egyptians used fire for sterilisation greeks burned
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• Egyptians used fire for sterilisation
• Greeks burned sulphur for fumigation
• Advanced techniques
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To Prevent • Transmission of diseases.
• Spoilage of food.
• Contamination of pure cultures in laboratories.
• Interference of unwanted microbes in industrial process.
• Research studies.
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Choice of Anti microbial agents depends on
• Type of microbe
• Stages of growth and number
• Surroundings
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Classification of Sterilization:
Sterilization
Physical methods. Chemical methods.
METHODS/AGENTS OF STERILIZATION
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Physical agents: 6
Sunlight
Drying
Heat
Dry heat: flaming, incineration, hot air
Moist heat: pasteurization, boiling, steam
under pressure.
Filtration: candles, asbestos pads,
membranes
Radiation
Ultrasonic vibrations.
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Sun light:
• Sun light: – Active germicidal effect
due to its content of ultraviolet rays .
– Natural method of sterilisation of water in
tanks, rivers and lakes.
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Drying:
Moisture is essential for growth of bacteria.
Drying in air has deleterious effect on many bacteria.
However, spores are unaffected.
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Heat:
of proteins.
• Most reliable method of sterilization and should be the method
of choice.
Dry Heat & Moist Heat.
The factors influencing sterilization by heat:
• Nature of heat-dry or moist
• Temperature and time
• Number of microorganisms present
• Characteristics of organisms –species, strain, sporing capacity
• Type of material from which organism have to be eliminated.
• Killing effect is due to protein denaturation, oxidative damage
and toxic effect of elevated level of electrolytes.
• Killing effect of moist heat due to denaturation and coagulation
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Thermal Death Time:TDT “Minimum time required to kill a suspension of organisms
at a predetermined temperature in a specified environment.
Thermal death time is inversely proportional to temperature.
TDT is increased in presence of organic substance, proteins, nucleic acid, starch, gelatin , sugar , fats, oils.”
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Heat : • Dry heat:
• 1.Red heat
2.Flaming
3.Incineration
4.Hot air oven
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Dry heat: 1. Red heat:
Materials are
held in the flame
of a bunsen
burner till they
become red hot.
» Inoculating wires or loops
» Tips of forceps
» Needles
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Dry heat: 2. Flaming:
Materials are passed through the
flame of a bunsen burner without allowing them to become red hot.
» Glass slides
» scalpels
» Mouths of culture tubes
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Incineration: • Materials are reduced to
ashes by burning.
• Instrument used was incinerator.
• Soiled dressings
• Animal carcasses
• Bedding
• Pathological material
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12/2/2012 Dr.T.V.Rao MD
Dry-Heat Sterilization
Disadvantages
–Less reliable than autoclaving
–Large temp difference may arise within
device.
–sharp instruments get dulled
–Many materials do not tolerate dry heat
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Hot air oven:
Hot air ovens are electrical devices used in sterilization.
The oven uses dry heat to sterilize articles.
Generally, they can be operated from 50 to 300C .There is a
thermostat controlling the temperature.
This is the most widely used method of sterilization by dry
heat.
Items: glassware, forceps, scissors, scalpels, all-glass syringes,
swabs, liquid paraffin, dusting powder, fats, grease.
(Materials should be properly arranged to allow free
circulation of air )
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Precautions:
Glass wares should be dry.
Oven should not be over loaded.
Articles are to be arranged in a manner to allow free circular
of air.
Door of the Oven should be opened after it cools down
(2Hours).
Temerature (c) Holding time(in
minutes
160 45
170 18
180 7.5
190 drsudeeshs
1.5 [email protected] 17
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Advantages & Disadvantages: They do not require water and there is not much pressure build up
within the oven, unlike an autoclave, making them safer to work with.
Suitable to be use in a laboratory environment.
They are much smaller than autoclaves but can still be as effective.
As they use dry heat instead of moist heat, some organisms like
prions, may not be killed by them every time.
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Moist heat:
Moist heat can be categorized into 3 groups:
Temperature below 100 C
Temperature at 100 C
Temperature above 100 C
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21
Below 100
Pasteurization: Developed by Louis Pasteur to
prevent the spoilage of beverages. Used to
reduce microbes responsible for spoilage of beer, milk, wine, juices, etc.
Classic Method of Pasteurization: Milk was
exposed to 65oC for 30 minutes.
High Temperature Short Time Pasteurization
(HTST): Used today. Milk is exposed to 72oC for 15
seconds.
