Binder BJ, Chisholm SW, Olson RJ, Frankel SL, Worden AZ (1996) Dynamics of picophytoplankton, ultraphytoplankton and bacteria in the central equatorial Pacific. Deep-Sea Research Part Ii-Topical Studies in Oceanography 43 :907-931
Pico- and ultraplankton are known to contribute significantly to overall biomass and primary productivity in the ’’high nutrient-low chlorophyll’’ waters of the equatorial Pacific. In order to understand the dynamics of this community on ecologically relevant time-scales, we examined the abundance, distribution and cellular characteristics of Prochlorococcus, Synechococcus, eukaryotic ultraphytoplankton and heterotrophic bacteria during two 20-day time-series at 0 degrees N, 140 degrees W in the spring and fall of 1992 (JGOFS time-series cruises, TS-I and TS-II). Prochlorococcus was numerically dominant among the autotrophic groups considered, with mean cell concentrations in surface waters on the order of 1.4 x 10(5) cells ml(-1). Synechococcus and ultraphytoplankton abundances were 17-30-fold lower than those of Prochlorococcus, and heterotrophic bacterial abundances were 5-7-fold higher (during TS-I and TS-II, respectively). Daily cell abundances for all groups varied by factors of 1.5-2 within each time-series. Depth-integrated Prochlorococcus abundance averaged over each time-series was 25% lower during TS-II relative to TS-I ; ultraphytoplankton abundance was 42% higher during the same period. Prochlorococcus and ultraphytoplankton both contributed significantly to the estimated total autotrophic biomass ; Synechococcus contributed relatively little. Estimated total photosynthetic pico- plus ultraplankton biomass was on average 30% higher than heterotrophic bacterial biomass. Changes in the fluorescence and light scatter properties of individual Prochlorococcus cells were observed during the passage of a tropical instability wave during TS-II, and are hypothesized to reflect a physiological response among these cells to that event. Examination of bulk properties alone (e.g. cell numbers or total red fluorescence) would not have revealed these physiological changes. Lower bounds for Prochlorococcus-specific growth rates were calculated based on the DNA distributions of these populations at dusk. These rates were maximal at 15 or 30 m depth, where they approached one doubling per day. Changes in Prochlorococcus forward angle light scatter (FALS) from dawn to dusk were well correlated with these estimates of specific growth rate, an observation that allowed us to relate measurements of FALS to cell volume for Prochlorococcus. Copyright (C) 1996 Elsevier Science Ltd.
Blanchot J, Rodier M (1996) Picophytoplankton abundance and biomass in the western tropical Pacific Ocean during the 1992 El Nino year : Results from flow cytometry. Deep-Sea Research Part I-Oceanographic Research Papers 43 :877-895
Natural populations of phytoplankton from the western tropical Pacific Ocean were analyzed by flow cytometry from a transect along 165 degrees E between 20 degrees S and 7 degrees N. The abnormal hydrological situation corresponded to a weak Fl Nino event, with no equatorial upwelling and a marked nutrient ridge centered on 10 degrees S. Prochlorophytes dominated numerically everywhere along the vertical, whatever the depth, in the 0-160-m layer (96% of cell abundance). Paradoxically, the highest concentrations, up to 4.4x10(5) cells ml(-1), were found in oligotrophic waters (<0.1 mu M NO3). In contrast, the highest concentrations of orange cyanobacteria and red-fluorescing picoeukaryotes were observed when nitrate was present in the photic layer, i.e. around 10 degrees S (up to 6.4x10(4) cells ml(-1) and 1.3x10(4) cells ml(-1)), and, to a lesser extent in the vicinity of the deep nitracline north of 8 degrees S. Along the transect we encountered two hydrological situations, characterized by different community structures. The first one, found from 15 degrees S to 7 degrees N, except at 10 degrees S, was a two-layer structure (Typical Tropical Structure, TTS) defined by a strong pycnocline in the upper 180 m and a well-marked nitracline. In this region, Prochlorococcus and picoeukaryotes co-dominated the 180-m integrated fluorescence and carbon biomass, but Prochlorococcus were the major component in the upper nitrate-depleted layer, while picoeukaryotes dominated the underlying rich layer. Inversely, Synechococcus were a relatively minor contributor to fluorescence (similar to 4%) and phytoplankton biomass (<1%) in comparison to the other cell types. The second structure observed in the southernmost part of the transect (20 degrees S-16 degrees S) was defined by the absence of a density gradient, and therefore by deep vertical mixing. In this case, the concentration of Prochlorococcus in the upper nitrate-depleted layer was reduced, whereas Synechococcus percentage contribution in the upper 180 m was significantly higher than in the TTS (>30% of total fluorescence and similar to 4% of carbon biomass). According to our results, we discuss the expected role of each phytoplankton group in the regenerated and new production. Finally, we discuss the importance of cell size as a factor in the expected roles of the different phytoplankton groups in the carbon sink. Copyright (C) 1996 Elsevier Science Ltd.
