receiver chamber in a 2 mL Eppendorf tube, and place the tube
on ice for 30 min.
2. Add 4 μL of PI, and place the tube on ice for 5 min.
3. Centrifuge the tube at 5500 rpm (4100 Â g) for 5 min at 4
C.
4. Remove the supernatant, and place one drop of the cell pellet
onto a clear microscope slide.
5. Observe under a fluorescence microscope, first taking a brightfield image, and then a blue fluorescent image at multiple places
on the slide (Fig. 5).
6. Use Image J software to count all of the bacteria (electroporated and nonelectroporated) on the bright-field images, and
the green (YOPRO) reversibly electroporated bacteria and the
red-orange (YOPRO and PI) irreversibly electroporated bacteria on the fluorescent images.
7. Calculate the corresponding percentages of reversibly electroporated bacteria by dividing the number of green bacteria by
the total (bright-field) number of bacteria, and the percentage
of irreversibly electroporated bacteria by dividing the number
of red-orange bacteria by the total number of bacteria.
4 Notes
1. Typically the solutions are used on the same day that they are
prepared.
2. Methanol and trifluoroacetic acid are toxic, and therefore,
solutions should be prepared under a fume hood with
proper personal protective equipment. Before addition of trifluoroacetic acid, the tin chloride solution (pH~7) is milky
white due to the precipitation of tin(II) hydroxide. Upon
addition of the acid, the solution turns clear, and the precipitate
is dissolved.
3. Ammonium hydroxide is corrosive. The silver nitrate solution
turns brown due to the precipitation of silver(I) hydroxide.
Further addition of ammonium hydroxide makes the solution
transparent again due to the formation of a soluble Ag(NH 3 ) 2
+
complex.
4. Formaldehyde is toxic; therefore, the gold plating solution
should be prepared under a fume hood with proper personal
protective equipment.
5. All equipment and solutions used should be sterile. Autoclave
for 15 min at 121
C.
6. The LB (Lennox) microbial medium powder contains 10 g/L
tryptone, 5 g/L yeast extract and 5 g/L sodium chloride.
52
Juliette Experton et al.
on ice for 30 min.
2. Add 4 μL of PI, and place the tube on ice for 5 min.
3. Centrifuge the tube at 5500 rpm (4100 Â g) for 5 min at 4
C.
4. Remove the supernatant, and place one drop of the cell pellet
onto a clear microscope slide.
5. Observe under a fluorescence microscope, first taking a brightfield image, and then a blue fluorescent image at multiple places
on the slide (Fig. 5).
6. Use Image J software to count all of the bacteria (electroporated and nonelectroporated) on the bright-field images, and
the green (YOPRO) reversibly electroporated bacteria and the
red-orange (YOPRO and PI) irreversibly electroporated bacteria on the fluorescent images.
7. Calculate the corresponding percentages of reversibly electroporated bacteria by dividing the number of green bacteria by
the total (bright-field) number of bacteria, and the percentage
of irreversibly electroporated bacteria by dividing the number
of red-orange bacteria by the total number of bacteria.
4 Notes
1. Typically the solutions are used on the same day that they are
prepared.
2. Methanol and trifluoroacetic acid are toxic, and therefore,
solutions should be prepared under a fume hood with
proper personal protective equipment. Before addition of trifluoroacetic acid, the tin chloride solution (pH~7) is milky
white due to the precipitation of tin(II) hydroxide. Upon
addition of the acid, the solution turns clear, and the precipitate
is dissolved.
3. Ammonium hydroxide is corrosive. The silver nitrate solution
turns brown due to the precipitation of silver(I) hydroxide.
Further addition of ammonium hydroxide makes the solution
transparent again due to the formation of a soluble Ag(NH 3 ) 2
+
complex.
4. Formaldehyde is toxic; therefore, the gold plating solution
should be prepared under a fume hood with proper personal
protective equipment.
5. All equipment and solutions used should be sterile. Autoclave
for 15 min at 121
C.
6. The LB (Lennox) microbial medium powder contains 10 g/L
tryptone, 5 g/L yeast extract and 5 g/L sodium chloride.
52
Juliette Experton et al.