Vital Stain Fluorescein Diacetate Research Articles

Enumerating viable phytoplankton using a culture-based Most Probable Number assay following ultraviolet-C treatment

Ballast water management systems (BWMS) must be tested to assess their compliance with standards for the discharge of organisms, for example in the ≥ 10- and < 50-μm size category, which is dominated by phytoplankton. Assessment of BWMS performance with the vital stains fluorescein diacetate + 5-chlorofluorescein diacetate, required by regulations in the USA, is problematic in the case of ultraviolet-C (UVC) radiation. This is because UVC targets nucleotides—and thus reproduction, hence viability—rather than membrane integrity, which is assayed by the stains. The Serial Dilution Culture-Most Probable Number (SDC-MPN) method, long used to enumerate fragile phytoplankton from natural communities, is appropriate for counting viable phytoplankton. We developed QA/QC “best practice” criteria for its application as a robust and repeatable assay of viable cells in cultures of phytoplankton before and after experimental treatment, then constructed dose-response curves for UVC-induced loss of viable cells in 12 species of phytoplankton from seven divisions. Sensitivity to UVC, expressed as the dose required to reduce viability by 99%—the criterion for type approval of treatment systems—varied more than 10-fold and was not correlated with cell size. The form of the dose-response curves varied between taxa, with most having a threshold dose below which there was no reduction in viability. Analysis of the patterns of growth indicates that if recovery from treatment occurred, it was complete in 1 or 2 days in > 80% of cases, long before the assays were terminated. We conclude that the SDC-MPN assay as described is robust and adaptable for use on natural phytoplankton.

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.

Refining a method for ascospore viability testing in overwintering chasmothecia of Erysiphe necator

The vital stain fluorescein diacetate (FDA) has long been used to assess the viability of Erysiphe necator ascospores in chasmothecia. To determine the viability of animal, plant and other cells, more complex staining procedures, for example simultaneous double-staining procedures based on the use of FDA and propidium iodide (PI), have also been widely used. This paper reports a refined, detailed version of this method and its limitations, where PI was replaced with ethidium bromide (EtBr), to be used in testing the viability of E. necator ascospores during overwintering in chasmothecia.

Evaluating the response of freshwater organisms to vital staining

The unintentional introduction of nonindigenous species by ballast water discharge is one of the greatest threats to biodiversity in freshwater systems. Proposed international regulations for ballast water management will require enumeration of viable plankton in ballast water. In this study we analyze the efficacy of vital stains in determining viability of freshwater taxa. The efficacy of vital stains fluorescein diacetate (FDA) and FDA+5-chloromethylfluorescein diacetate (CMFDA) was evaluated with freshwater macroinvertebrates, zooplankton, and phytoplankton. Macroinvertebrates were cultured in laboratory, while plankton were collected from Hamilton Harbour and ballast tanks of commercial vessels. Organisms were subjected to various treatments (i.e., heat, NaClO, and NaOH) to establish efficacy of stains for viable and non-viable organisms. No significant difference in accuracy rate was found between stains, regardless of treatment, within groups of organisms, indicating that the addition of CMFDA is superfluous in the sample region studied. False positive errors, in which dead organisms fluoresced similarly to live organisms, occurred in most groups and were significantly different between test groups. False positive error rates were 2.3% for phytoplankton, 20% for ballast water zooplankton, 35% for Hamilton Harbour zooplankton and 47% for macroinvertebrates. Response to stains varied between taxonomic groups. Low (< 10%) false positive error rates were observed with phytoplankton, soft-bodied rotifers, oligochaetes, and Bosmina spp., while rates between 20% and 50% were observed for Daphnia spp., Hexagenia sp., and Chironomus riparius. False positive rates of copepods, Hyalella azteca, and Hemimysis anomala were between 70% and 100%. The FDA/FDA+CMFDA vital staining methods provide useful tools for viability analysis of freshwater phytoplankton, soft-bodied invertebrates and zooplankton, and may be used for viability analysis of the ≥ 10 µm to < 50 µm size fraction in compliance testing of ballast water. However, viability analysis of larger freshwater crustaceans with vital stains should be undertaken with caution.