Buma AGJ, VanHannen EJ, Veldhuis MJW, Gieskes WWC (1996) UV-B induces DNA damage and DNA synthesis delay in the marine diatom Cyclotella sp. Scientia Marina 60 :101-106
The effect of UV-B on the occurrence of DNA damage and consequences for the cell cycle were studied in the marine diatom Cyclotella sp. DNA damage was quantified by immunofluorescent detection of thymine dimers in nuclear DNA of single cells using flow cytometry. A total UV-B dose (biologically effective dose BE(DNA300nm)) of 1.05 kJ m(-2) caused detectable damage in about 20% of the exposed population. In the period after the UV-B treatment, when the culture was exposed to PAR (450 mu mol m(-2)s(-1)) only, thymine dimers were removed ; after 8 hours none of these photoproducts remained. Cellular DNA content measurements and quantification of the fraction of recently divided cells revealed that the DNA synthesis rate as well as the rate of cell division were reduced during and shortly after UV-B exposure. Apparently, UV-B irradiation extends the cell cycle of Cyclotella sp. in the S (DNA synthesis) phase until the dimers are removed.
Burton RS (1996) Molecular tools in marine ecology. Journal of Experimental Marine Biology and Ecology 200 :85-101
Molecular tools have diver se applications in marine ecology. In microbial systems, DNA sequences of rRNA and other genes have identified a variety of novel lineages of bacteria inhabiting marine environments that have resisted traditional culture methods. However, relatively few natural populations have been characterized due to the rather labor-intensive methodologies employed. Recent technological developments such as in situ PCR and flow cytometry promise to greatly enhance the speed at which microbial taxa can be identified and enumerated in field collected water and substrate samples ; such advances will allow future work to employ the spatial and temporal field sampling required to monitor the impact of natural and anthropogenic changes in the environment. This approach also holds promise for examining physiological status of field collected cells, garnering information on such elusive parameters as growth rates and the extent of nutrient limitation under natural conditions. Studies of macrobiota have similarly benefited from the use of molecular approaches to species identification. This has been particularly true with regard to distinguishing among larval forms of closely related taxa which are nearly identical morphologically. Genetic variation within species assayed by molecular tools has been useful in examining the stability of populations through time and in assessing patterns of recruitment to geographically separated populations. Enhanced understanding of these ecological problems will also require intensive spatial and temporal monitoring of both larval and adult populations. Often, the newer techniques based on DNA sequence variation have practical advantages over allozyme techniques : e.g., PCR allows assay of minute quantities of DNA that may come from ethanol preserved samples. However, when ample allozyme variation exists to address a given issue, these older techniques may be favored on a variety of criteria, including spaed and cost. Hence, choice of methodology should be based on the expected efficiency of a given approach to a specific problem rather than the apparent sophistication of the method itself.
