Tissue Engineering

iii. Aseptic Technique

The term ‘aseptic technique’; refers to practices done to maintain sterility during some procedures. Good aseptic technique is very important in tissue engineering (and many other biological sciences) because infections can cast doubt on or ruin a lot of work.

Many operations that require sterility are done under a sterile hood, which is specially designed to provide a microbe-free workspace. These hoods are designed so that there is laminar flow (i.e. smooth, un-turbulent) of well-filtered air, which prevents the influx of microorganisms into the workspace.

Figure: Illustrations of two types of laminar flow hoods: horizontal and vertical. The horizontal flow hood draws in air from above, filters it, then forces it out the front of the working area of the hood. The vertical flow hood is more complicated, drawing air through the front of the working area into the vent system at the front of the working surface. 80% of this air is recirculated and the remainder expelled.

Figure: Photograph of a vertical laminar flow hood with intake vents in the front of the working surface and in the back wall of the working surface.

Do it right the first time by following these rules:

Video of some basic aseptic techniques

Always wear gloves and change them frequently.
If possible always use a laminar-flow hood when working with media, cells, and organs. Make sure to wipe down the surface of the hood with 70% ethanol before working in it. Make sure the hood is uncluttered and that there is nothing obstructing the vents.

Figure: Photo shows cleaning the hood with 70% ethanol before use.
Try to keep your working area as far away from drafts and traffic as possible.
Wipe down everything - bottles, caps, pipettes, and work benches before and after completion of your task. Make sure to wipe thoroughly after a spill.
Make sure you have everything you need before you start working. This prevents you from having to get up and prevents the agitation of the air surrounding your work area.
Avoid pouring. Pouring creates aerosols which can carry contamination to unwanted places. Use a pipette instead.
Open all bottles at a 45 º angle and recap containers after pipetting.

Figure: Photo shows holding media bottle at 45 degree angle for pipetting.
Flame all glass pipettes (or use new sterile glass pipette) and open bottles before using them.
If you touch a pipette to anything other than what you are working with, discard it. Never re-use a pipette.

Avoiding Cross Contamination of cell culture:

When working with more than one cell type at a time, cross-contamination of one cell type with another can be a problem. To insure this does not occur follow these rules:

Never work on more than one cell line at a time.
Never use the same pipette for different cell lines.
Never put a pipette back into medium after you have used it to pipette cells.

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