Getting Bacteria from an OD0.8 to an OD 0.1 - (Jun/15/2013 )
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?
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?
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 ..
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
I second Leelee's comment - check out the Beer-Lambert Law...
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).