Button DK, Robertson BR, Juttner F (1996) Microflora of a subalpine lake : Bacterial populations, size and DNA distributions, and their dependence on phosphate. Fems Microbiology Ecology 21 :87-101
In Lake Zurich, a deep subalpine mesotrophic lake, phosphate was low or limiting at 0.2 to 1 mu M relative to combined nitrogen at 50 mu M. Heterotrophic bacteria were responsible for 53% of the observed microbial wet biomass in our depth profile while phytoplankton, largely Planktothrix (Oscillatoria) rubescens, contributed most of the remainder. Most cell carbon was contributed by this carbon-sufficient cyanobacterium. A material balance indicated that most of the phosphate was sequestered by the bacteria due to a higher phosphate content and specific affinity for this nutrient. Size distributions of the heterotrophic bacteria were narrow ; 90% of organisms were from 0.06 to 0.66 mu m(3) in volume. Several subpopulations of bacteria were resolved by flow cytometry, and bivariate fluorescence (DAPI-DNA) and light scatter (cell-size) histogram profiles varied with depth. One or two of these subpopulations appeared to be bacteria with sufficient cytoplasmic constituents to produce a normal light-scatter signal but retained only a small amount of DNA ; an apparent content of 200 kbp or 5% of a usual oligobacterial genome. These helped increase the oligobacterial population to 6 X 10(6) ml(-1). Application of published specific affinities and measured nutrient concentrations to formulations of system kinetics led to the conclusion that growth rates of the heterotrophic bacterial fraction were carbon limited with cell size, and thus populations were controlled by grazing. The depth profile indicated that phototrophs affected phosphate concentrations in a significant way. Considerations of nutrient uptake kinetics suggested that much potential capacity remained in the dissolved phosphate pool to support additional phytoplanktonic biomass. Computations led to the conclusion that, if phosphate is generally limiting in lakes, then additional mechanisms exist which limit populations of phytoplankton to sufficiently small values to allow phosphate accumulation to observed levels. Bacterial biomass then depends on the organic carbon from these phosphate-controlled organisms.
Cailliau C, Claustre H, Vidussi F, Marie D, Vaulot D (1996) Carbon biomass, and gross growth rates as estimated from C-14 pigment labelling, during photoacclimation in Prochlorococcus CCMP 1378. Marine Ecology-Progress Series 145 :209-221
The C-14 labelling of chlorophylls and carotenoids is increasingly used to evaluate phytoplanktonic biomass and growth rates in oceanic systems. Rigorous testing of the technique in the laboratory, however, is necessary prior to its application in the field. A Mediterranean clone of Prochlorococcus, a photosynthetic prokaryote which is an important component of the autotrophic biomass in oligotrophic environments, was subjected to shifts in light intensity. Particulate organic carbon (POC) was monitored by CHN analysis, pigments by HPLC and Prochlorococcus and heterotrophic bacteria concentrations by flow cytometry. Using a combination of HPLC and on-line radioactivity detection, C-14 labelling kinetics of divinyl-chlorophyll a (Dv-chl a) and zeaxanthin were followed. Prochlorococcus changed its Dv-chl a content markedly in response to change in light intensity, but not its zeaxanthin content, which remained nearly constant around 1.07 fg cell(-1) regardless of the irradiance. Pigment synthesis rates were correctly estimated from their C-14 incorporation rates whatever the Light level. From POC measurements and cell concentrations, the Prochlorococcus carbon content was estimated to be 49 fg C cell(-1). Moreover, under both constant and shifted (high to low and vice versa) Light conditions, Prochlorococcus growth rate (as computed from variations in cell. density) was much better estimated from zeaxanthin than from Dv-chl a labelling rates.
