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tupike 12-02-2011 07:22 PM

"in between cultures"
 
Dear all,

I have the following problem: why do so many protocols (if not all) demand you to make a "in between culture" (or start culture).

When I am growing bacterial cells, the protocol tells me to inoculate 10ml of broth with a colony from a fresh agar plate.
Then I need to grow this till I reach an OD 0.6 , if so, I need to inoculate 500ml of broth and use these cells.

Now, I wonder: can I simply skip this "in between step" of inoculating 10 ml?

I see no reason why to do this.

Is it perhaps because when I use this in between step the bacteria will grow faster since I add bacteria in the exponential phase in the 500ml?
(although, this in between step takes lots of time too)

(it cant be because I need to make sure the bacteria are expressing the protein/enzyme to cope with the antibiotics, because I pick them from a fresh agar plate..., I can understand it if I would use an old plate or something, but when using a fresh palte:confused:)

If I could, I would try it out myself (skipping this step) but since my supervisor checks everything I do, I cant risk doing it and he hasnt really given me a good answer to why it is..

Any ideas?

mmorgan 12-03-2011 12:59 PM

Re: "in between cultures"
 
Quote:

Originally Posted by tupike (Post 437494)
Dear all,

I have the following problem: why do so many protocols (if not all) demand you to make a "in between culture" (or start culture).

When I am growing bacterial cells, the protocol tells me to inoculate 10ml of broth with a colony from a fresh agar plate.
Then I need to grow this till I reach an OD 0.6 , if so, I need to inoculate 500ml of broth and use these cells.

Now, I wonder: can I simply skip this "in between step" of inoculating 10 ml?

I see no reason why to do this.

Is it perhaps because when I use this in between step the bacteria will grow faster since I add bacteria in the exponential phase in the 500ml?
(although, this in between step takes lots of time too)

(it cant be because I need to make sure the bacteria are expressing the protein/enzyme to cope with the antibiotics, because I pick them from a fresh agar plate..., I can understand it if I would use an old plate or something, but when using a fresh palte:confused:)

If I could, I would try it out myself (skipping this step) but since my supervisor checks everything I do, I cant risk doing it and he hasnt really given me a good answer to why it is..

Any ideas?

If you're doing protein expression the starter culture is a critical step.

If you're just growing bugs to do a Maxi-prep you can skip it, although I find you get the best yields if you do a starter culture. I don't really pay attention to the OD600 when I make starter cultures for Maxis though. Just put 3 colonies into 3 separate 10ml cultures in the morning, grow until the end of the day, pick the one that looks like it's growing the best and chuck it into your large overnight culture.

You can just put a single colony directly into your 500ml overnight culture, but every now and again you may have a colony that doesn't grow. That's why setting up a few starter cultures is a good idea. Plus the bacteria grow much better if you start them in a small volume and then put that into a larger volume.

Jorge1907 12-03-2011 02:28 PM

Re: "in between cultures"
 
It is to establish control on your experirment, here working to a specific concentrstion as measured by optical optical density. It increases opportunity for reproducibility between experiments by control of a simple potential variable.
When given choice, drop the shortcuts and pursue rigor in your work.

tupike 12-03-2011 02:53 PM

Re: "in between cultures"
 
Quote:

Originally Posted by mmorgan (Post 437518)
If you're doing protein expression the starter culture is a critical step.

If you're just growing bugs to do a Maxi-prep you can skip it, although I find you get the best yields if you do a starter culture. I don't really pay attention to the OD600 when I make starter cultures for Maxis though. Just put 3 colonies into 3 separate 10ml cultures in the morning, grow until the end of the day, pick the one that looks like it's growing the best and chuck it into your large overnight culture.

You can just put a single colony directly into your 500ml overnight culture, but every now and again you may have a colony that doesn't grow. That's why setting up a few starter cultures is a good idea. Plus the bacteria grow much better if you start them in a small volume and then put that into a larger volume.

Ok, I understand what you mean, however, I am lost when you say:
"If you're doing protein expression the starter culture is a critical step."
What do you mean? You dont really explain it..?

Or do you refer to what I said about the expression of for example antibiotics?


Quote:

Originally Posted by Jorge1907 (Post 437521)
It is to establish control on your experirment, here working to a specific concentrstion as measured by optical optical density. It increases opportunity for reproducibility between experiments by control of a simple potential variable.
When given choice, drop the shortcuts and pursue rigor in your work.

Aha, I see, its to have a reproducibility!
Its not really about making short cuts, its about why they do it.
If there is no real reason, then I dont see the point in doing it... but now I know why they do it...


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