How to passage bacteria ? - (Aug/22/2015 )
Hi, I want to perform a plasmid stability test for a recombinant bacteria and hence have to passage the bacteria (for 30 generations).
For each generation, I will count the cfu on the antibiotic selective agar plate and compare with normal agar plate.
My question is, how do I passage bacteria in this case?
1. Pick a single colony from previous passage, grow in 5ml broth overnight, and take 100 ul to plate the bacteria.
2. Pick a single colony from previous passage, resuspend in 100 ul broth and plate the bacteria.
Which is better?
Normally how do you do it?
I am not sure what you mean with the second example.
Pick a single colony , resuspend in broth and then what? Plate right after ? Whats the point then?
Or you mean you will grow it in the broth first?
Pito,
Second example I plate it right away and let it grow for another 48 hours, and do the same for next passage.
Isn't it more appropriate? For the first example if I picked a colony and grow on broth first,isn't it considered another passage.
I want to passage bacteria from plate to plate.
picking a single "colony" and "diluting" it in broth is not really a good idea, I mean: its just 1 colony, not really a lot of cells to spread out....
Why do you not just restreak it every X days? From plate to plate? Its pretty much the same as your "broth" example.
A typical colony, grown from a single cell, is probably near 30 generations, probably more like 25. So, streaking out for single colonies once will do what you require.
I think he means with 30 generations literally 30 passages as in "30 times on a plate"
phage434 on Sun Aug 23 15:21:14 2015 said:
A typical colony, grown from a single cell, is probably near 30 generations, probably more like 25. So, streaking out for single colonies once will do what you require.
Well, that's (in my opinion) a very peculiar definition of a "generation."
phage434 on Sun Aug 23 19:53:45 2015 said:
Well, that's (in my opinion) a very peculiar definition of a "generation."
well... if its just about 1 time plating them out and thats it.. seems weird to as an experiment.
I think he just wants to keep plating them over a certain time period and see what the effect is...
(there is a famous experiment on this, where a professor is just replating E.coli over and over , every day.. for years to check for mutations. I can not remember the name at the moment... I think he is trying to do something like that).
Sorry for the incorrect use of the word "generation".
Yes, I am trying to grow it for 30 passages.
I do not have microbiology background and I am confused in how to plate the bacteria from previous passage to next.
If I do not first resuspend the single colony in liquid form, will I be able to plate/spread it? I want to do a cfu counting for each passage.
Meg P. Anula on Mon Aug 24 03:27:39 2015 said:
Sorry for the incorrect use of the word "generation".
Yes, I am trying to grow it for 30 passages.
I do not have microbiology background and I am confused in how to plate the bacteria from previous passage to next.
If I do not first resuspend the single colony in liquid form, will I be able to plate/spread it? I want to do a cfu counting for each passage.
If you want to do a CFU then you can not just restreak it.
you will have to dilute it in liquid. But just taking 1 colony and diluting that in some H2O ... to me it seems a bit weird
I think you will have to introduce a growth stage in between in liquid. So growth in liquid, spin down, plate and count CFUs.. but it seems a bit odd to do this as an experiment.