7. Add 2 μL of PI to reach a concentration of 3 μM PI in each
tube, and place the tube on ice for 5 min. PI is added more than
30 min after the bacteria solution had passed through the
membrane to allow the transiently created (reversible) pores
in the viable bacteria to close. As a result, only irreversibly
electroporated bacteria contained PI.
8. Measure the PI emission fluorescence intensity of each sample
at emission wavelength of 618 nm, using 540 nm light for
excitation.
9. Calculate the corresponding concentration of bacteria irreversibly electroporated or dead using PI emission fluorescence
intensity (see Note 19).
10. Calculate the percentage of irreversibly electroporated bacteria
by dividing the concentration of dead bacteria, obtained from
PI fluorescence intensity, by the concentration of bacteria that
passed through the membrane, obtained from the OD600.
11. Finally, calculate the percentage of reversibly electroporated
bacteria by subtracting the percentage of irreversibly electroporated bacteria (step 10) from the percentage of electroporated bacteria (step 6).
Second method of analysis by fluorescence microscopy (Fig. 5):
1. After electroporation, when all the bacteria have passed
through the membrane, collect 1 mL of the solution in the
Fig. 5 Microscope images of the bacteria in a drop of receiver solution after (a) an electroporation experiment
with a commercial electroporator with a voltage pulse of 2.5 kV, and (b) our flow-through electroporation
method with voltage pulses of 4 V. In each case, bright field and fluorescence images are overlaid. The bright
field images show all the bacteria. The fluorescence images show the electroporated bacteria. With a
commercial electroporator, 2% of the bacteria were found to be reversibly electroporated (YOPRO green),
and 7% irreversibly electroporated (YOPRO green and PI red). With our flow-through method, 22% of the
bacteria in the receiver solution were found to be reversibly electroporated, and 17% irreversibly
electroporated
Low-Voltage Flow-Through Electroporation Membrane and Method
51
tube, and place the tube on ice for 5 min. PI is added more than
30 min after the bacteria solution had passed through the
membrane to allow the transiently created (reversible) pores
in the viable bacteria to close. As a result, only irreversibly
electroporated bacteria contained PI.
8. Measure the PI emission fluorescence intensity of each sample
at emission wavelength of 618 nm, using 540 nm light for
excitation.
9. Calculate the corresponding concentration of bacteria irreversibly electroporated or dead using PI emission fluorescence
intensity (see Note 19).
10. Calculate the percentage of irreversibly electroporated bacteria
by dividing the concentration of dead bacteria, obtained from
PI fluorescence intensity, by the concentration of bacteria that
passed through the membrane, obtained from the OD600.
11. Finally, calculate the percentage of reversibly electroporated
bacteria by subtracting the percentage of irreversibly electroporated bacteria (step 10) from the percentage of electroporated bacteria (step 6).
Second method of analysis by fluorescence microscopy (Fig. 5):
1. After electroporation, when all the bacteria have passed
through the membrane, collect 1 mL of the solution in the
Fig. 5 Microscope images of the bacteria in a drop of receiver solution after (a) an electroporation experiment
with a commercial electroporator with a voltage pulse of 2.5 kV, and (b) our flow-through electroporation
method with voltage pulses of 4 V. In each case, bright field and fluorescence images are overlaid. The bright
field images show all the bacteria. The fluorescence images show the electroporated bacteria. With a
commercial electroporator, 2% of the bacteria were found to be reversibly electroporated (YOPRO green),
and 7% irreversibly electroporated (YOPRO green and PI red). With our flow-through method, 22% of the
bacteria in the receiver solution were found to be reversibly electroporated, and 17% irreversibly
electroporated
Low-Voltage Flow-Through Electroporation Membrane and Method
51