Does the Petri dish lid orientation on workbench affect aseptic technique?

Question

When examining an incubated plate, it is rather clear that holding the plate in one hand and the lid in another is best for aseptic technique, or simply resting the lid on the plate as shown below.

However, I can't seem to find an answer for what to do with your Petri dish lid when doing manipulations like plating. It's pretty obvious that the above practice is, well, practically impossible.

Thus, I always went under the assumption that when placing the Petri dish lid on the workspace, the orientation of the lid did not particularly affect risk of contamination, assuming that you're technique is indeed aseptic (ethanoled workbench, Bunsen burner, etc). However, discussing with collegues has resulted in mixed answers. Some say it doesn't matter, some say the lid should be facing up, some say down.

When doing manipulations like plating, is there a common practice for exactly how to place your Petri dish lid?

Answer 1

DISCLAIMER: I am a computational biologist and haven't touched a Petri dish since I was an undergraduate biology student.

I would expect that the best thing to do would be to place the lid face down. When placed so on the bench, it is only the tip of the lid that comes into contact with your (presumably sterile-ish) bench. If you place it face up, the interior of the lid will come into contact with the air of the lab. Despite the presence of the bunsen, I would expect that to gather more contamination.

Since the lid is larger than the dish itself, the lip of the lid never touches the dish. Therefore, if the lip of the lid touches the bench and becomes contaminated that is not a big problem since that contamination should be blocked from spreading to the actual culture. If, on the other hand, the lid were placed face up, the interior of the lid (which hangs just above your culture when closed) will have come into contact with possibly contaminated air and could then cause contaminants to drop onto your culture.

Answer 2

Working with petri plates both microbially and in tissue culture, there is one rule of thumb to follow. Contamination falls. Your main enemy is time, how long the medium is exposed (bar actually touching the surfaces). Ethanol DOES NOT sterilize the working surface. Think of it as a convenient scrubbant which evaporates quickly. The flame DOES NOT work to sterilize the air (except in the heated column above it, where you can't work). The flame creates an updraft near your workspace (see the rule of thumb), reducing falling contamination.

Placing the lid on a "clean" working surface is a common and reasonable practice. If you are coordinated, holding the lid in the non-dominant hand can also be done. If you are using a 'hockey stick' spreader (dip in alcohol, then flame, then spread a dispensed drop after cooling), there may be a better way.

Get a good petri plate turntable (might have to lubricate it with dry graphite). Dry your petri plates for a while until no free moisture is visible in the dish. Obtain some small volume glass pipettes (say 1 ml), with smooth tips, and sterilize them.

When you are ready to plate your suspension, place your petri dish on the turntable, remove the lid and give a good spin. Dispense 0.2-1 ml of your suspension onto the medium directly, working the tip on the medium surface from the center to the edge, keeping a wet edge (surface tension). If you press too hard or hold the pipette too vertical, it digs in. Quick to learn this though. Replace the lid, and rest the plate a little while on the bench. A perfect spread (slight spiral) every time. Okay, a little practice helps. No flame, no alcohol, more free time for beer. Moisture content in the media is the only issue, but drying the plates inverted in a standard microbial incubator is easy enough.

I've used this method for years, with no major contamination issues.

Answer 3

In my experience, I have always used some kind of selection marker (typically antibiotics). In this case, it doesn't matter at all because any miniscule amount of foreign contamination would not be expected to carry an antiobitic resistance gene.

Alternatively, when following sterile bench technique by wiping the benchtop with ethanol and working near a flame, then it also does not matter. The ethanol will take care of surface contaminants, and the flame keeps the air around it from being contaminated. Also, when working with selection markers, the flame is usually unnecessary if the rest of your technique is aseptic.