Simultaneous Gram and viability staining on activated sludge exposed to erythromycin: 3D CLSM time-lapse imaging of bacterial disintegration

The effect of erythromycin on activated sludge bacteria according to their Gram type was investigated with 3-dimensional Confocal Laser Scanning Microscopy (CLSM) time-lapse imaging. The fluorescent stains SYTOX Green and Texas Red-X conjugate of wheat germ agglutinin stained dying bacteria and Gram+ bacteria respectively. Time-lapse imaging allowed an understanding of the staining mechanism and the measurement of the death rate. In presence of erythromycin (10 mg/L), Gram+ bacteria had a higher mortality rate than the Gram− bacteria. This result suggests that antibiotic in wastewater could change the activated sludge bacteria composition, according to their Gram type by selecting the bacteria which are the least sensitive to the antibiotics. However bacterial death was followed by bacterial disintegration leading to a decrease in the fluorescence. Results suggested that the viability indicators based on membrane integrity should be used with a correct sampling method, which can give the initial quantity of living bacteria.