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

Correct laminar-flow hood laid out

Incorrect laminar-flow hood laid out

Work Surface

1. A completely clear surface

2. Swab the surface with 70% alcohol

3. Bring into the surface only the items you require

4. Remove everything that is not required

5. A wide space in the center of the bench to work on

6. When using the horizontal laminar flow, there is no obstructionsbetween the central work area and the HEPA filter

7. Work within your range of vision

8. Mop up any spillage immediately & swab the area with 70% alcohol

9. Remove everything when you have finished

Personal Hygiene

1. Washing will reduce loosely adherent microorganisms, which arethe greatest risk to the tissue culture

2. Tie your long hair back

3. Caps, gowns, face masks are required under Good ManufacturingPractice [but they are not necessary under normal conditions]

4. Do not talk (when working on an open bench)

Reagents and Media

1. Outside surface of the bottle is not sterile

2. Swab with 70% alcohol when they come from the refrigerator orfrom a water bath

Cultures

1. Cultures imported from another laboratory [High Risk !!]

2. They should be handled separately from the rest of your stocks andkeep free of antibiotics until they are shown to be uncontaminated

3. Antibiotics should not be used routinely

Sterile Handling

1. Swabbing:--Swab with 70% alcohol (work surface, bottles, flasks)

2. Flaming--Flame glass pipettes and the neck of bottles and screw caps before and after opening and closing a bottle

3. Handling bottles and flasks--Do not keep bottles vertical when open

4. Pipetting--Disposable plastic pipettes should be double wrapped

5. Pouring--Do not pour one sterile container into another

Potential risks during inserting a pipette

Holding the cap

Waste beaker

“prevents beaker from splashing back contents”

Petri dishes & Multiwell plates

1. Large surface

2. Risk of touching the rim of the dish

3. The lid is laid down

4. Medium filling the gap between the lid and the dish

5. Higher risk of contamination in the humid atmosphere of a CO2 incubator

“Prone to contamination”

Protocol 6-1Aseptic technique in vertical laminar flow