Carr MR, Tarran GA, Burkill PH (1996) Discrimination of marine phytoplankton species through the statistical analysis of their flow cytometric signatures. Journal of Plankton Research 18 :1225-1238
Flow cytometry is a research technique for the rapid analysis of phytoplankton abundance and distribution in marine waters. Although the technique is inherently much faster than optical microscopy for counting phytoplankton, its capability for analysing individual taxa is restricted, due largely to the lack of suitable data analysis protocols. These protocols. which use univariate and bivariate plots, can typically differentiate a maximum of three or four phytoplankton taxa under laboratory conditions. We present here two multivariate statistical techniques, quadratic discriminant analysis (QDA) and canonical variate analysis (CVA), used to identify 32 species of phytoplankton from how cytometric data. CVA was shown to be a useful graphical technique for analysing and displaying data, while QDA was successful at discriminating over two-thirds of the phytoplankton species, with classification rates >70%. QDA was also shown to be more than Two orders of magnitude faster than conventional flow cytometric analyses for discriminating and enumerating phytoplankton species. We discuss the potential of multivariate analysis, and ways of developing these techniques for the detailed analysis of complex natural phytoplankton populations in the marine environment.
Cid A, Fidalgo P, Herrero C, Abalde J (1996) Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry. Cytometry 25 :32-36
Flow cytometric measurements were used to investigate the toxic action of copper on some Phaeodactylum tricornutum membrane systems. Throughout the time of metal exposure, the percentage of viable cells decreased as copper concentration increased. The forward scatter signal increased as a result of copper exposure. After 72 h of metal exposure, cultures with 0.5 and 1 mg l-1 of copper showed an important increase in the peroxidase activity in comparison with control cells. Cells cultured with copper presented alterations in the membrane potential, increasing as copper concentration increased, after 96 h of metal exposure. Results obtained in this work showed that copper induced a degenerative process in P. tricornutum cells, closely related with alternations or disorders in membrane systems.
Cid A, Herrero C, Abalde J (1996) Functional analysis of phytoplankton by flow cytometry : A study of the effect of copper on a marine diatom. Scientia Marina 60 :303-308
Changes in cell volume, chlorophyll a fluorescence, cell viability, peroxidase activity, intracellular pH, and cytoplasmic and mitochondrial membrane potential of the marine diatom Phaeodactylum tricornutum stressed by copper were analyzed by flow cytometry. Results demonstrate that flow cytometry is a very useful tool in microalgal functional studies.
Cunningham A, Cid A, Buma AGJ (1996) Technical discussions II - Flow cytometric analysis. Scientia Marina 60 :299-300
In this paper the potencial of flow cytometry as applied to the aquatic life sciences is discussed. The use of flow cytometry for studying the ecotoxicology of phytoplankton was introduced. On the other hand, the new flow cytometer EUROPA was presented. This is a multilaser machine which has been specially designed to have a high volumetric throughput and wide dynamic range : purpose-built modules allow single cell diffractometry and video imaging in flow which help greatly with morphological analysis and taxonomic identification.
Deere D, Porter J, Pickup RW, Edwards C (1996) Survival of cells and DNA of Aeromonas salmonicida released into aquatic microcosms. Journal of Applied Bacteriology 81 :309-318
The survival of the bacterial fish pathogen Aeromonas salmonicida, and persistence of its DNA, were monitored in aquatic microcosms using selective culture and most probable number PCR. Bacterial cells and naked DNA were released into natural non-sterile microcosms consisting of lake sediment overlayered with lake water. Two different types of surface sediment were used. One was sandy in character, taken from the shoreline whilst the other was a littoral loamy surface mud. Inoculated cells and naked DNA became undetectable from water overlayers within 4 weeks of release. Colony counts of Aer. salmonicida declined below detectable limits after 4 weeks in loamy sediment or 7 weeks in sandy sediment ; however, naked DNA and DNA from released cells remained detectable for more than 13 weeks.
