Tip:
Highlight text to annotate it
X
Successful cell culture depends heavily on keeping the cells free from microorganism
contamination. This can be accomplished with proper knowledge of sterile environments,
working slowly and deliberately, and following all of the guidelines for sterile technique.
Always wear basic personal protective equipment, or PPE, when working in the laboratory. Talk
to the safety team at your institute for your required PPE. Also, remember to review the
MSDS information before working with any media or reagents.
Wear closed toe shoes, and clothes that cover your legs.
Washing your hands before handling cells or performing culture work removes bacteria and
microscopic dead skin particles. Dead skin can be a potential source of contamination.
A 70% ethanol wash kills microorganisms that could contaminate the cultures. Cleanliness
is one of the most important parts of sterile technique. Always clean the hood before and
after use. Spray alcohol should not be used in any area where a flame is being used, due
to the fire hazard. The outside of containers carry dust and contaminants.
Remember to clean each item placed in the hood with ethanol. You may choose to spray
the item before placing it in the hood, or immediately after as demonstrated. As you
clean the item and place into the cell culture hood, put it in the correct position. Enter
the hood with your hands in a forward motion, try not to sweep across the front and disrupt
the air flow. Proper set-up of the cell culture hood includes
not over-crowding the work surface. Not only does this increase the risk of contamination
through accidental touching, but it also interrupts the air flow through the chamber, which will
not maintain the sterile field. For this same reason, it is important to keep the front
sash in the lower position when working. The basic idea is to keep your media and reagents
sterile by only touching them with sterile objects. Using sterile media, reagents and
supplies is a big step in keeping your cultures free from contamination. Notice how the cell
culture hood is set up to make the following actions easier. The Pipette Aid is on the
right side so you can easily control the pipetting in your right hand. Reagents are in the center
back so you can easily open the bottles and pipette from them. It is very important to
have the set up in this manner, so you are not crossing your hands or supplies over top
of sterile items. Any movement over the top of a sterile item can result in contamination.
We’ll now demonstrate the basic technique of handling a pipette and a bottle of medium
while aseptically supplementing DMEM with Glutamax.
When opening the pipette package, be sure the pipette does not touch anything non-sterile.
Grasp the pipette high on the neck, insert the pipette into the Pipette Aid, turn the
desired measurement marks toward you and then discard the wrapper. It may take some practice,
but it is a very important first step. Discard any pipette you accidentally contaminate.
Only open your media, reagents, and supplies in the cell culture hood. Opening these items
outside the sterile field will result in contamination. When holding the cap, it is important not
to touch the inside edge or you could contaminate it. Replace the cap as soon as possible, in
order to decrease your risk of contamination. If you must set the cap down in order to free
your hand, set it down with the interior surface facing down.
When pipetting, try not to touch the pipette to anything non-sterile, particularly the
outside of containers, or contamination could result. Each pipette should be used one time
and discarded (or washed if using glass pipettes). Reusing pipettes by dipping directly into
another media bottle, or by leaving it standing in a media bottle, increases your chances
of spreading contamination. Gently mix the contents after supplementation.
Labeling the bottle after supplementation is good laboratory practice.
Sometimes you will need to transfer large volumes of liquid from one container to another.
The best way to transfer is always to use a sterile pipette, but with practice, it is
possible to aseptically pour instead. You should pour quickly and deliberately, using
the front corner of the bottle to channel the liquid and improve the speed and accuracy
of your pour. Any spills should be wiped with ethanol immediately.
When passaging cells or changing their medium, the basics of aseptic technique are the same:
only open the containers in the sterile field, do not cross your arms or other items over
an open flask, don’t rush (but work at a good pace and with deliberate motions), do
not re-use pipettes, and do not use items that were inadvertently contaminated.
When finished, make sure everything is closed tightly before removing from the cell culture
hood. Anything opened outside the sterile field will now be non-sterile and should not
be used for cell culture work. Wipe down the work surface with ethanol again and straighten
up the hood before you leave.