Effects of volatile substances of Streptomyces globisporus JK-1 on control of Botrytis cinerea on tomato fruit

Volatile substances produced by Streptomyces globisporus JK-1 grown on autoclaved wheat seed inhibited Botrytis cinerea growth and development both on media and in inoculated tomato fruit. The volatiles suppressed mycelial growth of various plant pathogens in vitro, especially that of B. cinerea and Sclerotinia sclerotiorum. Conidial germination and sporulation of B. cinerea were also suppressed. Disease incidence and severity on wound-inoculated tomato fruit (Lycopersicon esculentum) were inhibited when fumigated with 120g wheat seed culture of S. globisporus JK-1 per liter of airspace in treatment containers. Suppression of the infection process of B. cinerea on tomato fruit was observed via scanning microscopy, showing inhibition of conidial germination and of appressorial formation on tomato fruit, as well as abnormal morphology of appressoria and conidia. The viability of the conidia obtained from the volatile-treated and non-treated disease lesions was tested with the vital stains fluorescein diacetate (FDA) and propidium iodide (PI). Conidia fumigated with 30, 60 or 120g/L wheat seed culture of S. globisporus JK-1 at 20°C for 6days showed 46.0%, 69.8%, or 80.9% reduction in viability, respectively. Transmission electron microscopy of fumigated and untreated B. cinerea showed excessive vesiculation or thickened cell walls in exposed conidia and increased vesiculation or strong retraction of plasma membrane in exposed hyphae. These results provide a better understanding of the mode of action of volatiles from JK-1 on B. cinerea. The inhibition growth of B. cinerea both in vitro and in vivo showed that volatiles from S. globisporus JK-1 have the potential for control of postharvest grey mold of tomato fruits through fumigant action.

Estimating propagule pressure and viability of diatoms detected in ballast tank sediments of ships arriving at Canadian ports

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 425:47-61 (2011) - DOI: https://doi.org/10.3354/meps08999 Estimating propagule pressure and viability of diatoms detected in ballast tank sediments of ships arriving at Canadian ports Maria Célia Villac*, Irena Kaczmarska Department of Biology, Mount Allison University, 63B York St., Sackville, New Brunswick, E4L 1G7, Canada *Email: celiavillac@gmail.com ABSTRACT: This research uses the concept of propagule pressure (number of individuals introduced and number of introduction attempts) to investigate human-mediated bioinvasion patterns. We quantified diatoms in the sediments of ballast tanks of commercial ships arriving on both Canadian coasts during 2007 to 2009. Diatom cell concentrations varied from non-detected to 105 cells g–1 wet weight (1011 cells per tank). Although the lowest values were often found in tanks that underwent ballast water exchange, the highest concentrations (109 to 1011 cells per tank) were detected in all voyage categories: transoceanic with ballast exchange (TOE), and intra-coastal with exchange (ICE) and without exchange (ICU). For the west coast, 36% of tanks carried detectable quantities of diatoms and there was no statistical difference between ship categories. For the east coast, 60% of tanks contained diatoms; ICU represented a bioinvasion pattern based on more frequent events with consistently lower cell concentrations, whereas ICE and TOE corresponded to less frequent events, though more variable in cell concentrations. Diversity reached 40 taxa per tank, including resting stages and cells that were supposedly growing vegetatively. New records may lead to introduction hypotheses that ought not to be accepted uncritically. Cell viability was tested using the vital stain fluorescein diacetate; parallel counts of protoplasm integrity and chlorophyll autofluorescence revealed that all 3 indicators gave results within the same order of magnitude. Inoculation of 0.2 to 0.5 ml of the slurry into culture media led to the growth of diatoms, even of taxa not initially detected. Within 7 d, cultured assemblages reached cell concentrations equivalent to 1.8 to 4.4 doublings of the original inoculation. KEY WORDS: Diatoms · Biological invasion · Ballast sediment · Propagule pressure · Phytoplankton viability Full text in pdf format Supplementary material PreviousNextCite this article as: Villac CM, Kaczmarska I (2011) Estimating propagule pressure and viability of diatoms detected in ballast tank sediments of ships arriving at Canadian ports. Mar Ecol Prog Ser 425:47-61. https://doi.org/10.3354/meps08999 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 425. Online publication date: March 14, 2011 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2011 Inter-Research.