Donoghue AM, Garner DL, Donoghue DJ, Johnson LA (1996) Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress fluorescent staining and flow cytometry. Theriogenology 46 :153-163
A study was conducted to evaluate the integrity of turkey sperm plasma membrane subjected to various hypo-osmotic conditions, and to develop a test to determine the percentage of viable spermatozoa capable of withstanding hypo-osmotic stress after in vitro storage. Semen from 10 toms was collected and pooled twice weekly for 6 wk, and each trial was repeated 6 times. For Trial I, spermatozoa were subjected to varying osmotic solutions by suspension in 100, 80, 60, 40, 20 or 0% PBS in distilled water (297 to 19 mosm/kg H2O) and stained to assess membrane integrity with Calcein-AM (GAL) and propidium iodide (PI). The CAL detected viable spermatozoa (green fluorescence) while the PI shined dead cells (red fluorescence). Spermatozoa were evaluated microscopically and by flow cytometry. The percentage of viable spermatozoa, as determined by flow cytometry, was not different from that in 100% PBS (76.4+/-3.8) to 20% PBS (74.1+/-3.5). Fewer viable spermatozoa, however, were detected in 0% PBS (61.1+/-4.8, P < 0.05). The percentages of swollen tails observed for viable (green stained) spermatozoa were 0, 4.5, 6.5, 24.3, 50.5 and 100% for 100, 80, 60, 40, 20 and 0% PBS, respectively. Semen was also evaluated fresh or after 24 h in vitro storage at 5 degrees C in PBS or H2O (Trial II). The percentage of viable spermatozoa was not different for fresh or in vitro-stored spermatozoa in PBS. For spermatozoa stored 24 h in vitro, the percentage of viable cells was lower in H2O (48.0+/-5.1) than in PBS (66.1+/-5.6, P<0.05). Subjecting in vitro-stored sperm cells to hypo-osmotic stress before fluorescent staining resulted in detection of labile spermatozoa not accounted for by staining alone, indicating that the turkey sperm membrane is more susceptible to damage after cold storage.
Edvardsen B, Vaulot D (1996) Ploidy analysis of the two motile forms of Chrysochromulina polylepis (Prymnesiophyceae). Journal of Phycology 32 :94-102
In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (alpha) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (beta) contained cells with double the DIVA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, of both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation : authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable : cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis Includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.
Eglinton TI, Boon JJ, Minor EC, Olson RJ (1996) Microscale characterization of algal and related particulate organic matter by direct temperature-resolved mass spectrometry. Marine Chemistry 52 :27-54
In this paper we report a preliminary investigation of laboratory algal cultures and natural populations of marine particles utilizing Direct (i.e. ’’in-source’’) Temperature-resolved Mass spectrometry (DT-MS). With this technique the potential exists to characterize microgram quantities of particulate organic matter (POM) at the molecular level. Two particular aspects of the approach set DT-MS apart from previous pyrolysis (Py)-MS investigations. First, temperature resolution allows characterization of both desorption (biolipid) and pyrolysis (biopolymeric) products in a single measurement. Second, an improved sample inlet configuration enables compounds exhibiting a broad range of polarities and molecular weights to be transmitted to the analyzer. The above features, coupled with the rapid analysis time (typically 5 min/sample) and amenability to statistical data reduction procedures, renders DT-MS well suited for profiling or mapping molecular-level variations in POM composition at a spatial and temporal resolution hitherto impractical using conventional biochemical assays. To illustrate the versatility of the approach and extent of (bio)chemical information available from DT-MS, we have analyzed a series of algal cultures together with selected POM samples collected in sediment traps and filtered from hydrocasts. We have also exploited the inherent sensitivity of DT-MS, to obtain information on compositional variability between and, more significantly, within particle size classes. Results of the latter, which was attained by conducting DT-MS measurements on particles sorted by flow cytometry, reveal substantial internal variations in chemical composition.
