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Getting Bacteria from an OD0.8 to an OD 0.1


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#1 biology_06er

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Posted 15 June 2013 - 07:06 PM

Hi there,

Say I have 50mL of bacteria at an OD0.8 and I need to get bacteria to a reading of 0.1..this is what I've been doing in the past but just want to confirm that what I've been doing in correct.

So from my 50mL, I took 1mL, spun and then resuspended the pellet in 1mL of PBS. (I now have 1mL of bacteria at OD0.8 in PBS)
I took 125uL from this and added to 875uL of PBS (OD has gone down 8x now)

Is this correct--would this now give me 1mL of bacteria at an OD0.1?

Thanks,
biology_06er

#2 leelee

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Posted 15 June 2013 - 10:24 PM

What do you need the bacteria for?

The reason I ask is that when I am making competent bacteria, it is vital that the OD reading be on the original culture- as this is an indication of growth phase and will affect the quality of my end product cells (their transformability).
So if I did what you did, it would be pointless, as the cells would still have grown to the 0.8 density. If that makes sense?

#3 biology_06er

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Posted 15 June 2013 - 10:34 PM

It's because I want to add a certain about of bacteria to my assay..I know roughly how many are contained in an OD0.1 so from there I can add the correct volume ..

#4 leelee

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Posted 15 June 2013 - 11:03 PM

Ah ok.

In terms of consistency between your experiments- this is probably fine. In that you will be adding the same number of cells each time (regardless of what this number is).

But, and I *should* know this, is the relationship between OD and cell number linear (I have a feeling it isn't?).
Because then the number of cells in your dilution (1/8 of an OD 0.8 culture) won't be the same as the number of cells in 1ml of a culture at OD of 0.1

#5 bob1

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Posted 16 June 2013 - 12:25 PM

I second Leelee's comment - check out the Beer-Lambert Law...

#6 phage434

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Posted 16 June 2013 - 07:14 PM

Yes, it is linear. An OD of 0.8 has 8x the number of cells than an OD of 0.1 (to the extent that the cells are absorbing rather than reflecting, and modulo a whole lot of other factors making OD measurements very crude and inconsistent between instruments, especially).




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