Use of substrate responsive-direct viable counts to visualize naphthalene degrading bacteria in a coal tar-contaminated groundwater microbial community

A microscopy-based method was developed to distinguish naphthalene-degrading bacteria within the microbial community of a coal tar-contaminated groundwater system. Pure cultures of Pseudomonas putida NCIB 9816-4 were used to develop the substrate responsive-direct viable count (SR-DVC) method. Cells were concentrated on membrane filters, placed on agar plates of Stanier's minimal basal salts media containing antibiotics (nalidixic acid, piromidic acid, pipemidic acid, and cephalexin), and exposed to vapors of naphthalene. Following brief incubation, samples were fixed in 2% formaldehyde and examined by epifluorescent microscopy. Pure cultures displayed the expected cell elongation response to the SR-DVC assay and required a minimum incubation time of 9 h for differentiation of elongated cells. When applied to groundwater samples from the study site, naphthalene responsive cells in the groundwater community were easily distinguished from unresponsive cells and debris (350+/-180 substrate responsive cells/ml, relative to negative controls with no added growth substrate). In an attempt to reduce background counts of elongated bacteria and fungi, the SR-DVC procedure was modified by adding a wash step prior to incubation and a fungal inhibitor, cyclohexamide, to the plates. When groundwater samples were subjected to the modified procedure, only cells in washed samples showed a significant response to naphthalene (150+/-25 cells/ml), indicating the presence of inhibitory substances in the groundwater. Variations in response of the groundwater microbial community to the two SR-DVC procedures suggest that subsurface conditions (microbial and chemical composition) vary temporally. SR-DVC allows the phenotypes of individual naturally occurring cells to be assessed.