According to the previous method, ruthenium red staining assessed EPS production (Adnan et al., 2020 (link)). Cell suspensions (106 CFU/ml) of E. coli O157:H7 and different concentrations of Ech were cultured in a 96-well plate for 24 h at 37°C. The plate was washed with PBS (pH = 7.2), stained with 0.01% ruthenium red (Yuanye, Shanghai, China), and then incubated at 37°C for 1 h. 0.01% ruthenium red was used to fill the wells without biofilm was used as blank. 0.01% ruthenium red was used to fill the wells with biofilm and without Ech was used as a positive control. The absorbance was performed at 450 nm using Multiskan Go Reader after the liquid carrying the residual stain was transferred to new 96-well plates (Thermo Fisher Scientific, USA). EPS inhibition was calculated as follows formula (Adnan et al., 2020 (link)):
Whereas:
AB = absorbance of the blank.
AS = absorbance of the sample.
AP = absorbance of the positive control.
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