Frankel DS, Frankel SL, Binder BJ, Vogt RF (1996) Application of neural networks to flow cytometry data analysis and real-time cell classification. Cytometry 23 :290-302
Conventional analysis of flow cytometric data requires that population identification be performed graphically after a sample has been run using two-parameter scatter plots. As more parameters are measured, the number of possible two-parameter plots increases geometrically, making data analysis increasingly cumbersome. Artificial Neural Systems (ANS), also known as neural networks, are a powerful and convenient method for overcoming this data bottleneck. ANS "learn" to make classifications using all of the measured parameters simultaneously. Mathematical models and programming expertise are not required. ANS are inherently parallel so that high processing speed can be achieved. Because ANS are nonlinear, curved class boundaries and other nonlinearities can emerge naturally. Here, we present biomedical and oceanographic data to demonstrate the useful properties of neural networks for processing and analyzing flow cytometry data. We show that ANS are equally useful for human leukocytes and marine plankton data. They can easily accommodate nonlinear variations in data, detect subtle changes in measurements, interpolate and classify cells they were not trained on, and analyze multiparameter cell data in real time. Real-time classification of a mixture of six cyanobacteria strains was achieved with an average accuracy of 98%.
Fujihashi K, Kawabata S, Hiroi T, Yamamoto M, McGhee JR, Nishikawa S, Kiyono H (1996) Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes. Proc Natl Acad Sci U S A 93 :3613-3618
In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.
Guelorget O, Martin JL, Lefebvre A, Courties C, Perthuisot JP, Supangat A, Fuchs J, Suastika M (1996) Open sea paralic ecosystems south of Java Sea : Environmental approach by flow cytometry. Journal of Coastal Research 12 :256-270
The organization and functioning of several sites of shrimp production along the northern Java and Sumatra coasts have been studied through the following parameters : usual hydrological parameters, total suspended matter, suspended organic matter, suspended mineral matter, planktonic chlorophyll a and phaeophytin contents. Additionally, the structure and composition of phytoplanctonic populations was analysed by Bow cytometry. From obtained data on the site of Jepara (which is representative of all sites south of Java Sea), it appears that all coastal environments including open marine environments undergo a high confinement level. This is depicted by the drastic simplification of the natural as well as aquacultural ecosystems ; the natural benthic macrofauna is almost completely absent and the other organisms are only procaryotes which grow in such ecosystems at the continental edge of the paralic realm. Studied systems are rather stable through time and display gradients from offshore towards the most restricted parts of the aqua cultural structures, i.e., the semi-intensive and intensive ponds, These gradients move following the tidal cycles. A studied site in Sumatra along the Malacca straits displays similar gradients but a lesser confinement which is indicated by the presence of more diversified phytoplanctonic populations. In all studied sites, the intensive aquacultural practices tend to increase the confinement level not only for the aquacultural installations themselves but also for the offshore environments.
Kaprelyants AS, Mukamolova GV, Davey HM, Kell DB (1996) Quantitative analysis of the physiological heterogeneity within starved cultures of Micrococcus luteus by flow cytometry and cell sorting. Applied and Environmental Microbiology 62 :1311-1316
A high proportion of Micrococcus luteus cells in cultures which had been starved for 3 to 6 months lost the ability to grow and form colonies on agar plates but could be resuscitated from their dormancy by incubation in an appropriate liquid medium (A. S. Kaprelyants and D. B. Kell, Appl. Environ, Microbiol. 59:3187-3196, 1993). We used flow cytometry and cell sorting to study populations of bacteria that had been starved for 5 months. These cells could be stained by the fluorescent lipophilic ration rhodamine 123, but such staining was almost independent of metabolically generated energy in that it was not affected by uncouplers. Two populations could be distinguished, one with a lower degree of rhodamine fluorescence (a degree of fluorescence referred to as region A and containing approximately 80% of the cells) and one with a more elevated degree of fluorescence (region B, approximately 20% of the cells). Subsequent incubation of starved cells in fresh medium in the presence of the antibiotic chloramphenicol (to which M. luteus is sensitive) resulted in the transient appearance of cells actively accumulating rhodamine 123 (and fluorescing in region B) and of larger cells exhibiting a yet-greater degree of fluorescence (region C). These more fluorescent cells accounted for as much as 50% of the total population, under conditions in which the viable and total counts were constant. Thus, metabolic resuscitation of at least one-half of the cells takes place under conditions in which cryptic growth cannot play any role. Sorting experiments revealed that the great majority of the viable cells in the starved population are concentrated in regions B and C and that the extent of rhodamine staining under conditions of starvation therefore reflects the physiological state of the cells, Physical separation of these cells from cells in region A resulted in an increase (of approximately 25-fold) in the viability of cells in regions B and C and of the population as a whole. Resuscitation of dormant cells in a most-probable-number assay in the presence of supernatant taken from growing M. luteus revealed the resuscitation of cells from regions B and C but not from region A. It is suggested that initially dormant (resuscitable) cells are concentrated in regions B and C.