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• Below 100- Moist heat
Pasteurization-63 for 30minutes
–Used for milk, ice cream, yogurt, and fruit juices
» Heat-tolerant microbes survive
–Pasteurization of milk
» Holder method with cooling 13
» Flash pasteurization 72 for 15-20secs with quick cooling
Non sporing pathogens- mycobacterium, brucelle ,salmonellae
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Inspissation: Inspissation: Fractional sterilization Water jacketed copper box Eg. Lowenstein Jansen medium
Heating at 80-85°C for half an hour daily on three consecutive days
Serum or egg media are sterilized
Mesophilic non sporing bacteria
Replaced by tyndallisation
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Vaccine bath: Heating at 60°C for an hour daily in vaccine bath for several
successive days.
Serum or body fluids can be sterilized by heating at 56°C for an hour daily for several successive days.
Washer disinfectors—71 for 3min.
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A temperature at 100°C
1. Boiling 2. Tyndallisation
3. Steam sterilisation
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1. Boiling for 10 – 30 minutes may kill most of vegetative forms but spores with stand boiling.
2.Tyndallization : Intermittened sterilization: John Tyndall
Steam at 100C for 30 minutes on three successive days
Used for egg , serum and sugar containing media.
3.Steam sterilizer : Arnold sterilizer-Streaming steam
Steam at 100°C for 90 minutes.
Used for media which are decomposed at high temperature.
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Temperatures above 100°C
Autoclave : steam under pressure
Temperature increases with pressure
In a closed system
-Steam above 100°C has a better killing power/penetrating power than dry heat.
-Bacteria are more susceptible to
moist heat.
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Components of autoclave:
• Components of autoclave:
– Consists of vertical or horizontal cylinder of
gunmetal or stainless steel.
– Lid is fastened by screw clamps and rendered air tight by an asbestos washer.
– Lid bears a discharge tap for air and
steam, a pressure gauge and a safety
valve.
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Figure 9.6 Autoclave-overview
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Autoclave: Closed Chamber with High Temperature and
Pressure
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Filtration:
• Useful for substances which get • damaged by heat.
• To sterilize sera, sugars and antibiotic • solutions.
• To obtain bacteria free filtrates of • clinical samples.
• Purification of water.
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Several Types of Filters
Types of filters:
1. Candle filters
2. Asbestos disc filters
3. Sintered glass
filters
4. Membrane filters
5. Air filters
6. Syringe filters
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The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 μm (filtration membranes
are most popular for this purpose).
The filtering Depends on Pore Size
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Candle filters
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Berkefeld Filter:Germany This is made up of diatomaceous earth pressed in the shape of a candle. On the basis of porosity they are variously classified into V (veal) N (normal), W (dense) etc. Mandle filters; American Diatomaceous earth,asbestos and plaster of Paris
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Chambered or porcelain filters (French) This type of filter is made of unglazed porcelain. There are different types of this filter depending on their pore size. The grades are L1, L1a, L2, L3, L5, L7, L11, and L13. L1 is the coarser and L13 is the finest in the order. These filters are used for removing organisms from fluid to obtain bacterial toxin.
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2.Asbestos Filters Inorganic Filter: It consists of pad of porcelain or ground glass mounted in filter flask. It is used in sterilization of serum during media preparation. Eg: Seitz Filter -disposible
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4. Sintered Glass Filter: Fritted glass filters This is made up of finely ground glass which is subsequently fused to make the small particles adhere to each other. This filter is sterilised after use, avoiding temperature extremes.
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Membrane filters They are composed of inert cellulose acetate or poly carbonate. This filters are used in lab & industries for sterilization of fluids. In Microbiology lab, it is used for isolation, enumeration & identification of bacteria from water sample.
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• HEPA filter are used with laminar flow to produce dust & bacterial free air. Similarly, they are used to treat air entering surgical units, pharmaceutical fillings rooms.
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Exhaust HEPA filter
High-velocity
air barrier
viewscreen
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Radiations : • Radiations :
•Ionizing radiations
•Non - Ionizing
radiations
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Forms of Radiation
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–Nonionizing radiation •Wavelengths greater than 1 nm
•Excites electrons, causing them to make
new covalent bonds
– Affects 3-D structure of proteins and nucleic acids
•UV light causes pyrimidine dimers in DNA
•UV light does not penetrate well
•Suitable for disinfecting air, transparent fluids, and surfaces of objects
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Non-Ionising radiation:
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1. Infra red rays
2. Ultraviolet (UV) rays
– Infra red is used for rapid mass
sterilisation of syringes and catheters.
– Ultraviolet radiation is used for disinfecting enclosed areas such as
bacterial laboratory, inoculation hood, laminar flow and operation theatres.
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Ionising radiations:
– Ionizing radiations:
1. X rays
2. Gamma rays
3. Cosmic rays
• Gamma radiation are
commercially used for sterilisation of disposable
items. (cold sterilisation)
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Ultrasonic vibrations • High frequency Sounds beyond the range of human
ear
• When propagated in fluids ,cause the formation of microscopic bubbles or cavities. Need liquid medium
• Appears to boil
• Cavities collapse and send out shock waves.
• Cavitation and Cavitron Device.