Difference between revisions of "Team:NUS Singapore-Sci/Wetlab/Interlab"

This year, our team took part in the 2018 InterLab Study, which aims to standardise the fluorescence levels of bacteria transformed with different devices conferring it fluorescence. This is accomplished through the use of a plate reader. The plate reader we used was Synergy H1 Hybrid Multi-Mode Reader (courtesy of SynCTI?), which could read both absorbance and fluorescence.

For all the following measurements, we did not use pathlength correction, and kept the temperature setting to room temperature (20-25°C). The bandpass width is fixed at 16 nm. Bottom optics are used.

Before cell measurements were taken, three different calibration protocols were done.

1) OD600 Reference Point - LUDOX Protocol

LUDOX CL-X (45% colloidal silica suspension) was used to convert the Absorbance at 600 nm as obtained from the plate reader into a comparable OD600 measurement as obtained from a spectrophotometer. Here, pathlength correction is especially important to be switched off so as to not provide correction for the volume of sample in the 96-well plate.

Table 1. Results of Abs600 of LUDOX CL-X and H2O.

As shown in Table 1, the conversion factor to convert Abs600 measured using the plate reader to OD600 readings is by multiplying by 3.706.

2) Particle Standard Curve - Microsphere Protocol

A dilution series of monodisperse silica microspheres were prepared in the 96-well plates and the Abs600 measured. The microspheres have size and optical characteristics that mimic that of cells, so with a known amount of microspheres in each well, the Abs600 data will be able to give an estimate of the number of cells present in the well.

Table 2. Results of Abs600 for increasing particle numbers.

From the results as in Table 2, a particle standard curve (Figure 1) is plotted. A graph of Abs600 against particle count per 100 uL is shown in Figure 1.

A log scale particle standard curve was also plotted, where the values for particle count per 100 uL were applied the logarithmic number to obtain the graph as in Figure 2.

3) Fluorescence standard curve - Fluorescein Protocol

This calibration protocol was done to compare fluorescence values obtained from the test devices between teams, as different plate readers report different arbitrary units of fluorescence. Here, fluorescein is used in place of green fluorescent protein (GFP) as it is cost-effective, easy to prepare, and has similar excitation and emission properties to GFP. The excitation maximum used was 494 nm and the emission maximum used was 525 nm.

A dilution series of fluorescein were prepared in the 96-well plates and the fluorescence measured. This will enable us to convert the fluorescence readings obtained for the cells later on in the experiment to actual fluorescein (and hence GFP) concentrations.

Table 3. Fluorescence values against concentration of fluorescein.

Based on the raw fluorescence values obtained in Table 3, a fluorescein standard curve of fluorescence against fluorescein concentration was plotted (Figure 3).

A log scale particle standard curve was also plotted, where the values for particle count per 100 uL were applied the logarithmic number to obtain the graph as in Figure 4.