Koch AL, Robertson BR, Button DK (1996) Deduction of the cell volume and mass from forward scatter intensity of bacteria analyzed by flow cytometry. Journal of Microbiological Methods 27 :49-61
Forward scatter signals (0-20 degrees) from a flow cytometer can be converted into cellular volumes or cellular wet or dry biomasses by the calculations presented here. The results are applicable to bacteria, and including very small bacteria (0.02-0.6 mu m(3)), rods as well as cocci. The physical limitations and the mathematical basis of the calculations are discussed to justify the application of the proposed formula for practical use in microbial ecology and physiology of extremely small aquatic bacteria as well as the more well-studied forms. Although the method was developed for the small bacteria present in natural populations that are difficult to size by alternative methods, it will also be useful for laboratory cultures of bacteria when present in very low concentrations as well as for the characterization of subpopulations and different growth phases. Bacteria, in general, are small enough so that inclusion of the phase shifts of the light passing through the bacteria (the Mie theory) is superfluous. That theory is, however, useful for instrumental calibration with latex microspheres and for some blue-green algae. Even the interference between rays scattered from different parts of the cell (the Rayleigh-Gans theory), although significant, is minor, particularly from elongated particles which are oriented by the fluidics. The computer programs, provided here, correct for intraparticle interference and generate cell volumes (or wet or dry masses) from the forward scatter intensity. The result of the calculations of the light scattered into the collection system of the flow cytometer is presented. From analysis of these values a rule is developed to relate the observed intensities, when scaled by an empirical constant and raised to a power between 0.5 and 0.75, to the cell biomass.
Lange M, Guillou L, Vaulot D, Simon N, Amann RI, Ludwig W, Medlin LK (1996) Identification of the class prymnesiophyceae and the genus Phaeocystis with ribosomal RNA-targeted nucleic acid probes detected by flow cytometry. Journal of Phycology 32 :858-868
Target regions specific for the class Prymnesiophyceae and the genus Phaeocystis (Har.) Lag. were identified from 18S ribosomal RNA coding regions, and two complementary probes were designed (PRYMN01 and PHAEO01). Detection of whole cells hypbridized with these probes labeled with fluorescein isothiocyanate was difficult using epifluorescence microscopy because autofluorescence of the chlorophylls seriously interfered with the fluorescence of the probes. In contrast, flow cytometry proved very useful to detect and quantify the fluorescence of the hybridized cells. Hybridization conditions were optimized, especially with respect to formamide concentration. Both probes were tested on a large array of both target and nontarget strains. Positive and negative controls were also analyzed. Specificity was tested by adding a competing nonlabeled probe. Whereas probe PHAEO01 seems to have good specificity, probe PRYMN01 appeared less specific and must be used with stringent positive and negative controls.
Marie D, Vaulot D, Partensky F (1996) Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes. Appl Environ Microbiol 62 :1649-1655
Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes. Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4’, 6-diamidino-2-phenylindole (DAPI). YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA. They are highly sensitive to ionic strength. Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals. After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining. These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E. coli. However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.
