A method for quantification of Rhodococcus chlorophenolicus in activated sludge by immunofluorescence microscopy

Immunofluorescence microscopy was used to quantify the strain Rhodococcus chlorophenolicus PCP-I added to activated sludge samples. Various pretreatment techniques were tested in a factorial design experiment for deflocculation of the activated sludge to optimize the recovery of R. chlorophenolicus. The best results were obtained by addition of 0.1% V/V Tween 20, 0.5 min sonication, followed by appropriate sample dilution to obtain 10–50 cells in the microscope counting field (corresponding with a content of 7–35 106 cells in the 3.5 cm2 slide). The recovery of R. chlorophenolicus was more than 90% compared with control samples without activated sludge. This method makes it possible to study biodegradation kinetics of the chlorophenol-degrading bacterial strain when present in mixed cultures, such as in activated sludge.