Figure 4. Observation of cell morphology using SEM. (a, b)
Control culture. (a) ×5.0k, (b) ×10.0k. (c, d) IC50culture. (a) ×5.0k, (b) ×10.0k. Culture was carried out at 37 ℃ and 200
rpm for 12 h in test tubes.
Furthermore, the cells cultured on glass slides were observed using
CLSM. The cells were stained with Calcofluor White (blue fluorescence),
SYTO 9 (green fluorescence) and PI (red fluorescence) and were examined
using CLSM. Calcofluor White has high specificity to specifically bind
to β-1,3 and β-1,4 polysaccharides in cellulose and chitin (Zhao et al.,
2020), which are the major components of EMs (Cui et al., 2019). SYTO 9
could stain the cells with intact cell membranes and PI could stain the
cells with damaged cell membranes (Takenaka et al., 2001). All samples
were stained with the three dyes. As shown in Fig. 5, when indoxyl was
absent, blue fluorescence was concentrated and strong. This phenomenon
indicates that large amounts of EMs were formed. In the presence of
indoxyl, blue fluorescence decreased compared with the control group,
and the higher concentration of indoxyl resulted in a weaker blue
fluorescence, indicating that the presence of indoxyl can inhibit the
formation of EMs. Moreover, indoxyl decreased green fluorescence and
increased red fluorescence, indicating the destruction of the cell
membrane by indoxyl. When indoxyl was 0.6 g/L, no emission was detected,
indicating that S. aureus could not grow under such a condition.