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How to maintain sterility while preparing petri dishes

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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How to maintain sterility while preparing petri dishes

Postby animus » Thu Jun 13, 2013 10:42 pm

I'm a newbie in this area and just prepare and observe bacteria in petri dishes (and then under the 'scope) for my own learning experience.

I thought I was being sufficiently sanitary while heating the agar and pouring it into the plates. But before I even got the chance to start up bacteria in one plate to isolate in the other (both plates were supposed to be sterile), there were many colonies growing in both of them.

I heat up the agar by boiling it and I held the plates in boiling water just before pouring the agar into them. I try to open the covers a minimal amount while pouring the agar. I put a rubber band around each once I'm done pouring. I'm not sure what else I can do to get a sterile environment in the dishes.

Any tips? Also, once I do get to putting bacteria in them, how will I do that sterilely also? I have inoculating loops that I'll sterilize before using but that also requires opening the lids.
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Postby Cat » Sat Jun 15, 2013 4:26 pm

First, you need to autoclave agar and plates (if glass) or buy sterile (if plastic). Second, if you "held the plates in boiling water just before pouring the agar into them", your plates are wet at the moment you pour agar that increases contamination risk (microbes from the outside of the plate can swim inside the plate). Plates should be dry.
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Re: How to maintain sterility while preparing petri dishes

Postby Amphibitile » Sun Jun 23, 2013 8:11 pm

As Cat said water only really increases the risk of contamination. Buy sterile petri dishes and only open then in a sterile environment, from your results I presume you aren't doing that. The main issue with petri dish contamination is airborne bacteria, if you get a Bunsen burner and work closely with it on a blue flame (not too close of course) it will increase the sterility of the area. This should hopefully reduce contamination, I wish you luck with your dishes.
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Postby animus » Mon Jun 24, 2013 1:35 am

Thanks Cat and Amphibitile.

Maybe I'll just bring them with me to the university in the fall and use their equipment. Not exactly willing to invest in an autoclave yet! Maybe I'll try the burner idea. Or just buy some disposable sterile plates. Thanks again. :)
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Postby biohazard » Mon Jun 24, 2013 7:12 am

Like others already said, autoclaving the agar and using sterile plastic or autoclaved Petri dishes is essential. In addition to that, pouring the agar should be done in a laminar flow hood, which essentially eliminates the issue of airborne bacteria or other contaminants. For some of our courses we prepare like 1000 agar plates this way and in most cases none of them have signs of contamination. When the lids are closed and the plates are packed into a plastic bag they also stay sterile for several weeks in the refrigerator.

Sterile plastic loops (or alternatively Bunsen-sterilized metal ones) are good for plating the bacteria. This we have done simply on the
desktop without any real contamination issues. Actually by far the most common contamination from the air is mold and that requires much longer incubation times than bacteria do, so you can grow your bacteria and do whatever experiments you want to do on them before the mold manages to grow.

I am actually quite surprised to hear that you managed to get "many colonies" by doing what you said. Are you sure that the colonies are not from spore-forming bacteria that survived the heating and were already in the agar? Because like I said you don't easily get many colonies simply from the air (unless the air at your location is very different from what we have over here!).
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Postby biohazard » Mon Jun 24, 2013 8:56 am

Just to add to my previous post:

I was a bit in a hurry when replying, so I noticed only now that you are not doing this in a university lab or similar, so maybe the laminar hood falls into the same category as the autoclave :P

Anyway, now as I realized that the agar was not autoclaved but boiled, it could indeed be the source of the contamination - some bacterial spores can withstand several minutes of boiling temperatures and then grow on your plates when they have cooled down. I don't remember by heart, but I think you need up to 15 or even 30 mins of boiling time to kill off all the endospores.

Properly heated agar poured on sterile plastic dishes should give you sterile agar plates in most cases even if done without autoclaves and hoods. Plating the bacteria shouldn't be a problem: even if you open the lids for as long as several minutes, you are likely to get no contaminating bacteria from the air (our 100+ students do so dozens of times in our labs and we see airborne contaminants very, very rarely - the contamination is almost always from poorly sterilized loops or from their own butter-fingers!). That being said, the lids are best kept open as little as possible still - after all there is always a chance that something drops on your plate from the air.

If the contaminating colonies were identical to each other, it might suggest a spore-forming bacterium, such as some Bacillus spp. Multiple different colonies would indicate some wider problem in your working conditions.

By any chance, were the contaminating colonies large, whitish, slimy and had somewhat uneven borders ? :)
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Postby animus » Thu Jun 27, 2013 12:18 pm

Thank you for that information! Excellent! I think I've found where I've gone wrong—the agar itself. I only boiled it for a few minutes each time I've tried so there were very likely some contaminants still in there. After I used the bottle the first time, there were some strange things growing on the surface. It had a greenish, mucousy look. Next time I will try boiling it for the 15+ minutes you mentioned.

I think the contaminating colonies were nearly identical; they were small, though. Probably the size of...I don't know; probably had half the diameter of a pencil eraser. Yes, whitish. Some of the colonies were rounded while others were not.
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Postby biohazard » Fri Jun 28, 2013 10:04 am

When talking about bacterial colonies something around the size of half the pencil eraser is already fairly large. A small colony would be the size of a pin-prick or similar. Well, difficult to say without further details, but it could be that you have some Bacilli contaminating your agar or other supplies - many of them they are very common in e.g. soil and they form resilient spores that can withstand even boiling temperatures for some time.
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Postby animus » Sat Jun 29, 2013 3:11 am

Oh, okay! There is so much to learn. I love it. Thanks.
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