Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.     

Deep-red fluorescent imaging probe for bacteria

A versatile deep-red fluorescent imaging probe is described that is comprised of a bis(zinc(II)-dipicolylamine) targeting unit covalently attached to a pentamethine carbocyanine fluorophore with Cy5-like spectroscopic properties. A titration assay based on fluorescence resonance energy transfer is used to prove that the probe selectively associates with anionic vesicle membranes whose composition mimics bacterial cell membranes. Whole-body optical imaging experiments show that the probe associates with the surfaces of both Gram-positive and Gram-negative bacteria cells, and it can target the site of bacterial infection in a living mouse. In vivo accumulation at the infection site and subsequent clearance occurs more quickly than a structurally related near-infrared bis(zinc(II)-dipicolylamine) probe. The fact that the same deep-red probe molecule can be used for spectroscopic assays, cell microscopy, and in vivo imaging studies, is an important and attractive technical feature.     

A fluorescent probe for a phosphonium ion efflux system in bacteria

Abstract The interaction of a fluorescent probe with three species of bacteria shows properties consistent with the probe acting as a substrate for an efflux system that can also transport phosphonium ions and related compounds.     

Pan and sentinel lymph node visualization using a near-infrared fluorescent probe

A number of different types of agents have been employed to aid in the visualization of lymph nodes, particularly the sentinel lymph node, and to decrease the tissue destruction associated with the diagnosis of nodal metastases. The current study was performed to see if a novel macromolecular near-infrared fluorescent (NIRF) probe could be used to visualize lymph nodes after intravenous administration (pan-node visualization) or subcutaneous administration (sentinel node visualization), and serve as method for guiding dissection with interventional radiologic and surgical procedures. Cy5.5-PGC, the near-infrared dye Cy5.5 coupled to a protected graft copolymer (PGC), was injected (i.v. or s.c.) into nude mice. Twenty-four hours later white light and NIRF images were obtained on (i) the live animal, (ii) a partially dissected animal, and (iii) tissue specimens. With Cy5.5-PGC administered intravenously, axillary nodes were visualized from outside a living mouse. With partial dissection, iliac and aortic nodes were visible as concentrated foci of high-intensity NIRF signals. With subcutaneous injection in the front extremity, axillary and brachial nodes draining the injection site were easily visualized. NIRF imaging provides a nonradioactive method of visualizing lymph nodes through layers of tissue that can be employed with intravenous or subcutaneous injection.     

Development of multicolour digital image analysis system to enumerate actively respiring bacteria in natural river water

AIMS: To develop a rapid and simple method for the assessment of metabolic activity of bacteria in natural environment. METHODS AND RESULTS: A rapid and simple multicolour digital image analysis system for enumerating viable bacteria based on active fluorescent staining has been developed. This system can accurately differentiate actively respiring bacteria and non-respiring bacteria by distinctive colour information in the digital image captured by an epifluorescence microscope equipped with low magnification objective lens. An algorithm to distinguish bacteria from considerable detritus, which produced bright fluorescence in different colours, by colour segmentation has also been developed. This system was applied to river water samples, and the total and respiratory active bacterial counts by digital image analysis were highly related to those by epifluorescence microscopy (r2 = 0.96 and 0.93, respectively). CONCLUSION: This system allowed the rapid and simple differentiation of bacteria from detritus and concurrent assessment of their metabolic activity, with results being available within 1 h. SIGNIFICANCE AND IMPACT OF THE STUDY: The low magnification image analysis allows more rapid and simple quantification of bacteria and could considerably decrease image data amount and acquisition time. This system could be easily applied to the rapid analysis of microbes in various environments.     

CTC staining and counting of actively respiring bacteria in natural stone using confocal laser scanning microscopy

