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How do you grow an isolated colony of e.coli from scratch?

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 do you grow an isolated colony of e.coli from scratch?

Postby mbyyas » Tue Nov 21, 2006 1:02 pm

Hello there! I need help as I guess most do on this forum.

Essentially would anyone be able to provide me with a detailed description in the lab protocal to grow an isolated e.coli from scratch. What I mean is exactly how to I sart growing the colony in a glycerol stock solution overnight? found many a method that vary.
Do I have to freeze the e.coli and glycerol solution once mixed or can I jump straight into leaving in a shaking water bath overnight at 37 degrees C. Can I then proceed, from that overnight culture, to streak the colony on an agar plate, exact method would be nice. Would this plate be where I first inttroduce an agar mixture combined with the right antibiotics such as ampicilin or do I add them with the glycerol stock before cultivating overnight?
From the colonies streaked do I then find a single colony from that plate and once again reintroduce into a liquid medium? and exactly how to I proceed from here to end up with three to for aliquots or vials of a stock single colony e.coli culture stored at -70/80 degrees C from which to carry on with my research?
Please help
My e-mail address is mbyyas@nottingham.ac.uk If you have any journals or lab protocols to file you wouldnt mind sharing.

kind regards

Alexander Sano-Davies
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Postby canalon » Tue Nov 21, 2006 5:23 pm

What is your starting point:

a colony on a plate? Then pick it and streak it on a fresh plate using 2 or 3 crossing lines changing toothpick (or whatever picking tool) each time. You should keep selective agent (if any, like amp) in the fresh plate. Grow overnight at an appropriate temp.
Depending how likely a colony is to be mixed with something you might want to repeat this one or two times before being sure that your colony is pure. Then grow overnight in liquid medium with selective agent. add the same volume of 30% glycerol and freeze.

from glycerol? streak on selective plate with multiple streaks and pick one isolated colony. Same thing as above.
Patrick

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Postby mbyyas » Tue Nov 21, 2006 10:51 pm

My starting point is a pre frozen sample of glycerol and bug stock, 50 micro litres, the end point i am trying to achieve is 8 similar stocks of glycerol and bug solution of a single cloned colony of e.coli. In other words I wmust prove through my lab work that I have started with a e.coli stock that may or maynot be all identical, and end up with 8 stocks of identical, dna wise, ecoli. I know i have to streak the ecoli to begin with, grow overnight on plates, and then from the plates isolate a single cell colony, insert into a 10ml LB liquid broth with ampicilin and kanamycin? grow while shaking again overnight, from this I have a glycerol stock. It is now the expression in LB medium that is presenting the issue for me. I know I have to add 20 micro litres of the glycerol stock to arround 100ml of LB with the corresponding amount of ampycilin and kanamacyn and again shake to grow overnight, but the following day, from research ive been doing, most procedure recomend preparing 4/6L! of LB in 2 or 3 flasks to add starter culture too! and then grow to arround 0.6 OD, is this correct! that much LB! and how do i measure the OD of such a vast volume, do I have to take periodical samples from the micture to test or can I measure the OD of the entire conical flask!? In terms of harvesting methods i have found mention centrifuging the mixture at 5000rpm, how do you do that with a 2L conical flask? from then on i seem to be ok, in terms of suspension of cell pelets and the sonication of the suspension. If you could answer any of these gaps then please help, and thank you so much for the replies so far, and any further replies made, I very much appreciate this guys/girls. Thank you
Kind Regards
Alex
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Postby mikki » Wed Nov 22, 2006 7:28 am

Using A-septic technique, flame sterilize your inoculating loop and transfer your organism to a nutrient agar or bhi plate (not sure but I think ecoli will grow on any media). To do a streak plate, which is used to isolate a mixed culture into a pure culture (in the form of colonies) use the four-quadrant method. Inoculate quadrant 1, flame sterilize loop and wait 10 seconds to cool, then streak from quadrant 1 into 2, flame sterilize loop and wait 10 seconds, steak from quadrant 2 to 3. Be sure you zigzag 3 lines without going back into the previous quadrant.

So you would streak organism from quadrant 1 to 2, streak back into quadrant 1, streak out into quadrant 2 again, streak back into quad 1, streak back into quad 2, then make 3 zigzag streak lines without touching quad 1. When you have done all 4 sides then invert plate and incubate for 24 to 48 hours at 37C.
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Postby canalon » Wed Nov 22, 2006 6:47 pm

Well don't worry that much. It is easier than you describe.

So you know how to get an isolated colony. Mikki's method is great but expensive I usually isolate 6 different colonies per plate :) and in BHI or LB 16h is more than enough to get nice E. coli colonies.

In liquid you are going to get a higher survival rate if you use a colony in exponential or late exponential phase (OD~0.6) but you will have more than enough with a saturated culture. So do not worry about OD. What you simply want is to have a 15% vol/vol proportion of glycerol in your culture in the end, or if you prefer 15 µl glycerol for 100µl of final suspension. Considring that glycerol is a pain to pipet I suggest you prepare a solution of 30% or 45% glycerol, sterilize it (filter is best) and add the correct volume to your culture to have the 15% glycerol.

Alternatively you can pellet the bacteria (1ml in 1.5ml tubes at 13000g for 1 min) and resuspend them in a medium (LB is fine) containing selective agents (amp and kan) and 15% glycerol.
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