Calibration polystyrene bead standards
To account for green fluorescence and light scattering and transmittance without having to grow the bioreporter bacteria every time, we have made the following calibration standards.
Polysytrene spheres are quite expensive. Until there are alternatives...
We need non-fluorescing beads as well as green-fluorescent beads of similar sizes.
Here, for non-fluorescent beads, we used fluorescent beads that were lying around that are excited below the 488nm excitation wavelength of eGFP.
It also helps if the beads are charged (often chemically modified to have charges on the surface, and not hydrophobic) so they are less likely to clump in a physiological buffer.
|Approx. mean particle size||1 micrometer||1 micrometer|
|Source||Duke Scientific B0100||Sigma-Aldrich L1030|
First, we make sure that (A) the absorbance of your stock solutions is in the range that is interesting for the bioreporter test, and (B) both the non- and green-fluorescing beads give approximately the same absorbance readings.
Use a physiological buffer like PBS.
Dilute the Beads
- Using what the manufacturer tells us as a ball-park (1% and 2.5% solids), first dilute with buffer so that they are both 1%.
- Check the absorbance (optical density, OD)
- Match the absorbance on the 2 beads emperically (by trial and error)
- If you see clumps in the beads, try to filter them out.
- The bioreporter test bacteria OD is 0.3-0.6, so make sure the calibration has OD above and below
Make the Standard
Scattering and Transmittance Standard
This standard is used to calibrate the scattering (blue LED) and the transmittance (red LED).
We use the 'non-fluorescent' beads at varying dilutions.
*OD was calibrated with a 'lab standard' instrument
eGFP Fluorescence Standard
This standard is used to calibrate the green fluorescence (blue LED).
Here, the Fluorescence varies while the total scattering and transmittance is kept constant (OD=0.6). This is a "mock" bioreporter assay - where each of the vial has more or less the same number of bacteria, but depending on the eGFP expression, there is more fluorescence. While the bacteria would all have some green fluorescence, here, we have different proportions of green fluorescent beads.
|Vial||Green bead stock No.2 [ml]||'Non-fluorescing' bead stock No.2 [ml]||x concentration|