A method was established for staining and counting of actively respiring bacteria in natural stone by using the tetrazolium salt 5-cyano-2,3-ditolyltetrazolium chloride (CTC) in combination with confocal laser scanning microscopy (CLSM). Applying 5 mM CTC for 2 h to pure cultures of representative stone-inhabiting microorganisms showed that chemoorganotrophic bacteria and fungi-in contrast to lithoautotrophic nitrifying bacteria-were able to reduce CTC to CTF, the red fluorescing formazan crystals of CTC. Optimal staining conditions for microorganisms in stone material were found to be 15 mM CTC applied for 24 h. The cells could be visualized on transparent and nontransparent mineral materials by means of CLSM. A semi-automated method was used to count the cells within the pore system of the stone. The percentage of CTC-stained bacteria was dependent on temperature and humidity of the material. At 28 degrees C and high humidity (maximum water holding capacity) in the laboratory, about 58% of the total bacterial microflora was active. On natural stone exposed for 9 years at an urban exposure site in Germany, 52-56% of the bacterial microflora was active at the east, west, and north side of the specimen, while only 18% cells were active at the south side. This is consistent with microclimatic differences on the south side which was more exposed to sunshine thus causing UV and water stress as well as higher temperatures on a microscale level. In combination with CLSM, staining by CTC can be used as a fast method for monitoring the metabolic activity of chemoorganotrophic bacteria in monuments, buildings of historic interest or any art objects of natural stone. Due to the small size of samples required, the damage to these objects and buildings can be minimized.     

Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

An amphiphilic fluorescent probe for the visualization of histamine in living cells

A novel fluorescent probe composed of two moieties, Nile Red and an iminodiacetic acid-Ni(2+) complex, for the detection of histamine in living cells is described. The probe was successfully applied to visualizing histamine in RAW264 cells, representing the first demonstration of the imaging of histamine itself in living cells.     

DNA binding fluorescent proteins for the direct visualization of large DNA molecules

Fluorescent proteins that also bind DNA molecules are useful reagents for a broad range of biological applications because they can be optically localized and tracked within cells, or provide versatile labels for in vitro experiments. We report a novel design for a fluorescent, DNA-binding protein (FP-DBP) that completely ‘paints’ entire DNA molecules, whereby sequence-independent DNA binding is accomplished by linking a fluorescent protein to two small peptides (KWKWKKA) using lysine for binding to the DNA phosphates, and tryptophan for intercalating between DNA bases. Importantly, this ubiquitous binding motif enables fluorescent proteins (K_d = 14.7 μM) to confluently stain DNA molecules and such binding is reversible via pH shifts. These proteins offer useful robust advantages for single DNA molecule studies: lack of fluorophore mediated photocleavage and staining that does not perturb polymer contour lengths. Accordingly, we demonstrate confluent staining of naked DNA molecules presented within microfluidic devices, or localized within live bacterial cells.     

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe

Background

Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking.

Use of 5-cyano-2,3-ditolyl tetrazolium chloride for quantifying planktonic and sessile respiring bacteria in drinking water.

Direct microscopic quantification of respiring (i.e., viable) bacteria was performed for drinking water samples and biofilms grown on different opaque substrata. Water samples or biofilms developed in flowing drinking water were incubated with the vital redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and R2A medium. One hour of incubation with 0.5 mM CTC was sufficient to obtain intracellular reduction of CTC to the insoluble fluorescent formazan (CTF) product, which was indicative of cellular respiratory (i.e., electron transport) activity. This result was obtained with both planktonic and biofilm-associated cells. Planktonic bacteria were captured on 0.2-microns-pore-size polycarbonate membrane filters and examined by epifluorescence microscopy. Respiring cells containing CTF deposits were readily detected and quantified as red-fluorescing objects on a dark background. The number of CTC-reducing bacteria was consistently greater than the number of aerobic CFU determined on R2A medium. Approximately 1 to 10% of the total planktonic population (determined by counterstaining with 4,6-diamidino-2-phenylindole) were respirometrically active. The proportion of respiring bacteria in biofilms composed of drinking water microflora was greater, ranging from about 5 to 35%, depending on the substratum. Respiring cells were distributed more or less evenly in biofilms, as demonstrated by counterstaining with 4,6-diamidino-2-phenylindole. The amount of CTF deposited in single cells of Pseudomonas putida that formed monospecies biofilms was quantified by digital image analysis and used to indicate cumulative respiratory activity. These data indicated significant cell-to-cell variation in respiratory activity and reduced electron transport following a brief period of nutrient starvation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells

Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time.     

Terbium as a fluorescent probe for DNA and chromatin.

