Abstract
Flavobacterium psychrophilum has emerged as one of the most significant bacterial pathogens in salmonid aquaculture worldwide. We have been studying the detection of harmful bacteria using immunomagnetic separation and flow cytometry (FCM). In this study, we used fluorescent magnetic beads and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC). Bacteria were specifically collected using fluorescent magnetic beads with only one antigen-antibody reaction. CTC turns into a red fluorescent formazan that is detectable by FCM. F. psychrophilum cells were stained with CTC and labeled with fluorescent magnetic beads. Double-stained bacteria (red fluorescence by CTC and green fluorescence from fluorescent magnetic beads) were detected by FCM. Bacterial cell numbers were determined by FCM and compared with those measured by a traditional colony counting method in the range of 102–108 cells/ml. The FCM assay could provide a bacterial cell count within 1 min and the total assay time, including sample preparation, was less than 3 h.
Highlights
Sensors and Materials, Vol 24, No 6 (2012)Measurements are made to determine total cell counts, total culturable cell counts, and the presence of specific viable bacterial cells, such as pathogens
CTC is reduced by bacteria to a water-insoluble, red fluorescent formazan product, which allows for the quantification of the metabolic activity of bacteria under both aerobic and various anaerobic conditions.[5,6] Several factors, can affect CTC reduction during incubation.[7,8,9] CTC counts are commonly determined by flow cytometry (FCM),(10–12) epifluorescence microscopy can be used to overcome the rapid fading of the fluorescence signal,(13) yielding equal or higher counts.[14]
The CTC concentration was adjusted to 2 mM, on the basis of the report of Yamaguchi et al[12] For E. coli cultures, tryptone soya broth was used for CTC assays,(13) but we chose cytophaga broth, which is the medium used for F. psychrophilum
Summary
Sensors and Materials, Vol 24, No 6 (2012)Measurements are made to determine total cell counts, total culturable cell counts, and the presence of specific viable bacterial cells, such as pathogens. Tetrazolium dyes are reduced from a colorless complex to a brightly colored, intracellular formazan precipitate by components of the electron transport system and/or a variety of dehydrogenase enzymes present in active bacterial cells. CTC is reduced by bacteria to a water-insoluble, red fluorescent formazan product, which allows for the quantification of the metabolic activity of bacteria under both aerobic and various anaerobic conditions.[5,6] Several factors, can affect CTC reduction during incubation.[7,8,9] CTC counts are commonly determined by flow cytometry (FCM),(10–12) epifluorescence microscopy can be used to overcome the rapid fading of the fluorescence signal,(13) yielding equal or higher counts.[14]
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