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I've been working on what I think might be a special strain of E. coli, I've been streaking it on LB agar plate and exposing it to varied times of UVC via the germicidal hood. I'm wondering if how I streak, as in too heavily, vs very lightly, or diluted culture, etc would make much effect in survival. If I'm putting too much onto the plate, the e coli could be self-shielding, or some ecoli dying off, but the rest underneath being protected from the light. I've done several generations of surviving colonies, I'm now comparing to wild type to see if my streaking method results in survival in both. This test will also rule out if the lamp is failing and my surviving colonies are just the tougher ecoli compared to the growth on the control. tldr Should I lightly streak or dilute my culture to ensure all ecoli are being hit with the UVC?

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  • $\begingroup$ What about using a suspension in liquid broth. Put it in a shaker, put it under UV light. $\endgroup$ – Almeisan Mar 4 '16 at 3:56
  • $\begingroup$ Wouldn't dilution be more quantifiable for replication purposes? $\endgroup$ – James Mar 4 '16 at 4:35
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I work with e. coli myself though I do not know if your special strain has anything drastically different than my own so I'll just give advice that I've gotten from post docs and other PhDs on how to handle cultures.

(Note: This is methodology that I can't really find a "source" for so take with a grain of salt and change for your needs)

Typically when streaking I try to ensure that I make the streaks light enough so there aren't colonies within vicinity of each other. It's hard to really give a quantitative answer on how to do this so you will have to experiment on your own so your streaks aren't laden with massive amounts of colonies next to one another. It should look like consecutive "beads" on a string. This is generally what I try to accomplish with my colonies. Keep in mind I am focused on cloning plasmids so I'm not really concerned about experimenting on the e. coli themselves, just getting individual populations that do not have mixed variations of my plasmid. This is why having colonies with good separation is important for my particular project.

As for making sure they are all being affected by the UVC, I'm not entirely sure if having them to heavily grouped together would "shield" from UV. I feel as though UV can easily penetrate through e. coli colonies but I guess its not out of the question. Best bet is to keep your colonies well spread out, enough so you can pickup a single colony with a pipette tip and nothing else. This will make any genetic experiments you may want to do a lot easier as well. Which I think would be interesting if you are interested in the mutations occurring.

Is there something about your project that requires you to streak these out on agar plates? I saw comments suggest liquid broth. Maybe that's easier to do to make sure everything is exposed and then streak them out on a plate to see growth and/or test for mutations.

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