1. Ch6
Aseptic Technique
“Contamination of microorganisms (bacteria, mycoplasma, yeast, fungus) remains a major problem in tissue culture”
[see Table 19-1]
Routes & Prevention to Contamination

2. Correct laminar-flow hood laid out

3. Incorrect laminar-flow hood laid out

4. Work Surface
A completely clear surface
Swab the surface with 70% alcohol
Bring into the surface only the items you require
Remove everything that is not required
A wide space in the center of the bench to work on
When using the horizontal laminar flow, there is no obstructions between the central work area and the HEPA filter
Work within your range of vision
Mop up any spillage immediately & swab the area with 70% alcohol
Remove everything when you have finished

5. Personal Hygiene
Washing will reduce loosely adherent microorganisms, which are the greatest risk to the tissue culture
Tie your long hair back
Caps, gowns, face masks are required under Good Manufacturing Practice [but they are not necessary under normal conditions]
Do not talk (when working on an open bench)

6. Reagents and Media
Outside surface of the bottle is not sterile
Swab with 70% alcohol when they come from the refrigerator or from a water bath

7. Cultures
Cultures imported from another laboratory [High Risk !!]
They should be handled separately from the rest of your stocks and keep free of antibiotics until they are shown to be uncontaminated
Antibiotics should not be used routinely

8. Sterile Handling
Swabbing: --Swab with 70% alcohol (work surface, bottles, flasks)
Flaming --Flame glass pipettes and the neck of bottles and screw caps before and after opening and closing a bottle
Handling bottles and flasks --Do not keep bottles vertical when open
Pipetting --Disposable plastic pipettes should be double wrapped
Pouring --Do not pour one sterile container into another

9. Potential risks during inserting a pipette

10. Holding the cap

11. Waste beaker
“prevents beaker from splashing back contents”

12. Petri dishes & Multiwell plates
“Prone to contamination”
Large surface
Risk of touching the rim of the dish
The lid is laid down
Medium filling the gap between the lid and the dish
Higher risk of contamination in the humid atmosphere of a CO2 incubator

14. Aseptic technique in vertical laminar flow
Protocol 6-1
Aseptic technique in vertical laminar flow