Effects of thymol fumigation on survival and ultrastracture of Monilinia fructicola

Postharvest treatment of stone fruit with thymol vapours controls brown rot, caused by Monilinia fructicola. While the mechanism of the fungicidal action is not known, the gross effect of the thymol vapours appeared to be on the fungal spores and surface mycelia. Harvested plums were inoculated with conidia of M. fructicola and incubated for 4–6 days at 21 °C to allow for sporulation. Sporulating fruit was treated with thymol vapours from 0 to 8 μg mL −1. The viability of the conidia obtained from thymol treated and untreated brown rot lesions was tested with four vital stains. M. fructicola conidia were immediately affected with a 50% reduction in viability after treatment with 2 μg mL −1 thymol as detected by the vital stains fluorescein diacetate, propidium iodide, fluorescent dye DiOC 6(3) and the commercial product FungoLight™. Conidia treated with 8 μg mL −1 thymol exhibited 17–23% survival. One millimeter square sections of the infected fruit skin with the associated surface mycelial mass were prepared for transmission electron microscopy. The electron micrographs display the intra and intercellular ultrastructure of the fungal conidia. Thymol crystallized on the outer surface of the fungal cell wall. The cytoplasm of the thick walled spores did not appear changed from the untreated spores. In contrast, all other fungal fine structures on the surface of the lesion were severely affected by thymol vapours. Sections of germ tubes, appressoria and surface hyphae exposed to thymol vapours were characterised by disrupted and disorganised cytoplasmic organelles. The fine structures of the fungal intra- and intercellular hyphae inside the plum tissue were unaffected by thymol treatments.

Cell viability in natural phytoplankton communities quantified by a membrane permeability probe

A nonstaining method, based on the digestion of dead cells, the membranes of which are permeable when exposed to an enzymatic cocktail (DNAse I and Trypsin), was used to identify living cells in cultured and natural phytoplankton populations. The cell digestion method was applied to cultures of nine phytoplankton species, encompassing a broad taxonomic range. The percentage of living cells obtained with the enzymatic digestion test was found to be consistent with that obtained with the vital stain fluorescein diacetate, which could be applied to only six of the species used. Use of the cell digestion method showed the phytoplankton community viability in Blanes Bay (NW Mediterranean littoral) to be very low at the beginning of the summer (34% and 31% of diatoms and cyanobacteria cells were alive, respectively), when oligotrophic conditions prevailed. The phytoplankton viability increased slightly by midsummer, with the entire cyanobacteria population, which dominated the community, composed of living cells, and most (81%) of the diatom cells were living in the fall. The patterns in cell viability revealed by the cell digestion method were consistent with the seasonal variability in phytoplankton lysis rates and the shifting contribution of different groups to the phytoplankton community.

Effects of darkness on multi‐excitation in vivo fluorescence and survival in a marine diatom

Surveys of the California Current System in 1993 revealed high concentrations of photosynthetic pigment biomass at ∼200‐m depth, well below the euphotic zone. The deep fluorescence feature contained an estimated 2.2 × 104 metric tons of carbon and contained ∼2.5 times the amount of chlorophyll observed in surface waters directly above it. Deep phytoplankton assemblages may be a signature of water mass subduction, suggesting the possibility of using phytoplankton as water mass tracers. These field observations led to a laboratory study of the fluorescence characteristics of autotrophic cells as possible indices of acclimation to extended periods of darkness. In vivo multiexcitation Chl a fluorescence of the diatom Thalassiosira weissflogii was monitored for 2 months of total darkness. Numbers of living and dead cells were determined using the vital stain fluorescein diacetate (FDA). By the end of the dark incubation period, in vivo Chl a fluorescence and fluorescence per cell had leveled off to ∼45% and 65% of initial values, respectively. The contribution of accessory pigments to Chl a fluorescence, expressed as multiexcitation fluorescence ratios, was higher in the dark than prior to transfer to darkness but showed no significant changes during 2 months of darkness. The FDA assay indicated that ∼85% of the cells were alive for at least the first 3 weeks during the first dark experiment and for the entire 2 months of second dark incubation. Cell numbers decreased to 65% of initial values and then grew exponentially upon reexposure to a light: dark photoperiod. Our results for T. weissflogii suggest that extended light limitation of photosynthesis does not preclude the survival of subducted phytoplankton assemblages and the consequent accumulation of Chl a at depths below the euphotic zone. If these results extend to natural assemblages, it is not possible to estimate advective time scales based on a maximum persistence time of pigment fluorescence below the euphotic zone. Nevertheless, the deep phytoplankton assemblage we observed provides evidence for water mass subduction and suggests that large, intermittent pulses of phytoplankton carbon are a part of cross‐shelf exchange and vertical flux from surface waters to depth in this region.

