During this calibration part we obtained two particle standard curves which are important for proper cell measurement. However, we can observe a curve in the log scale graph (Fig. 1), although it should have a 1:1 slope. We assume that this inconsistency could have been due to pipetting errors or an oversaturated detector.

3. GRAPHING A FLUORESCEIN FLUORESCENCE STANDARD CURVE

In the last part of the calibration we prepared a dilution series of fluorescein in four replicates and measured the fluorescence. During this calibration part we generated a standard curve of fluorescence for fluorescein concentration.

Fig. 3 Standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration.

Fig. 4 A standard curve of fluorescein generated by measuring the fluorescence of serial dilution stock (µM). Fluorescence is plotted against the fluorescein concentration on a logarithmic scale.

During this calibration part we generated a standard curve of fluorescein. Standard curves (linear and on a logarithmic scale) have a 1:1 slope which ensures us that there were no significant mistakes during this calibration part and the data can be used for cell measurement. This allows us to successfully convert cell based readings to an equivalent fluorescein concentration.

Cell Measurements

For cell measurements we used the same settings that we used in our calibration measurements. At first, according to the standard protocol we transformed cells with 8 different plasmids (Tab. 1). We picked 2 colonies from each transformation plates and inoculated in 5-10 mL LB medium + Chloramphenicol. We grew the cells overnight (16-18 hours) at 37 °C and 220 rpm. After that we diluted the cultures to a target Abs₆₀₀ of 0.02. We took samples from these diluted cultures prior to incubation and after 6 hours of incubation measured Abs600 (Fig. 5) and fluorescence (Fig. 6).

Fig. 5 Graph comparing the raw Abs₆₀₀ prior incubation and at hour 6 for each colony using each control/device

Fig. 6 Graph comparing the raw fluorescence prior to incubation and at hour 6 for each colony using each control/device

Comparing absorbance and fluorescence of cells prior to incubation and after 6 hours we can observe that absorbance as well as fluorescence were more intense after 6 h of incubation as it was expected. Based on the assumption that one bacterial cell gives rise to one colony, colony forming units per 1 mL of an OD₆₀₀ = 0.1 culture was calculated by counting the colonies on each plate with fewer than 300 colonies and multiplying the colony count by the Final Dilution Factor on each plate The results are shown in Tab. 3.