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STERILIZATION
AND ASEPTIC Dr. Nida Dastagir

TECHNIQUES
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• 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
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 • A process which removes all kinds of
living organisms including bacterial
spores
• Sterilization methods vary in their

Sterilization severity and the properties of the

items to be sterilized
• Microorganisms also vary in
sensitivity/suitability for different
methods of inactivation or removal

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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

Problems and artifacts

Contamination of
in research and
other cultures
diagnostic studies

In worst case,
complete destruction
of cell culture

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Elements of Aseptic Techniques

Quiet Area Work Personal Reagents Cultures

Surface Hygiene and Media
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Quiet Area
• A separate sterile room - if there is no laminar flow cabinet.
• A quiet corner of the laboratory - if not possible to work in separate room.
• With laminar flow, an area should be selected that is free from air currents from doors,
windows, etc.
• No through traffic and no equipment that generates air currents
• Air conditioners should be positioned so that air currents do not compromise the functioning of
the hood.
• Restricted activity to tissue culture
• Microbiological cultures should be excluded from the tissue culture area.
• Area - clean and free of dust and should not contain equipment other than that connected with
tissue culture.
• Nonsterile activities, such as sample processing, staining, or extractions, should be carried out
elsewhere
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Work Surface

• Work surface should be completely clear.

• Surface swabbing - 70% alcohol
• Put only those items on the surface which are particularly required.
• Arrange work area- easy access to all items.
• Work on clear surface – there should be no obstruction between central work
and HEPA filter - better to work under laminar flow hood.
• Spillage - quickly mop it.
• Clear and clean the work surface after finishing the work
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Hands should be washed properly

before and after working in cell
culture.

Proper PPEs should be worn to

Personal avoid shedding of skin and dust from
clothes.
Hygiene
Do not talk when working aseptically.

Do not work in tissue culture lab if a

person is suffering from any
infection.
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• Commercial reagents and media – already

Reagents sterilized - but they can become

contaminated while handling.
and Media • Sterilize media and reagents using
sterilization procedure (e.g., autoclave,
sterile filter).
• Media and reagent bottles should be
decontaminated with 70% alcohols when
bottles are removed from water bath or
refrigerator.
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Cultures

• Imported Cultures - high risk, because they may have been

contaminated either at the source or in transit.
• Imported cell lines should be immediately quarantined upon arrival.
• New cell lines should be handled separately from the rest of stocks
and kept free of antibiotics until they are shown to be uncontaminated
• Antibiotics should not be used routinely as they may suppress, but not
eliminate, some contaminations
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• Swabbing
• Capping

Sterile • Flaming
• Handling Bottles and Flasks

Handling • Pipetting
• Pouring
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Work surface should be

swabbed with 70% alcohol
before and after work.

Swabbing All bottles and tubes should be

swabbed when remove from
water bath.

Plates and flasks should be

swabbed when removed from
incubator.
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Pouring

• Do not pour from one sterile

container into another
• Major risk - generation of a bridge
of liquid between the outside of
the bottle and the inside -
contamination
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• When working on an open bench flame glass pipettes

and the necks of bottles and screw caps before and
after opening and closing a bottle
Flaming • Work close to the flame
• Flaming is not advisable when you are working in a
laminar-flow hood, as it disrupts the laminar flow,
which, in turn, compromises both the sterility of the
hood and its containment of biohazardous material
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Handling Bottles and Flasks

• Bottles and flask should not be kept open.

• Culture flasks should be laid down horizontally
when open and, like bottles, held at an angle
during manipulations.
• In laminar flow, bottles can be left open and
vertical, but do not let your hands or any other
items come between an open vessel or sterile
pipette and the HEPA air filter.
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Pipetting
Standard glass or disposable plastic pipettes are the easiest way to manipulate
liquids.
Syringes may be used sometimes but they produce high shearing forces when
you are dispensing cells, and the practice also increases the risk of self-
inoculation.
Mouth pipetting should be avoided, as it has been shown to be a contributory
factor in mycoplasma contamination and may introduce an element of hazard to
the operator.
Disposable plastic pipettes should be double wrapped and removed from their
outer wrapping before being placed in a hood.

Always pipettes with cotton plugged should be used.

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Capping

• Deep screw caps are preferred to stoppers.

• Caps should be washed carefully from behind the inner rubber liner.
• Aluminum foil should be used to cover the screw cap to protect the neck of
the bottle from sedimentary dust
• There are two main types of caps that are in common use for glass bottles:
• (1) Aluminum or phenolic plastic caps with synthetic rubber or silicone
liners
• (2) Wadless polypropylene caps that are reusable (Duran); these caps are
deeply shrouded and have ring inserts for better sealing
 •Autoclaving - preferred to filtration - since it is
cheaper.
•Autoclaving needs less labor
Sterilization in •Autoclaving is uniformly effective
•Most of the media are available pre-sterilized
Cell Culture commercially.
•Heat labile constituents like serum, trypsin, proteins,
growth factors, etc. must be sterilized by filtration
through a 0.2-micron porosity membrane filters

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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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• Dry heat and Moist heat

Sterilization • Irradiation
• Filtration
Methods • Chemical Disinfection
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Methods of sterilization
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• 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.
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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).
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Working with Open Flame

• Burners should be on only for as long as needed for a particular

procedure.
• The blue flame of a hot Bunsen burner can be difficult to see.
• Never leave an open flame unattended, not even for a few seconds
• It is also necessary to remove flammable and combustible materials
from the vicinity of the flame
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Sterilization of
Equipment and
Apparatus
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Sterilization
of Liquids
 Filtration

• Sterilizing solutions without heating.

• Work by passing the solution through a
filter with a pore diameter that is too
small for microbes to pass through.
• For removal of bacteria, filters with an
average pore diameter of 0.2um is
normally used.
• Viruses and phages can pass through
these filters, so filtration is not a good
option if these are a concern.

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Filtration

• Sterilizing heat-labile solutions

• Numerous kinds of filter membranes are available
• Filters are also available in different sizes from syringe-fitting filters through small and
intermediate in-line filters to multi-disk and cartridge filters
• Low-protein-binding filters are available
• Syringe-tip filters - used for low-volume filtration
• Intermediate-sized filters (for 50 ml–5 L) - used in-line with a peristaltic pump, or as bottle-top
filters used with a vacuum line and a regular medium bottle - vary in size from 13- to 50-mm
diameter
• Large-capacity cartridge filters (for 20–500 L) are usually operated in-line under positive
pressure from a reservoir
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What is Filter-aid?
• To remove impurities from the medium
• Prevent blockage of filters
• Chemically inert
• Low specific gravity so remain suspended in
liquid
• Recoverable
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Ultraviolet Light

• 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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1 2 3 4
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.

Safety Aspect in Cell Cultures