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Sterilization and Aseptic Techniques-2023
Sterilization and Aseptic Techniques-2023
TECHNIQUES
• A set of procedure that are performed
under sterile condition
• Comprises all aspects of environmental
control, personal hygiene, equipment and
media sterilization, and associated quality
Aseptic control procedures
• Needed to ensure that a procedure is,
Techniques indeed, performed with aseptic, non-
contaminating technique.
• Designed to provide a barrier between the
microorganisms in the environment and
the sterile cell culture
• A process which removes all kinds of
living organisms including bacterial
spores
• Sterilization methods vary in their
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• Sterility - absence of any
contaminating organism apart from the
cells of interest.
• Bacteria, mycoplasma, yeast, and
fungal spores may be introduced via
the operator, the atmosphere, work
Aseptic surfaces, solutions, and many other
sources.
Techniques • Aims to exclude contamination by
establishing a strict code of practice
in cell Culture and ensuring that everyone using the
facility adheres to it
• Essential tools in research,
diagnostics and manufacturing of
various sterile biological products.
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What Contamination can do?
Affect the cell derived Compromise safety of
products e.g yield and cell derived
stability therapeutic products
In worst case,
complete destruction
of cell culture
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Elements of Aseptic Techniques
Sterile • Flaming
• Handling Bottles and Flasks
Handling • Pipetting
• Pouring
Work surface should be
swabbed with 70% alcohol
before and after work.
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• Microorganisms also vary with regard to their
sensitivity like
Bacterial endospores - resistant to heat
Prions - resistant to heat, irradiation and
Microbial detergents
Non-enveloped viruses - resistant to
Sensitivity organic solvents and detergents
Mycoplasma and viruses are not
removed by conventional 0.2µ sterilizing
filters
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Sterilization • Irradiation
• Filtration
Methods • Chemical Disinfection
Methods of sterilization
• Autoclaving (using steam under pressure) is a
rapid method for sterilizing almost anything
except heat-labile substances.
• A temperature higher than the boiling point of
Autoclaving water inside the autoclave (super-heating of
liquids) is achieved because the system is under
pressure
• Typical autoclaving conditions of 121◦C (250◦F)
for 15 minutes at 103 kPa (15 psi) are sufficient to
kill virtually all forms of life, including bacterial
endospores, and will inactivate viruses.
Oven Sterilization
Selected goods such as glassware, metal, and other objects that will not melt at temperatures
between 121◦C and 170◦C can be sterilized with dry heat (in a hot-air oven).
As the heat takes much longer to be transferred to the organism, a temperature of 160◦C for ≥2
hr or 170◦C for 1 hr is routinely used.
Cannot be applied for liquids, rubber, or any plastic objects.
It can be used for powders and other heat-stable items that are adversely affected by steam,
and it does not cause rusting of steel objects.
It is also a method of choice for the treatment of glassware used for work with ribonucleic acid
(RNA), since the process of baking not only kills organisms, but also inactivates any residual
RNA-degrading enzymes (RNases).
Working with Open Flame
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Filtration
• Many laminar flow hoods are equipped with ultraviolet (UV) lights, which
can help reduce contamination by inducing DNA damage to potential
contaminants.
• Such light is also harmful to laboratory workers.
• Never look directly at a UV light, as that can cause eye damage.
• The UV light can also cause skin burns (sun burns) to anyone in the room.
• Consequently, UV lights should always be turned off whenever the room is
occupied
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Switch on the laminar Cell cultures which Possibly keep Never use the same
flow cabinet 20 are frequently used cultures free of media bottles for
minutes prior to start should be sub- antibiotics in order to different cell lines.
working cultured and stored be able to recognized
as duplicate strains. the contamination.