Figure 3. Flow cytometric detection of the bacterial
populations in terms of side scattering and forward scattering without
(a) and with (b) Ag+ incubation. (c) Side scattering
intensity distribution of the bacteria with and without
Ag+ incubation. (d) Schematics of back-illumination
microscopy. (e) Bright-field image and (f) back-illumination image of
bacteria in a same region of interest.
We further seek to distinguish the individual ‘strongly reducing’
bacteria visibly. Bright field microscopy is first applied to the
bacteria mixture after Ag+ incubation and washing with
PBS buffers. Figure 3e shows that in the view over 100 bacteria is seen
which yet exhibit very different contrasts. While most bacteria appear
to be greyer than the background, a small portion shows somewhat even
darker which is not observed in the control group (see Figure S2).
Although bright field microscopy allows the observations of all the
bacteria in the optical field, the difference in the contrasts between
different bacteria is sometimes not very distinct (e.g. the upper left
inset in Figure 3e). To improve the situation, we have developed an
alternative imaging mode utilising the back reflection of a light beam
that is able to presumably better distinguish between different types of
bacterial cells. As shown in Figure 3d, the technique basically uses the
same light path as that in fluorescent microscopy where a light beam
illuminates the bacteria and reflect back to the camera, but in this
back-reflection mode the light is not restricted to the narrow range of
wavelength for fluorophore excitation. Consequently, Figure 3f insets
show a more significant contrast between these bacteria – much greater
optical intensity for the darker bacteria seen using the bright field
mode (Figure 3e insets), while far lower intensity for the rest
population. Moreover, the former type of bacteria is observed to be
apparently fewer than the latter in the back reflection imaging mode,
which is consistent with the results of the flow cytometric
measurements. We thus assume the imaging mode is more sensitive to the
internal silver nanoparticles. This imaging mode is therefore considered
to be a well-suited approach to discern the bacteria of a strong
capacity of silver metal metabolism from those of an average ability.