Microbial cell responses to a non-ionic surfactant. II. Effects as assessed by fluorescein diacetate uptake

Summary The effects of a non-ionic surfactant, Pluronic F-68, on uptake of fluorescein diacetate (FDA) into yeast cells as measured by increase in fluorescence atca. 500 nm have been studied. The rate of FDA uptake was increased almost 2.5 fold by incubating cells with up to 5% commercial grade pluronic but this depended on source and degree of purity. Dye uptake was reduced by pre-purification of pluronic but this again depended on source of material. None of the pluronic preparations had any significant effects on the rate of enzymemediated FDA hydrolysis by cell-free extracts.     

Potential problems with fluorescein diacetate assays of cell viability when testing natural products for antimicrobial activity.

There are two potential problems in the use of fluorescein diacetate (FDA) as a measure of cell viability. The first is the hydrolysis of FDA to fluorescein in the absence of live cells and the second is the quenching of fluorescence by assay solutions. We show that common media components such as tryptone, peptone and yeast extract all promote hydrolysis of FDA in the absence of live cells, as do Tris-HCl and sodium phosphate buffers. As a consequence, various microbiological media promote hydrolysis of FDA in the absence of live cells. Different media were also shown to reduce the amount of visible fluorescence of fluorescein. Diluting the medium decreases the background hydrolysis of FDA as well as increases the amount of visible fluorescence. Both problems should be considered when using FDA as an indicator of cell viability when testing natural products for antimicrobial activity.     

Applicability of the fluorescein diacetate method of detecting active bacteria in freshwater

Fluorescein diacetate (FDA) hydrolysis was evaluated as a means to detect actively metabolizing bacteria in freshwater. Fluorescein diacetate, a nonfluorescent derivative of fluorescein, can be transported across cell membranes and deacetylated by nonspecific esterases. Resultant fluorescein accumulates within cells and allows direct visualization by epifluorescent microscopy. Application of FDA to a variety of freshwater habitats yielded estimates of active cells ranging from 6–24% of the total population. These estimates were 49–61% lower than estimates of active cells obtained from measures of electron transport activity. The difference was attributed to low permeability of the fluorogen through the outer membrane of heterotrophic gram-negative cells. Data suggest that FDA hydrolysis as a means of detecting active bacteria may be limited to environments rich in eucaryotes and gram-positive cells.     

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.     

Macrophage activation by synthetic peptides. I. Kinetic changes in the transport of fluorescein anions across the plasma membrane of macrophages

As shown by cytofluorimetric technique, fluorescein anions formed in macrophages due to hydrolysis of fluorescein diacetate (FDA) release into the extracellular medium through the probenecid-inhibitable transport system of organic acids. Technical procedures have been elaborated to record separately the process of FDA hydrolysis characterising the activity of intracellular esterases, and the fluorescein anion transport representing secretion of organic acids by macrophages. It has been established that the tetrapeptide tuftsin stimulates the cell esterase activity without affecting the rate of fluorescein efflux. The peptide KPR (Lys-Pro-Arg) decreases both the esterase activity and the fluorescein anion efflux.     

Use of fluorescein diacetate for research on macrophage activation

Kinetic of changes in the fluorescence intensity of macrophages in a medium containing fluorescein diacetate (FDA) has been analysed. It is shown that under certain conditions the intensity of macrophages retains a constant level for rather a long time. The addition of stimulants such as lipopolysaccharide or tuftsin to the incubation medium leads to characteristic changes in the cell fluorescence level related to the increase in the activity of FDA intracellular hydrolyses and fluorescein efflux from macrophages. It is concluded that the kinetic of changes in the macrophages fluorescence intensity in a medium with FDA may serves as a test for detecting early functional changes upon macrophage activation.     

