CFU Assay

In order to determine the number of viable cells in a liquid culture, we used a Colony Forming Unit (CFU) assay. This simple assay involves the collection and serial dilution of samples of a liquid culture. These dilutions are then plated and colonies are counted. To determine the number of CFUs per milliliter, you use this formula:

CFU/mL = (# of colonies*dilution factor)/volume plated in mL’s

We used this assay to generate a standard curve representing the effect of ampicillin concentration on CFU/mL of liquid E. coli cultures with no antibiotic resistance.

Here is the curve we have generated:

We plan to use this curve to determine the concentration of ampicillin in media that we treat with our project.

Dialysis

In order to remediate antibiotics in liquids without releasing cells into the liquid, we used a 1000KD molecular weight cut off (MWCO) dialysis tube. By placing our beta-lactamase producing cells in this tube and placing this tube in an ampicillin containing liquid, we hope to release the beta-lactamase enzyme into the liquid and degrade the ampicillin.

We ran this test with 10ml (OD600 = 4.628) of cells containing an ampicillin resistant plasmid encoding beta-lactamase in the dialysis tube. We took samples of the ampicillin-containing media every hour and used those samples for another CFU assay.

It is clear that the concentration of ampicillin in the media is decreasing, but there is still work to be done to be sure that it is the dialysis treatment and not heat or pH having this effect. We will have those results come the Jamboree!