Terbium reacted with DNA and chromatin to form a complex in which terbium acted as a sensitive fluorescent probe. By measuring the narrow-line emission of Tb-3+ when DNA is selectively excited, the relative amount of Tb-3+ bound to the DNA can be calculated. Terbium was bound to DNA until one Tb-3+ was present for each phosphate group. After this point no more terbium was bound. TbCl3 was bound to chromatin in a linear manner until approximately 0.48 TbCl3 was added for each phosphate group in the chromatin-DNA solution. From these data it appears that 52% of the phosphate groups in chromatin were unavailable for binding. The binding of Tb-3+ to DNA can be reversed by prolonged dialysis against 0.5 M NaCl and chelating agents. The terbium ion is ideal in that it binds DNA tight enough so that completion of the reaction can be assumed but loose enough so that it can be removed by gentle means. Low concentrations of salt (up to 2 mM NaCl) enhance the quantum efficiency. Below pH 3 and above pH 7 the DNA-terbium complex will not form. Between pH 3 and pH 7 the quantum efficiency of the DNA terbium complex increases from either pH to a maximum at pH 5.5 to 5.6. Several biochemical uses for Tb-3+ ion are suggested.     

Design and evaluation of a novel fluorescent CB2 ligand as probe for receptor visualization in immune cells

Cannabinoid CB2 receptor has emerged as a very promising target over the last decades. We have synthesized and evaluated a new fluorescent probe designated NMP6 based on 6-methoxyisatin scaffold, which exhibited selectivity and K(i) value at hCB2 of 387 nM. We have demonstrated its ability to be an effective probe for visualization of CB2 receptor binding using confocal microscopy and a flow cytometry probe for the analysis of CB2 protein expression. Furthermore, NMP6 was easily obtained in two chemical steps from commercially available building blocks.     

Use of a fluorescent stain for visualization of nuclear material in living oocytes and early embryos

Hoechst dyes 33342 and 33258 were used to visualize pronuclei and nuclei of early preimplantation embryos. Murine one-cell zygotes exposed to dye stained rapidly over a range of concentrations (0, 0.02, 0.04, 0.1 or 0.2 micrograms/50 microliter of media). Development to morula and blastocyst in vitro was reduced (39/70, 56%; p less than 0.05) compared to controls (44/57, 77%) but not completely blocked. Porcine and bovine zygotes and embryos could also be stained but required incubation times up to 4 hr. Porcine embryos exposed to Hoechst 33342 had limited (p less than 0.01) in vitro development (29/74, 39%) compared to unstained controls (49/64, 76%). Hoechst dyes stain embryos from different species but suitably adjusted incubation times are required. Limited preimplantation development in vitro may be expected following staining and exposure to ultraviolet light.     

Membrane potentials in respiring and respiration-deficient yeasts monitored by a fluorescent dye

Changes in fluorescence of 3,3′-dipropylthiodicarbocyanine iodide which had been equilibrated with suspensions of the wild-type yeast Saccharomyces cerevisiae and of respiration-deficient mutants were followed. The changes have been attributed to changes of yeast membrane potentials, since the fluorescence with wild-type yeast could be affected in a predictable manner by uncouplers and the pore-forming agent nystatin. As in other systems, a rise of steady-state fluorescence was ascribed to depolarization and a drop of the fluorescence to hyperpolarization. (1) A considerable rise in steady-state fluorescence was brought about by addition of antimycin A or some other mitochondrial inhibitors to respiring cells. A major part of the composite membrane potential monitored in intact yeast cells appeared to be represented by the membrane potential of mitochondria. (2) Addition of D-glucose and of other substrates of hexokinase, including non-metabolizable 2-deoxy-D-glucose, induced a two-phase response of fluorescence, indicating transient depolarization followed by repolarization. Such a response was not elicited by other sugars which had been reported to be transported into the cells by a glucose carrier or by D-galactose in galactose-adapted cells. The depolarization was explained by electrogenic ATP exit from mitochondria to replenish the ATP consumed in the hexokinase reaction and the repolarization by subsequent activation of respiration. (3) In non-respiring cells only a drop in fluorescence was induced by glucose and this was ascribed to an ATP-dependent polarization of the plasma membrane. (4) Steady-state fluorescence in suspensions of respiration-deficient mutants, lacking cytochrome a, cytochrome b, or both, was high and remained unaffected by uncouplers and nystatin. This indicates that membranes of the mutants may have been entirely depolarized. A partial polarization, apparently restricted to the plasma membrane, could be achieved by glucose addition.     

Use of tannic acid for the ultrastructural visualization of periplasm in gram-negative bacteria

Tannic acid mordanting reveals the periplasm, the area between the outer membrane and the inner membrane of gram-negative bacteria, Rhizobium sp., Escherichia coli and Enterobacter aerogenes, as an electron-dense layer continuous with the inner leaflet of the outer membrane. The method involves 18 hr of tannic acid treatment after fixation in aldehyde prior to osmium tetroxide postfixation, followed by conventional electron microscopy.