3.1. Fluorescence analysis of the superoxide radical level in
individual bacteria
The SR levels of individual bacteria were analysed using fluorescence
microcopy. Experimentally, the mature bacteria (OD=1.0) were first
resuspended in PBS buffer solutions and imaged using bright field
microscopy. Figure 1a shows a total number of nearly 200 separate
entities of bacteria in the view. Next, dihydroethidium (DHE) was added
into the bacterial solution for use as a specific fluorescence probe to
uncover the SR levels with a positive response. The DHE-uptaken bacteria
were then immoblised on to a sodium alginate-modified coverglass and
imaged using fluorescence microscopy at an excitation light wavelength
of 510 nm. Consequently, Figure 1b presents all the ca. 200
bacteria observed in Figure 1a which now appear red, suggesting the
superoxide radical generation is active all over the bacterial colony.
The inset with a much better contrast than that in Figure 1a further
resolves the structure of each entity of the bacteria and shows their
predominant existence as tetracocci. Importantly, it is seen that within
the bacterial culture, their fluorescence (FL) intensities differ from
each other. The FL intensity distribution as shown in the Figure 1c was
obtained by measuring the integral intensity of the 2D area of each
bacteria. It is therefore clear that individual bacteria exhibit
different SR levels inside them, indicating the differences of their
antioxidant capacities within the colony.