Aseptic Technique
Chapter 6
Objectives of aseptic technique
• Contamination must be excluded
- Cultures are checked carefully by eye via a
microscope
- Cultures are maintained without antibiotics
- Reagents are checked for sterility before use
- Bottles of media or other reagents must not be
shared with other people or used for
different cell lines
- Maintenance of high sterile techniques
Elements of Aseptic Environment
• Work surface
• Clear the surface of the
hood
• Swab the surface with
70% alcohol
• Keep items related to
working experiment
• Swab between
procedures
Elements of Aseptic Environment
• Work Surface
• Keep a clear central
working space/area
• Pipette should not be
contaminated
• Mob any spillage and
swab with 70 % ethanol
• After experiment –
swab again
Elements of Aseptic Environment
• Personal Hygiene
• Hand washing removes microorganisms and
dead skin
• Surgical gloves may be worn
• Caps, gowns and face masks
• Tie back long hair
• Talking is permissible with a barrier between
you and culture
Elements of Aseptic Environment
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Reagents and media
Undergo strict quality control by companies
Outside surface of bottles might not be sterile
Bottles wrapped in polyethylene
Wrappings removed before use in hood
Unwrapped bottles swabbed by 70 % ethanol
Elements of Aseptic Environment
• Cultures
• Imported ones – contamination at source or
transit
• Should be quarantined
• Kept away or incorporated into main stock
• Antibiotics usage – suppress and not eliminate
contamination
Elements of Sterile Handling
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Swabbing
Swab work surface with 70% alcohol
Clean spillage
Swab bottles – cold storage/water
bath/incubators
• Label with alcohol resistant markers
Elements of Sterile Handling
• Capping
• Deep screw caps preferred to stoppers
• No detergent remains in rubber liners of caps
• Screw cap covered with aluminum foil to
protect neck of bottle from deposition of dust
Elements of Sterile Handling
• Flaming
• Open bench – flame
glass pipettes + neck of
bottles and screw caps
• Place open side down
on clean surface and
flamed before being
replaced
• Flaming not advisable
in hoods
Elements of Sterile Handling
• Handling bottles and flasks
• Open bench – Do not leave open vertical
bottles to avoid spillage
• Bottle racks – keep bottles tilted
• Hoods – Leave bottles vertical and open
• Hoods – No blockage between open vessel or
sterile pipette and HEPA air filter
Elements of Sterile Handling
• Pipetting
• Pipettes of sizes 1 ml, 5 ml,
10 ml, 25 ml and 100 ml is
available
• Unwrapping pipettes should
be done carefully
• Glass pipettes must be
sterilized before use in hood
• Mouth pipetting should be
strictly avoided
Laminar Flow Hood
• Horizontal Hood
• Airflow blows from the
side facing you, parallel
to work surface
• No recirculation of air
• Stable airflow and best
sterile protection to
culture and reagents
Laminar Flow Hood
• Vertical Hood
• Air blows down from
top of hood onto work
surface.
• Drawn through work
surface and either
recirculated or vented
• Protection to operator
• Avoids overspill in
work area
Different types of hoods
• Class I for simple and non-pathogenic
• Class II for potentially hazardous material
(human or primate-derived cultures, virally
infected cultures etc)
• Class III for known human pathogens
Cytotoxicity hood
• Protection against chemical and radiochemical
hazards
• Carbon filter trap in recirculating airflow or
hood with all effluent vented to outside the
building
Pressure and Air flow in a hood
• Pressure drop – Manometer
• Air flow – Anemometer
• Below 0.4 m/s (80 ft/min) – stability of airflow
is lost – sterility cannot be maintained
Routine maintenance checks of hoods
• Primary filters (3-6 months) – removed
• Horizontal-flow hoods – removal – discarded
or washed in soap and water
• Vertical- flow hoods – Biohazard hoods are
internal – only an engineer can replace
- Incinerated or autoclaved and discarded
Routine maintenance checks of hoods
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HEPA filter – once every 6 months
Should be monitored for airflow and holes
Monitoring done by engineers
Biohazard cabinets – bagging and disposing of
filters by incineration
Routine maintenance checks of hoods
• Weekly checks – spillage cleaned, sterilized
with 5 % phenolic disinfectant and 70 %
ethanol
• Do not let any material block the airflow.
Check regularly for any droppings
• Labs keep hoods running to keep area clean
• Ultraviolet light sterilizes but do not reach
crevices – alcohol by capillary action
Incubators
• Major source of contamination
• Should be cleaned regularly (weekly or
monthly)
• Washing racks or shelves by nontoxic
detergent – Decon or Roccall
• Traces of detergent removed by ethanolbefore placing back shelves
Incubators
• Fungicide – 2 % Roccall or 1 % Copper
Sulfate placed in humidifier tray
• Micropore filtration and laminar airflow to
inhibit circulation of microorganisms
Boxed Cultures
• Repeated
contaminations in
incubator
• Use sandwich boxes
• Enclose dishes, plates
and flasks with
slackened caps
• Swab 70% ethanol
Gassing with CO2
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