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

• 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

• 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

• 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 ethanol- before 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

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