper disinfectant concentration to
be used and to check if the used disinfectant is working
properly or not:
◦ Take 1ml from used disinfectant to 9 ml of nutrient broth.
Immediately transfer 0.02ml into 10 different area of well
dried nutrient agar.
◦ Incubate one plate at 37C for 3 days and the other at R.T
for 7 days.
◦ Growth in more than5drops- Failure of Disinfectant
◦ No growth ------- good disinfectant
◦ Growth in less than 5 drop --- success but needs
increasing the concentration.
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Phenol co-efficient test:
◦ Serial 2 fold dilution of both tested disinfectant and
phenol are inoculated with Salmonella typhi and
immediately re-subcultured on agar medium at 0
time, after 5 minutes and after 10 minutes.
◦ Good disinfectant gives real number after dividing
the MIC of the disinfectant against the phenol.
◦ 4/2= 2 Good
◦ 2/4= ½ Bad disinfectant.
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Time killing assay:
◦ Used to determine the efficiency of disinfectant
against bacteria during the times.
◦ Make serial dilution of disinfectant and subculture
the commonly isolated bacteria and immediate
culture on agar medium at 0 time, after 5 min and
10 min.
◦ Determine the last dilution of disinfectant that able
to kill M.O in a shortest time.
◦ Use 4X this concentration in your lab.