Fluorescence spectroscopy protocols to measure bacterial survival rate
LIVE/DEAD BacLight Bacterial Viability Kit from Invitrogen can be utilized to quantitatively analyze cell viability rates using a spectrofluorometer (Jasco FP-6500) by following appropriate fluorescence spectroscopy protocols. To measure cell viability rate of the sample in a microchip after a specific period of time, a peristaltic pump (P720, INSTECH Inc.) is used to pump solutions out of the microchip into a quartz fluorescence cuvette for the spectrofluorometer. The procedure is as follows,
i. Measure the fluorescence emission spectrum with an excitation wavelength of 470 nm and an emission wavelength ranging from 490 to 700 nm.
ii. Calculate the ratio of the integrated intensity of the portion of each spectrum between 510 to 540 nm to that between 620 to 650 nm, that is
iii. Calculate the ratio at different reaction time periods, for example 5, 10, 15, 30, 45 and 60 minutes. Correlate the ratio with the cell viability rate.
iv. Repeat the procedure several times (in our case, 5 times) for each period of time and calculate the average cell viability rate as well as minimum and maximum rates for each period of time. A plot of cell viability rate against reaction time can be obtained.
To correlate the green/red fluorescence ratios with cell viability rates, characterization is needed for pre-defined cell viability rates. The procedure is as follows,
i. Mix five different proportions of the bacterial suspensions in 1 cm quartz fluorescence cuvettes as shown in Table S1. The total volume of each of the five samples is 1 mL.
Table S1. Recipe of various proportions of live:dead cells for fluorescence spectroscopy
Ratio of live:dead cells / Live cell suspension (mL) / Dead cell suspension (mL)0:100 / 0 / 1
10:90 / 0.1 / 0.9
50:50 / 0.5 / 0.5
90:10 / 0.9 / 0.1
100:0 / 1 / 0
ii. Prepare a combined reagent mixture in a microfuge tube by adding 10 µL of SYTO 9 and 10 µL of propidium iodide. Add 3 µL of the mixture to each of the five samples and mix thoroughly by pipetting up and down several times. Incubate at room temperature in the dark for 15 min.
iii. Measure the fluorescence emission spectrum (excitation wavelength of 470 nm, emission wavelength of 490-700 nm) of each cell suspension in the spectrofluorometer as shown in Figure S1(a). Calculate the ratio of the integrated intensity of the portion of each spectrum between 510-540 nm to that between 620-650 nm for each bacterial suspension. Plot the ratio of integrated green fluorescence to integrated red fluorescence versus cell viability rate shown as black dots in Figure S1(b).
iv. Since green/read fluorescence ratio versus cell viability rate is considered linear, least-squares curve fitting is performed to obtain a relationship between the two parameters based on the result obtained in the last step. Cell viability for a specific green/read fluorescence ratio is determined using the black line shown in Figure S1(b).
(a)
(b)
Figure S1. Analysis of relative viability of E. coli suspensions by fluorescence spectroscopy. (a) Fluorescence emission spectra of various proportions of live and dead E. coli; (b) Green/Red fluorescence ratio versus cell viability with least-squares curve fitting.