The purpose of this InterLab measurement is to try to reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of optical density (OD).
Two orthogonal approaches were used as follows:
1. Converting between absorbance of cells to absorbance of a known concentration of beads;
2. Counting colony-forming units (CFUs) from the sample.
Calibration 1: OD600 Reference Point
We first used LUDOX CL-X (45% colloidal silica suspension) to determine the correction factor to covert Abs600 to OD600. Four replicates of the LUDOX and H2O were measured to determine the corrected absorbance and will be used as correction factor for OD600 of bacterial culture.
Table 1. Absorbance measurement of LUDOX and H2O and correction factor table.
Calibration 2: Particle Standard Curve
Serial dilutions of monodisperse silica microspheres were prepared in 96 well plate and Abs600 were measured. The measurement will be used to construct a standard curve of particle concentration, which uses the Abs600 measurements to estimate the number of cells.
Figure 1. Graph of particle standard curve in linear scale.
Figure 2. Graph of particle standard curve in logarithmic scale.
Calibration 3: Fluorescence Standard Curve
Serial dilution of fluorescein was prepared in a 96-well plate and the fluorescence was measured at 485 nm excitation and 525 nm emission. The measurement will be used to construct a standard curve of the fluorescence at different fluorescein concentrations which can convert the cell-based reading to an equivalent fluorescein concentration.
Figure 3. Graph of fluorescein standard curve in linear scale.
Figure 4. Graph of fluorescein standard curve in logarithmic scale.
On Day 1, E. coli K-12 DH5-α was used to transform with devices from distribution kit following the recommended protocol. On Day 2, two colonies from each of the plates were picked and were inoculated in 5 mL of LB medium with chloramphenicol overnight at 37°C and 220 rpm. On Day 3, the overnight cultures were diluted by 1:10 dilution. Abs600 were then measured and the cultured further diluted into Abs600 of 0.02 in a final volume of 12 mL of LB medium with chloramphenicol in a 50 mL falcon tube. 500 μL samples of the diluted cultures at 0 hours were pipetted into 1.5 ml microtubes (on ice) and the remained cultures were incubated at 37°C and 220 rpm for 6 hours. Later, another 500 μL samples of the incubated cultures were pipetted into 1.5 ml microtube (on ice) before all the samples were being transferred into 96 well plate and measured in plate reader of the Abs600 and fluorescence.
Table 2. Abs600 raw readings at zero hour.
Table 3. Abs600 raw readings at six hour.
Table 4. Fluorescence raw readings at zero hour.
Table 5. Fluorescence raw readings at six hour.
Net Abs600 Measurement
Grow rate of the cells were measure using Abs600. From Figure 4 below, device 5 show the slowest growth rate as the increase in Abs600 was the lowest.
Figure 5. Graph of Net Abs600 at zero hour.
Figure 6. Graph of Net Abs600 at six hour.
Net Fluorescein a.u. Measurement
Based on the plate reader we used, the positive control, device 1 and 4 showed fluorescein reading at significantly higher values than the rest of the samples after 6 hour of incubation.
Figure 7. Graph of Net Fluorescein a.u. at zero hour.
Figure 8. Graph of Net Fluorescein a.u. at six hour.
uM Fluorescein/OD600 Measurement
From Figure 10, the negative control, device 3 and 6 showed uM fluorescein/OD at values significantly lower than other samples, while device 1 show the highest value of uM fluorescein/OD.
Figure 9. Graph of uM Fluorescein/OD600 at zero hour.
Figure 10. Graph of uM Fluorescein/OD600 at six hour.
MEFL / Particle Measurement
From Figure 12, again, the negative control, device 3 and 6 showed MEFL/particle at value significantly lower than that of other samples, while device 1 showed the highest value.
Figure 11. Graph of MEFL/Particle at zero hour.
Figure 12. Graph of MEFL/Particle at six hour.
Devices 1 showed the highest fluorescein reading, while the net Abs600 indicated the cells only grew at moderate rate compared to others. Devices 1 also showed the highest value of uM fluorescein/OD and MEFL/particle at 6 hour of incubation. This might indicate that growth of cells with device 1 was inhibited. On the other hand, device 4 showed the highest Abs600 and the second highest fluorescein reading after device 1. Value of uM fluorescein/OD and MEFL/ particle after 6 hours of incubation in device 4 also only came after device 1. Therefore, we can conclude that device 4 showed the best fluorescein while maintaining growth rate.