Hello all,
I find myself in need to perform serial dilution of E.coli cells for a luminometric ATP assay.
Normally for serial dilution I would use PBS, but from what I understood the concentration of salts (Na and K) is too high, which interferes with luciferase activity.
Diluting in DI water should cause too much stress, right? Passing from LB growth culture to nothing could cause shock.
Is there a different buffer that keeps the cells alive and well while not providing a too high concentration of monovalent metal ions?
EDIT:
I came up with a possible solution. I could centrifuge 5 ml of the culture broth with 5 ml of 0.1 Peptone water (to retrieve the cells, remove salts and not to shock the E.coli too much) at 5000 rpm in Falcon tubes, discard the supernatant and resuspend the pellet in Peptone water, which I would use for subsequent serial dilution. The time between dilution and test would be ~ 1 hour, so the bacteria would not suffer too much from the change of nutrients/osmotic pressure?
Dear Riccardo,
I hope your are using 1X PBS for serial dilution. If that is affecting your luciferase activity due to high salt concentration, you may try for 0.1X PBS to carry out serial dilution and luciferase activity. Hope that helps.
1% glycerol solution maintains the right osmotic pressure for most bacteria.
Dear Riccardo,
I hope this link is useful
Article Saline catholytes as alternatives to phosphate buffers in mi...
Thank you all for your answer.
In the end i went with the 0.1% yeast extract water. It did work fine, I could get a good enough calibration curve and plot the bacterial concentrations.
In the coming days I will test with the other solutions.
- Badges
- Science method