Kinetic and temporal factors influence chilling injury to germinal vesicle and mature bovine oocytes

In this study we examined the effects of low, above freezing temperatures on the viability and functionality of bovine oocytes. Germinal vesicle (GV) stage and in vitro matured oocytes (MII) were exposed to various combinations of time (15 and 60 min) and temperature (4, 16, 23, and 39 degrees C). After being treated, the ability of oocytes to undergo maturation and fertilization in vitro was examined, as well as their viability assayed by two fluorescent probes, fluorescein diacetate (FDA) and 5-carboxyfluorescein diacetate (cFDA). Cooling GV oocytes to 16 degrees C for 15 min reduced the fertilization rate by more than 40%, compared with those left at 39 degrees C. Surprisingly, cooling oocytes to 4 degrees C reduced the fertilization rate by only 10% compared with control. Exposing GV oocytes to temperatures below 23 degrees C reduced their viability. Similar to the reduction in fertilization, the viability of GV oocytes after exposure to 16 degrees C was reduced by more than 50%, whereas exposure to 4 degrees C reduced it by only 9%. Viability measurements using FDA and cFDA gave comparable results and showed a similar trend. The viability of MII oocytes and of GV oocytes pretreated with butylated hydroxytoluene, following exposure to low temperatures, was higher compared with that of GV controls. We interpret these results as indicating chilling effects on membrane integrity. Improving the chilling resistance of bovine oocytes may facilitate their short- and long-term preservation.     

Analysis of enzyme kinetics in individual living cells utilizing fluorescence intensity and polarization measurements

BACKGROUND: The Cellscan mark-S (CS-S) scanning cytometer was used for tracing enzymatic reactions in the same individual cells under various physiological conditions over periods of minutes. On-line reagent addition and changes in the experimental conditions (buffers, ions, substrates and inhibitors) were performed. METHODS: Kinetic events were monitored by fluorescence intensity (FI) and fluorescence polarization (FP) measurements of fluorescein diacetate (FDA) and chloromethyl fluorescein diacetate (CMFDA) intracellular hydrolysis. FP measurements have been used to assess the intracellular marker's mobility restrictions. RESULTS: Kinetic measurement along 1000 s of FDA labeled individual Jurkat T cells, indicated variation of 65% for FI(t) and approximately 10% for FP(t). While FI increased linearly with time, FP(t) decreased nonlinearly and asymptotically, reaching a constant value. The FP(t) of CMFDA-labeled cells was different from that of FDA-labeled cells. Average cellular Km of 3.9 microM was calculated from individual cell FDA hydrolysis curves. CONCLUSIONS: (1) Analysis of the reaction kinetics of intracellular enzymes can be refined by using FP measurements of the products of fluorogenic substrates in addition to the FI measurements. (2) Subpopulations or individual cells could be classified according to their reaction rates. (3) A specific dependence of FP(t) on type of enzyme substrate is suggested.     

Fluorescein diacetate used as a vital stain for labeling living pollen tubes

Snapdragon and tobacco pollen treated with fluorescein diacetate (FDA, 10 μg/ml) for 40 min could germinate and grew well in FDA-free medium. The pollen tubes showed bright fluorescence of the protoplasm when they were living. Thus the FDA method can be used, in addition to its previous usage for viability test of cells, also for vital staining of pollen tubes, and may be valuable to the study of other living cells as well.     

Rapid flow cytometric method for viability determination of solventogenic clostridia

We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone–butanol–ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.     

Quantification of metabolic activity of cultured plant cells by vital staining with fluorescein diacetate

The metabolic activity of suspension cultures of Sonneratia alba cells was quantified by measurement of the hydrolysis of fluorescein diacetate (FDA). FDA is incorporated into live cells and is converted into fluorescein by cellular hydrolysis. Aliquots (0.1–0.75 g) of S. alba cells were incubated with FDA at a final concentration of 222 μg/ml suspension for 60 min. Hydrolysis was stopped, and fluorescein was extracted by the addition of acetone and quantified by measurement of absorbance at 490 nm. Fluorescein was produced linearly with time and cell weight. Cells of S. alba are halophilic and proliferated well in medium containing 50 and 100 mM NaCl. Cells grown in medium containing 100 mM NaCl showed 2- to 3-fold higher FDA hydrolysis activity than those grown in NaCl-free medium. When S. alba cells grown in medium supplemented with 50 mM NaCl were transferred to fresh medium containing 100 mM mannitol, cellular FDA hydrolysis activity was down-regulated after 4 days of culture, indicating that the moderately halophilic S. alba cells were sensitive to osmotic stress. Quantification of cellular metabolic activity via the in vivo FDA hydrolysis assay provides a simple and rapid method for the determination of cellular activity under differing culture conditions.     

