A LIVE/DEAD BacLightTM bacterial viability kit (Invitrogen, USA)
was used to discriminate between viable cells and dead cells in
sludge samples. This kit contains the green-fluorescent nucleic acid
stain, SYTO 9, and the red-fluorescent nucleic acid stain, propidium
iodide. According to the method and principle described in
the kit instructions, SYTO 9 and propidium iodide were mixed
evenly, after which the mixture was used for the sludge samples.
Consequently, bacteria with intact cell membranes (viable cells)
in sludge stained green fluorescence, whereas bacteria with damaged
membranes (dead cells) stain red fluorescence. For each sample,
more than 10 images were taken at random using a
fluorescence microscope (Eclipse 80i, Nikon, Japan). Quantification
of viable and dead cells was performed based on green and red fluorescence
intensity analyzed by ImageJ software.