13. It is important to completely remove any traces of fixative as
this might result in crystalline microstructures present in samples following dehydration. It is helpful to use a thin pipette tip
to completely remove liquid in wells after each wash step
following fixation.
14. For rapid procedures, each EtOH concentration can be used
once. For complete dehydration, it is advisable to incubate
samples in each EtOH concentration twice for 15 min each.
For example, after 15 min of incubation in 25% EtOH, fresh
25% EtOH is used for a second incubation before the next
concentration of 40% EtOH is used.
15. To analyze the internalization capability of bacteria, further
incubation after the invasion process is required. We have
observed that internalization of the invasive E. coli into the
cytoplasm takes place between one- and three-hours post invasion. Complete culture media containing 80 μg/mL gentamicin can be used to eliminate external bacteria in the first hour
following invasion. To observe internalization of E. coli at later
timepoints, cells should be maintained in complete media containing 20 μg/mL gentamicin until their fixation timepoint.
16. Cells will attach more readily to plastic coverslips compared to
glass coverslips. Care should be taken when rinsing cell monolayers growing on glass coverslips. Culture media, wash buffer,
and fixative solutions should be added gently to the side of
wells.
17. We have found that working with solutions chilled to 4
C
following sample fixation works best for preserving sample
intactness. Transitioning between cold and warm conditions
once the samples have been fixed increases the likelihood of
damaging the samples by introducing cracks in the cell membranes, and by detaching bacterial cells that would be otherwise properly attached to the membrane.
18. Both propidium iodide and acridine orange bind strongly to
DNA and passively to the cytoplasm and cell membrane. Cells
can be washed extensively if the passive staining of the cytoplasm and cell membrane is overwhelming compared to DNA
stained in the nucleus.
19. In place of specialized mounting reagents, a small drop of
glycerol can be used as a mounting reagent. Lint-free tissue
can be used to wipe away excess glycerol from the mounted
microscope slide. Coverslips should be prevented from moving
on the glass slide when mounted by gently dabbing excess
glycerol instead of wiping.
High-Resolution Imaging of Bacterial Invasion
39
this might result in crystalline microstructures present in samples following dehydration. It is helpful to use a thin pipette tip
to completely remove liquid in wells after each wash step
following fixation.
14. For rapid procedures, each EtOH concentration can be used
once. For complete dehydration, it is advisable to incubate
samples in each EtOH concentration twice for 15 min each.
For example, after 15 min of incubation in 25% EtOH, fresh
25% EtOH is used for a second incubation before the next
concentration of 40% EtOH is used.
15. To analyze the internalization capability of bacteria, further
incubation after the invasion process is required. We have
observed that internalization of the invasive E. coli into the
cytoplasm takes place between one- and three-hours post invasion. Complete culture media containing 80 μg/mL gentamicin can be used to eliminate external bacteria in the first hour
following invasion. To observe internalization of E. coli at later
timepoints, cells should be maintained in complete media containing 20 μg/mL gentamicin until their fixation timepoint.
16. Cells will attach more readily to plastic coverslips compared to
glass coverslips. Care should be taken when rinsing cell monolayers growing on glass coverslips. Culture media, wash buffer,
and fixative solutions should be added gently to the side of
wells.
17. We have found that working with solutions chilled to 4
C
following sample fixation works best for preserving sample
intactness. Transitioning between cold and warm conditions
once the samples have been fixed increases the likelihood of
damaging the samples by introducing cracks in the cell membranes, and by detaching bacterial cells that would be otherwise properly attached to the membrane.
18. Both propidium iodide and acridine orange bind strongly to
DNA and passively to the cytoplasm and cell membrane. Cells
can be washed extensively if the passive staining of the cytoplasm and cell membrane is overwhelming compared to DNA
stained in the nucleus.
19. In place of specialized mounting reagents, a small drop of
glycerol can be used as a mounting reagent. Lint-free tissue
can be used to wipe away excess glycerol from the mounted
microscope slide. Coverslips should be prevented from moving
on the glass slide when mounted by gently dabbing excess
glycerol instead of wiping.
High-Resolution Imaging of Bacterial Invasion
39