Seeding Plates Contamination

Hello,

I was seeding plates with OP50-1 w/strep and I got this nasty contamination. The plates seemed fine before and the contamination was originally restricted to the bacterial lawn, but has started growing to other parts of the plates. I grew the bacteria in LB media and used brand new M9 media when resuspending the bacteria pellet. I believe the contamination was in the seeding material, but I’m not sure why and I’ve never had contamination this bad.

I won’t be using that M9 anymore, but wanted to ask for any help/tips. Could it have come from the LB plate where I picked the colony from? I ask because I streaked an LB plate with the same seeding material that ended up contaminating all those plates. This was before I saw any contamination on them. On that note, after seeding I leave the plates out for 24 hours and then store them, would it be bad for me to leaving them out for longer to catch contamination like this sooner since it took longer for the contamination to grow/become visible when it was stored in the fridge.

Also new user, so it’s not letting me upload pictures!

Thank you for any help!

If you are not already doing so, I recommend taking a stab from a frozen stock of OP-50 and making a plate streaked for single colonies. Then every time you grow OP-50, start them from a single colony picked into your LB media; the plate started from the freezer stock can be kept wrapped in parafilm and aluminum foil (to protect the antibiotic from light) in the fridge. By the way, I use the same LB media that was used to grow the bacteria to distribute the bacteria to the NGM plates. Less work and less chance of contamination. I recommend doing this all in an area of the lab away from where you have been using any of the contaminated plates. I generally restart a plate with single colonies of OP-50 from frozen OP-50 every 6 months (or more frequently if I notice any contamination problems). Hope this is helpful.

Just some practical notes, to add to Janet Duerr’s comments:

I always use single colonies of bacteria streaked out on LB media (without antibiotic). For E. coli, I grow it overnight at 37C and then store it parafilmed in the refrigerator, where it remains viable for at least a month and probably two.

I pick single colonies and use them to inoculate LB media (or B broth), maybe 100 ml that had been autoclaved in a 200 ml glass bottle. I grow that overnight (and usually not much longer than that) in a 37C incubator, with the lid open a crack. I then tighten the lid shut and store it in the refrigerator, where it will be good for at least two months (if I’m not spotting enough plates to use it up before then); each time I take it out, I give it a good swirl to resuspend everything that’s settled out.

Every month I use the most recent bottle to make a new streak plate, and use the most recent streak plate to inoculate a new bottle. I’m not as responsible as Janet is about using refreshing from a frozen stock.