Distribution and Retention of Cleistothecia ofUncinula necatoron the Bark of Grapevines.

Clesitothecia of Uncinula necator were dispersed by late summer and early autumn rain to the bark of grapevines. Rain-dispersed ascocarps accumulated rapidly on bark during a 10-wk period and were then retained on bark through subsequent rain events between leaf abscission and budbreak the following spring. The density of populations on bark was significantly correlated (r = 0.72-0.88) with catches of cleistothecia in filter-paper funnels attached to the trunk of grapevines. The density of populations on bark during winter was not correlated with several measures of disease incidence and severity from the previous growing season. We concluded that while incidence and severity may determine the potential population available for dispersal, rain events determine the actual efficiency of transfer from infected organs to the bark of the vine. The percentage of ascocarps that reacted positively with the fluorescent vital stain fluorescein diacetate ranged from 50 to 62% and did not change significantly during overwintering until cleistothecia began to dehisce in spring. Therefore, the density and viability of populations of cleistothecia on bark at the time of budbreak appear to have been determined at the time of leaf fall the previous autumn and were not modified by subsequent environmental conditions. The densest aggregations of cleistothecia occurred on the cordons of cordon-trained vines, with successively lower densities occurring on the bark of the upper and lower trunks. The pruning and training system of vines of Vitis labrusca cv. Concord did not affect the density of populations of cleistothecia on bark

FREEZING STRESS AND OSMOTIC DEHYDRATION IN FUCUS DISTICHUS (PHAEOPHYTA): EVIDENCE FOR PHYSIOLOGICAL SIMILARITY1

ABSTRACTThe effects of osmotic dehydration and freezing on photosynthesis were studied in the brown alga Fucus distichus L. The data indicated that F. distichus exhibits similar physiological responses to both osmotic dehydration and freezing stress and that these responses resemble those in the literature for the effect of desiccation in air. Both stresses inhibited light‐limited (Psubsat) and light‐saturated (Pmax) photosynthesis measured immediately after plants were reimmersed in seawater. The degree of initial inhibition and subsequent recovery of photosynthesis were proportional to the severity of the dehydration or freezing treatment. Psubsat and Pmax recovered completely from osmotic dehydration for 3 h in 200% and 3 hr at – 10°C, but recovery was only partial following 3 h in 300%o or 3 h at – 15°C. In most cases, recovery was complete within 2 h following dehydration, with little further recovery occurring between 2 and 24 h posttreatment. No time‐dependent recovery occurred following severe freezing. Observations using the vital stain fluorescein diacetate suggested that the lack of complete recovery might be due to severe damage or death of a proportion of cells in the thallus. There were no clear effects of either osmotic dehydration or freezing on dark respiration (Rd), although Rd was stimulated in all emersed treatments (frozen plants and 5° C controls) immediately following reimmersion. Measurement of chlorophyll fluorescence induction kinetics indicated that both osmotic dehydration and freezing reduced the ratio of variable to maximum florescence (Fv/Fm), indicating a decrease in the quantum efficiency of photosystem I. Based on these data, we suggest that there are common cellular and physiological components involved in the response of fucoid algae to a range of water stresses. This hypothesis was supported by experiments that showed that osmoacclimation in hyperosmotic seawater (51%o)for 2 weeks increased the ability of F. distichus to recover from freezing at – 15° C. During acclimation, mannitol content increased under hyperosmotic conditions and decreased under hypoosmotic conditions. Changes in plasma membrane integrity, determined by fresh weight: dry weight ratio, and amino acid release following freezing indicated an increasing gradient of freezing tolerance from low to high salinity. However, none of these physiological changes fully explained the marked increase in the freezing tolerance of photosynthesis observed in plants acclimated under hyperosmotic conditions.

