Practical tips for picking colonies & starting starter cultures

When picking colonies, your goal is to take genetically-identical populations of bacteria (benefit of plates) and transfer them to a larger amount of food (media) (i.e. inoculate a new culture) - this time in liquid form. This allows you to grow lots and lots and lots of those cells so you can do things like:
“miniprep” them to isolate a plasmid out of them (that plasmid that - in addition to whatever recipe you want them to have - has the antibiotic resistance gene that allowed you to select for them)
now you have enough to check them via sequencing to see if they have a properly-cloned plasmid
for miniprep purposes, I typically do ~5 mL O/N (overnight) cultures (in LB + antibiotic)
transfer to a large amount of media and get them to make protein for based on the plasmid’s recipe
this might me LB or TB, somewhere between 5 mL & 50 mL depending on how big of a large-scale you want to do & how much you want to inoculate with
blog: bit.ly/picking...
The point of the plate was to isolate individual colonies (which should at least in theory be genetically identical but because bacteria divide so rapidly, there are bound to be some mutations). So you don’t want to “erase” all that hard work by picking up more than that single colony when you do the transfer. The most important part of picking colonies is to pick A colony (per growth) - not multiple (although, especially if you’re trying to check cloning, you might be picking multiple ones that you use to inoculate multiple separate growths).
Even if you can’t visibly see a colony next to the one you’ve got your aim on, there could be cells there, such as persister cells vying their time in a sort of hibernation mode. So avoid touching anything other than your colony - even the surrounding agar that “looks clean.” When you pick a colony, you just need to dip into it - look to see you actually did (there should be a dent in the dot and some gloop on your picker) but don’t worry about getting it all. Each colony can contain several billion bacterial cells!¹ And they’ll multiply rapidly once you give them more food & room to grow! So be careful not to “smear” when you select. Just go straight-down-ish.
To make it easier to pick single colonies:
choose isolated ones - preferably large ones, but isolated is more important
don’t let your plates overgrow or the colonies can start merging together (and the antibiotics can go bad)
also avoid satellite colonies, those small little colonies that can start to grow around bigger colonies. These colonies likely don’t actually have the plasmid, but they’re able to survive thanks to the antibiotics breaking down (either just because they’re unstable for long times or because you’re doing something like ampicillin selection where betalactamase gets secreted by the cells with the plasmid and destroys the antibiotics in the vicinity)
identify the colonies you want to pick before you dive in with your pipet tip or toothpick or inoculating loop or whatever your pick of picker & circle them (on the bottom, not the lid!)
hold the plates up to the light to get a better view
when you label the bottom of your plate, write in small text along the outer perimeter so you don’t block your view
open the plate (with Bunsen burner going) and look from a side view if you can’t tell if something is a colony or a bubble
and avoid those bubbles when pouring the plates! Stir in the antibiotics rather than shaking the media up to mix (you can autoclave with a stir bar in there!)
Some other tips:
get everything ready to go before you start picking. With a Bunsen burner going - away from flammable stuff…
add your antibiotic fresh to a sufficient volume of media for the number of cultures you want to start
transfer the antibiotic-spiked-media to the individual tubes/flasks
don’t worry about volumes being exact
label the tubes/flasks
with the culture tubes, keep the lids loose so you don’t have to fumble around with them when trying to stick your colony in them
if you’re using an inoculating loop that you heat sterilize, dissipate the excess heat on by touching a clean agar plate first so you don’t fry your colony!
if you’re doing colony PCR, you can inoculate a culture at the same time (being sure to have things cross-referenced) - if the colony PCR looks good, go ahead and grow up & miniprep that growth. If not, bleach & toss it.
¹source for billions of cells reference: Mashimo, K., Nagata, Y., Kawata, M., Iwasaki, H., & Yamamoto, K. (2004). Role of the RuvAB protein in avoiding spontaneous formation of deletion mutations in the Escherichia coli K-12 endogenous tonB gene. Biochemical and biophysical research communications, 323(1), 197-203. doi.org/10.101...
finished in comments

[@jancordelia]

Thank you so much ❤❤❤❤ i am starting to learn formally in a research lab today and these videos help a ton! Please keep making them ❤❤

[@thebumblingbiochemist]

Awesome! So glad I could help. Best of luck in your lab!

[@adwoabiotech]

I love that you explained the reason for using a plate rather than the liquid media.

[@thebumblingbiochemist]

a helpful resource: McCleary Lab Standard Techniques W.R. McCleary (adapted from - J.S. Parkinson, Univ. of Utah), Basic Methods in Microbial Genetics mmbio.byu.edu/https:/brightspotcdn.byu.edu/65/8c/112bcbbd44f584ca680bf5479cf0/mccleary-lab-protocols.pdf
more about molecular cloning: bit.ly/molecularcloningguide & ruclips.net/video/U0O2T7NBUgg/видео.html
more about minipreps: bit.ly/minipreps & ruclips.net/video/UydCrBN0vBE/видео.html
more about colony PCR: bit.ly/colony_PCR & ruclips.net/video/DUWYnXaZyAo/видео.html
more about satellite colonies: blog: bit.ly/satellitecolonies ; RUclips: ruclips.net/video/v71nHvoeg-4/видео.html
   
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@adwoabiotech]

My kind of teacher - no details left out ⭐⭐⭐⭐⭐