1. Aseptic Technique
Chapter 6

2. 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

3. 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

5. 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

6. Absolutely no talking while performing bench work to prevent ur saliva from falling into culture

7. 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
Vertical laminar hood has a barrier between you and culture – can talk but keep limited talking. If you have a cold then wear a mask to prevent spread of infection

8. 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
Polyethylene wrapped bottles keeps them clean and allows them to be placed in water bath to be warmed or thawed. Unwrapped bottles should be swabbed with ethanol after their removal from refrigerator or water baths

9. 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
Should be quarantined for contamination and kept separately from rest of your stocks.

10. 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

11. 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

12. 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
Screw caps should be flamed while opening and closing bottles. Screw caps must be held in hands during pipetting to avoid flaming or laying them down
Flaming will disrupt laminar flow (sterility of hood and its containment of biohazardous material). Open flames can damage high efficiency particulate air (HEPA) filter or melt some plastic interior fittings

13. 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

14. 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
Glass pipettes – insert cotton plug in top of glass pipette before sterilization to keep pipette sterile. The plug prevents contamination from bulb or pipetting aid and cross-contamination from pipette to bulb or pipetting aid. If plug becomes wet, discard the pipette into disinfectant for return to wash up. Plugging pipettes for sterile use and also their removal before washing is a tedious job. The disposable plastic ones come plugged. It is advisable to use individually wrapped pipettes as they are free from contamination rather than buying all plastic ones in a common packing which would lose the purpose of sterility if opened. They are free of chemical and microbial contamination and reduce washing requirements.

16. 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

17. 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

18. 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

19. Cytotoxicity hood
Protection against chemical and radiochemical hazards
Carbon filter trap in recirculating airflow or hood with all effluent vented to outside the building

20. 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
If resistance pressure drops in hood then airflow rate also drops

21. 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

22. 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

23. 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

24. 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

25. Incubators
Fungicide – 2 % Roccall or 1 % Copper Sulfate placed in humidifier tray
Micropore filtration and laminar airflow to inhibit circulation of microorganisms
Fungicide is not effective as it does not touch all surfaces

26. Boxed Cultures Repeated contaminations in incubator Use sandwich boxes
Enclose dishes, plates and flasks with slackened caps
Swab 70% ethanol

27. Gassing with CO2
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