Comparison of physiological responses of guard cell protoplasts of Nicotiana glauca isolated from leaves collected before dawn or at midday

We observed that guard cell protoplasts isolated from leaves collected at midday from Nicotiana glauca Graham (tree tobacco) did not give the same physiological responses to light as those isolated from leaves collected in early morning. Based on that observation, we attempted to determine whether there were significant differences between the physiological responses of guard cell protoplasts isolated from leaves collected before dawn (with closed stomata) and those isolated from leaves collected at midday (with open stomata). We isolated guard cell protoplasts from leaves collected before dawn and at midday and compared (1) rates of red and blue light‐induced pH changes in weakly buffered media caused by changes in their metabolism, (2) their rates of oxygen consumption in darkness and oxygen evolution in light and (3) relative rates of decay of variable chlorophyll a fluorescence in their chloroplasts. Studies with the vital stain fluorescein diacetate failed to reveal any significant differences in the viabilities of protoplast preparations from leaves collected before dawn and at midday. Furthermore, protoplasts from leaves collected at these times swelled to similar extents in an osmotic medium containing 10 µM fusicoccin and 5 mM KCI. Nevertheless, rates of light‐induced pH changes, rates of oxygen consumption and evolution and rates of decay of variable chlorophyll a fluorescence were all lower in preparations of guard cell protoplasts from leaves collected at midday than in preparations from leaves collected before dawn. Initial volumes of guard cell protoplasts isolated from leaves collected at midday were 150% of those of guard cell protoplasts isolated from leaves collected before dawn. We suggest that the differences in responses of guard cell protoplasts isolated from leaves collected before dawn and at midday may be caused by (1) nonoptimal isolation conditions for guard cell protoplasts prepared from leaves collected at midday, (2) the lower surface‐to‐volume ratio of guard cell protoplasts isolated from leaves collected at midday or (3) diurnal and/or circadian regulation of guard cell metabolism over the course of a day.

Desiccation Tolerance of Papaver dubium L. Pollen during Its Development in the Anther

Developing pollen of Papaver dubium L. becomes functional and desiccation tolerant at approximately 2 to 1 days prior to anthesis, coincident with degradation of starch and a doubling of the amount of sucrose, the primary soluble carbohydrate present. When anthers were taken from flower buds at 3 days before anthesis, pollen dehisced upon exposure to the ambient air. This dried pollen did not fluoresce with the vital stain fluorescein diacetate, had increased leakage of K(+), and did not swell properly in a germination medium. In contrast, pollen became functional and desiccation resistant when these young anthers were first incubated in a water-saturated atmosphere for 30 hours. Phospholipid composition revealed no major differences over the last 3 days of development. When this immature pollen was liberated mechanically and allowed to mature in humid air, starch degraded and sucrose content nearly doubled, and the grains became largely functional and dehydration tolerant. Large unilamellar vesicles were prepared from isolated phospholipids to study dehydration-induced fusion and leakage. When dried in the presence of increasing concentrations of sucrose, vesicle integrity was progressively retained. These data indicate that pollen maturation during the last 3 days of development occurred independently from the parent plant. Sucrose may play an essential role in the acquired tolerance to severe dehydration.

Determination, by fluorescein diacetate staining, of fungal viability during mycoparasitism

Trichoderma hamatum is a mycoparasite of Rhizoctonia solani. When stained with the vital stain fluorescein diacetate (FDA), both interacting fungi showed fluorescence during the first 24 h of incubation. Thereafter R. solani showed only a faint fluorescence while T. hamatum was still glowing up to 7 days. When each of these fungi were grown alone they showed a significant fluorescence for 4 days. This technique for determination of fungal viability during mycoparasitism may indicate that the initial attack occurs while the host is still alive.