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.