RAPID IN-SITU ASSESSMENT OF PHYSIOLOGICAL ACTIVITIES IN BACTERIAL BIOFILMS USING FLUORESCENT-PROBES

Two rapid in situ enumeration methods using fluorescent probes were us ed to assess the physiological activities of Klebsiella pneumoniae bio films on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetraz olium chloride (CTC) and rhodamine 123 (Ph 123), were chosen to perfor m this study. CTC is a soluble redox indicator which can be reduced by respiring bacteria to fluorescent CTC-formazan crystals. Ph 123 is in corporated into bacteria with respect to cellular proton motive force. The intracellular accumulation of these fluorescent dyes can be deter mined using epifluorescence microscopy. The results obtained with thes e two fluorescent probes in situ were compared to the plate count (PC) and in situ direct viable count (DVC) methods. Viable cell densities within biofilms determined by the three in situ methods were comparabl e and always showed approximately 2-fold higher values than those obta ined with the PC method. As an additional advantage, the results were observed after 2 h, which was shorter than the 4 h incubation time req uired for the DVC method and 24 h for colony formation. The results in dicate that staining with CTC and Rh 123 provides rapid information re garding cell numbers and physiological activities of bacteria within b iofilms.