Energy-dependent, carrier-mediated extrusion of carboxyfluorescein from Saccharomyces cerevisiae allows rapid assessment of cell viability by flow cytometry.

Carboxyfluorescein diacetate is a nonfluorescent compound which can be used in combination with flow cytometry for vital staining of yeasts and bacteria. The basis of this method is the assumption that, once inside the cell, carboxyfluorescein diacetate is hydrolyzed by nonspecific esterases to produce the fluorescent carboxyfluorescein (cF). cF is retained by cells with intact membranes (viable cells) and lost by cells with damaged membranes. In this report, we show that Saccharomyces cerevisiae extrudes cF in an energy-dependent manner. This efflux was studied in detail, and several indications that a transport system is involved were found. Efflux of cF was stimulated by the addition of glucose and displayed Michaelis-Menten kinetics. A Km for cF transport of 0.25 mM could be determined. The transport of cF was inhibited by the plasma membrane H(+)-ATPase inhibitors N,N'-dicyclohexylcarbodiimide and diethylstilbestrol and by high concentrations of tetraphenylphosphonium ions. These treatments resulted in a dissipation of the proton motive force, whereas the intracellular ATP concentration remained high. Transport of cF is therefore most probably driven by the membrane potential and/or the pH gradient. The viability of S. cerevisiae was determined by a two-step procedure consisting of loading the cells with cF followed by incubation at 40 degrees C in the presence of glucose. Subsequently, the fluorescence intensity of the cells was analyzed by flow cytometry. The efflux experiments showed an excellent correlation between the viability of S. cerevisiae cells and the ability to translocate cF. This method should prove of general utility for the rapid assessment of yeast vitality and viability.     

A Method for Producing Dust-, Streak- and Hole-Free Formvar Films in Laboratories Having High Atmospheric Humidity

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Optimisation of the fluorescein diacetate antibacterial assay

The fluorescein diacetate (FDA) antibacterial assay relies on the cleavage of fluorescein diacetate by metabolically active bacteria. The recent finding that microbiological media can lead to significant levels of cleavage has reduced the reliability of the assay. Using the nucleophilic scavengers N-ethylmaleimide and maleic anhydride, we have demonstrated that this abiotic cleavage is most likely due to nucleophiles such as cysteine and histidine commonly present in the media. To increase the reliability of the assay we have modified the original assay conditions to include use of dilute medium (peptone 0.2% w/v, yeast extract 0.1% w/v and NaCl 0.1% w/v) in a non-nucleophilic buffer and overnight incubation of the medium after addition of antibacterial agents. The optimised fluorescein diacetate assay has been used to determine the MIC of gentamicin, tetracycline and chloramphenicol for Escherichia coli, Staphyloccocus aureus and Pseudomonas aeruginosa and gave quantitative results that were reproducible and consistent with published data.     

Simultaneous determination of apoptosis and surface antigen expression in tumor adherent cells

