Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo

How to passage bacteria ?

passage bacteria plasmid stability

  • Please log in to reply
11 replies to this topic

#1 Meg P. Anula

Meg P. Anula

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 75 posts
1
Neutral

Posted 21 August 2015 - 10:59 PM

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?

 



#2 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 22 August 2015 - 04:09 AM

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?


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 Meg P. Anula

Meg P. Anula

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 75 posts
1
Neutral

Posted 23 August 2015 - 12:57 AM

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.



#4 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 23 August 2015 - 01:05 AM

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.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts
304
Excellent

Posted 23 August 2015 - 07:21 AM

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.



#6 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 23 August 2015 - 07:39 AM

I think he means with 30 generations literally 30 passages as in "30 times on a plate"

 

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.

 


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#7 phage434

phage434

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 2,747 posts
304
Excellent

Posted 23 August 2015 - 11:53 AM

Well, that's (in my opinion) a very peculiar definition of a "generation."



#8 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 23 August 2015 - 11:57 AM

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).


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#9 Meg P. Anula

Meg P. Anula

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 75 posts
1
Neutral

Posted 23 August 2015 - 07:27 PM

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.


Edited by Meg P. Anula, 23 August 2015 - 07:28 PM.


#10 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 24 August 2015 - 02:40 AM

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.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#11 Meg P. Anula

Meg P. Anula

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 75 posts
1
Neutral

Posted 24 August 2015 - 08:26 PM

I guess it makes sense to grow the bacteria in liquid form for few hours before plating.

 

When I referred to journal, it only mentioned to passage the bacteria for 30 times and count the cfu in the agar each passage and compare with control.

 

I am stucked at how to passage the bacteria (agar to agar).



#12 pito

pito

    Veteran

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,464 posts
100
Excellent

Posted 24 August 2015 - 11:07 PM

Best to just email the authors of that paper. What paper is it?

 

Restreaking will of course not be an option if you really want to count CFUs.


If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.






Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.