Hello, I work inducing aggregation of E. coli with polymers, I need to asses the metabolic activity and survivability of my bacteria under different polymers.
I am trying with FDA in DMSO, 5mg/mL. 1ug per every 10^6 cells.
How I am doing is to transfer the bacteria at a OD600=1(in LB, and some washed twice from the media) to 96 well plates, inoculate it with my polymers, then after that (without cooling or anything else) pipette the FDA. After that I analyze the fluorescence at ex:490nm and em:514nm.
Problem is that the fluorescence I am getting is really low, most of my cells should be metabolically active (at least the ones in LB), so I am doing something wrong.
I also have some questions about this assay:
1) Is it suitable to measure activity at different time points, like 2-4-6-8-24 hours? Or just to take a sample and measure at one point then discard the sample?
2) Do I need to get rid of the media?
3) Do I need to cool down the sample?
4) What is the lifespan of the die?
Hi Steve, thanks for taking time to clearing some questions.
My polymers are dissolved in Acetic Acid in water with a pH of 2.8, I add 47uL of this to a total volume of 1mL of either bacteria on LB or centrifuged and washed to get rid of the media. I am still tweaking the experiment, so far I experienced that if I do not neutralize the pH prior adding the FDA I won't get fluorescence.
José,
As Steve mentions, it is the hydrolysis product of FDA which is the source of fluorescence: fluorescein. The absorption and emission of fluorescein are strongly pH dependent, which may in part explain the response you mention. A useful paper that describes this pH dependence is J. Luminescence 10 [1975] 381-390; it shows how quantum yield varies significantly with pH. [I've been involved in the industrial manufacture of FDA years ago - I echo Steve's point that FDA will hydrolyse to fluorescein in the presence of water: its concentration along with pH and temperature are influences on rate of deacetylation.]
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