Apoptosis is a physiological, gene-directed form of cell death aimed at controlling cell proliferation in several biological conditions. It plays a crucial role in modulating tissue growth during embryonic development, cell turnover in adult life, and it seems to be the most frequent mechanism of tumor cell deletion by chemotherapy. Flow cytometry is a widely-used technique for checking apoptosis, permitting a multiparametric analysis. It is possible to follow the alterations occurring in the nucleus, mitochondria and plasmatic membrane during the different apoptotic stages using probes such as LDS-751, JC-1 or Annexin V. The potential of these probes to identify the early or late stages of apoptosis has been widely investigated in cells growing in suspension. In order to assess apoptosis in adherent cells, we tested a combination of fluorescein diacetate (FDA), a substrate for non specific esterase whose activity decreases during the early phase of apoptosis, and trypan blue in MCF-7 human breast cancer cells. Apoptotic cells showed a decrease in the green fluorescence emitted by fluorescein, the product of FDA hydrolysis, whereas necrotic cells emitted a red fluorescence due to the trypan blue staining. FDA-trypan blue double-staining was used to investigate the different kinetics of apoptosis induced by taxol, camptothecin and UV-B irradiation in MCF-7 cells. This method is rapid and simple, and can be used for monitoring the process of apoptosis from early stages in adherent cells, for the physical separation of apoptotic and live cells, and for immunophenotyping, including Fas expression.     

Staining with Fluorescein Diacetate Correlates with Hepatocyte Function

To establish the importance of fluorescein diacetate (FDA) as a viability stain for cultured hepatocytes. we hypothesized that FDA staining would correlate positively with hepatocyte viability and function. Mixtures of live and dead cells were stained with FDA and scanned by flow cytometry. A close correlation was observed between the live cell fraction and percent viability as determined by FDA staining (R² = 0.962). Hepatocytes were also sorted into low fluorescence and high fluorescence groups. Both albumin production and lidocaine metabolism (P-450 activity) were significantly increased in the high fluorescence group compared to the low fluorescence group. An automated, fluorescence-activated assay was useful for rapid assessment of hepatocyte viability. In addition. the intensity of green fluorescence following staining with FDA correlated well with two specific measures of hepatocyte function.     

Modification of the fluorescein diacetate assay for screening of antifungal agents against Candida albicans: comparison with the NCCLS methods

A modified fluorescein diacetate (FDA) assay has been compared with standard NCCLS broth macrodilution and broth microdilution methods for the detection of antifungal activity. The FDA assay was performed in a medium containing bacteriological peptone, NaCl, yeast extract and glucose (0.2%, 0.1%, 0.1% and 1% w/v, respectively) and buffered with 10 mM BES buffer. The MICs of amphotericin B, fluconazole, miconazole and flucytosine (representing three major classes of antifungal agents) obtained by the three methods were compared. The results obtained with the FDA assays correlated well with the NCCLS macrodilution method for MICs of amphotericin B, miconazole and fluconazole, but not for flucytosine. However, the MIC values of flucytosine obtained with the FDA assay were well within the quality control range for the two reference strains recommended by the NCCLS. The FDA assay described is an attractive alternative to the NCCLS methods for screening for antifungal agents, with the added advantage of objectivity of fluorescence measurement.     

The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.     

Carboxyfluorescein diacetate succinimidyl ester fluorescent dye for cell labeling

Our objective was to study the properties of the carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) and the methodology of cell labeling using CFDA-SE fluorescent dye. First, we analyzed the kinetics of CFDA-SE fluorescent dye intensity over time. Second, we determined the optimal concentration of CFDA-SE fluorescent dye for cell labeling. Third, we tested the toxicity of CFDA-SE fluorescent dye on labeled cells. Finally, we determined the optimal staining time of CFDA-SE fluorescent dye for cell labeling.The results show that the optimal concentration of CFDA-SE fluorescent dye for cell labeling varies according to different cell types. CFDA-SE fluorescent dye is non-toxic to cells as the cell death rate caused by CFDASE labeling is below 5%. The optimal cell labeling time was determined to be 8 min of incubation with CFDA-SE fluorescent dye. We concluded that the advantages of using CFDA-SE fluorescent dye for cell labeling are as follows: (1) the binding of CFDA-SE fluorescent dye to cells is stable; (2) CFDA-SE fluorescent dye is not toxic and does not modify the viability of labeled cells; and (3) CFDA-SE fluorescent dye is a suitable fluorochrome for cell labeling.     

Vital Fluorochromes as Tracers for Fungal Growth Studies

Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyanines DiIC₁₈(3) and DiOC₁₈(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma. harzianum Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence with at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC₁₈(3) and DiOC₁₈(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.