First, we put our bacteria in a kanamycin-LB solution to grow overnight, since we only wanted the kanamycin resistant ones to survive. To get a 50mg/ml solution, we diluted 0.5g of solid kanamycin into 0.5ml of ddH2O (double distilled water).
The following day, we diluted our colonies and put them on agar-LB-Kanamycin plates to be able to count them later.
We also made a glycerol stock to be able to store the bacteria at -80°C during a longer time. For that, we used:
- 0.5 ml of 40% gylcerol (it will prevent the water to expand while freezed, and preserve the bacteria)
- 0.5 ml of our sample bacteria (As & eGFP-only E.Coli)
We poured the glycerol in the vial first to avoid contamination, then gently vortex the solution and stored it on dry ice.
In parallel to the plating and glycerol stock making, we made some tests with our prototype with 6 dilutions of eGFP–bacteria (the data are still in processing). In addition, we did some control measures with a fluorometer.
The results below show a diminution of fluorescence in function of dilution following a linear behaviour (Fig 1) as expected.
- For the excitation (Fig 2) and emission (Fig 3) wavelengths, we can see that the maximum values are around 488 nm and 509 nm as expected (detection of eGFP). The greater dilutions are not visible, as they have each time a 10 times smaller signal.
These measures show that the bacteria is indeed expressing eGFP and that the fluorescence is dependent of the concentration.
Unfortunately, we had some problems with the measures for the prototype testing, so we will make some more in another wet-lab session.
For the bacterial counting, we used ImageJ, But the pictures we took were lacking contrast, so we draw black dots on the colonies. Then, we set the threshold (Image/Adjust/Threshold) so only the dots remain visible and let the programm count them (Analyze/Analyze particles). Do not forget to convert the image to 8-bit (Image/Type), so that the colors do not interfere with the processing.
The program counted 106 colonies.