Nair-Menon JU, Campbell GT, Blake CA (1996) Toxic effects of octylphenol on cultured rat and murine splenocytes. Toxicol Appl Pharmacol 139 :437-444
Alkylphenol polyethoxylates and alkylphenols, such as 4-tertoctylphenol (OP), are environmental contaminants. Because these compounds are toxic to aquatic animals, we studied the effects of OP on splenocytes removed from male Fischer 344 rats or male Balb/c mice and cultured in vitro. Cell viability was assessed by trypan blue exclusion after 5 or 27 hr of culture. Culture with 0.08% ETOH (vehicle) or any dose of OP did not alter total cell number or the percentage of viable cells after 5 hr. Culture of cells with two different alkylphenol polyethoxylates for 5 hr resulted in the loss of all cells. The percentages of viable rat or mouse cells after 27 hr of culture were decreased significantly by 10(-12) M OP or greater concentrations. The actions of OP, dexamethasone (DEX), and 17 beta-estradiol on rat splenocytes were compared. Dexamethasone was more toxic than OP after 24 hr of culture ; 17 beta-estradiol was not toxic. Dexamethasone and OP, but not 17 beta-estradiol, caused significant nuclear condensation after 3 hr of culture (acridine orange staining) or 4 hr of culture (propidium iodide staining). The toxicity of 10(-6) M OP, but not that of 10(-6) M DEX, was eliminated when mouse splenocytes were cultured in Ca2+ -free medium. Significantly more mouse splenocytes containing free 3’-OH DNA ends were detected by activated cell sorter analyses when the cells had been incubated for 4 hr with 10(-4) or 10(-6) M OP or 10(-6) M DEX. The results of these studies demonstrate that OP is toxic to cultured rat and mouse splenocytes and suggest that this toxic effect is exerted, at least partially, through Ca2+-dependent apoptosis.
Nystrom T, Larsson C, Gustafsson L (1996) Bacterial defense against aging : role of the Escherichia coli ArcA regulator in gene expression, readjusted energy flux and survival during stasis. Embo J 15 :3219-3228
Using two-dimensional gel electrophoresis and N-terminal amino acid sequencing analysis, we demonstrate that a mutant of the global regulatory protein ArcA fails to decrease the synthesis of the TCA cycle enzymes malate dehydrogenase, isocitrate dehydrogenase, lipoamide dehydrogenase E3 and succinate dehydrogenase in response to stasis, while the increased production of the glycolysis enzymes phosphoglycerate mutase and pyruvate kinase is unaffected. Microcalorimetric and respiratory measurements show that the continued production of TCA cycle enzymes in the (delta)arcA mutant is manifested as an elevated rate of respiration and total metabolic activity during starvation. The (delta)arcA mutant is severely impaired in surviving prolonged periods of exogenous carbon starvation, a phenotype that can be alleviated by overproducing the superoxide dismutase SodA. In addition, flow cytometry demonstrates that starving (delta)arcA mutant cells, in contrast to wild-type cells, fail to perform reductive division, remain large and contain multiple chromosomal copies. We suggest that the ArcA-dependent reduced production of electron donors and the decreased level and activity of the aerobic respiratory apparatus during growth arrest is an integral part of a defense system aimed at avoiding the damaging effects of oxygen radicals and controlling the rate of utilization of endogenous reserves.
Parpais J, Marie D, Partensky F, Morin P, Vaulot D (1996) Effect of phosphorus starvation on the cell cycle of the photosynthetic prokaryote Prochlorococcus spp. Marine Ecology-Progress Series 132 :265-274
The influence of phosphorus (P) starvation on the cell cycle of Prochlorococcus was studied for several strains from the Mediterranean Sea, the Atlantic and Pacific Oceans. P starvation could only be induced at high N/P ratio (greater than or equal to 150). In P-starved stationary phase, the fraction of cells in G(1) dropped markedly, while cells accumulated not only in G(2), but also in the DNA synthesis phase S, indicating that cells were not arrested at a precise cell cycle location but in all cell cycle phases. The percentage of P-starved cells in each phase varied slightly between strains. For the Mediterranean Sea strain CCMP 1378, cell cycle arrest in G(1) and G(2) could be reversed by addition of phosphates, while cells arrested in S could not resume their cycle and died. Such irreversible arrest in S had never been reported previously in phytoplankton. This suggests that cell cycle controls are not very tight in Prochlorococcus, since cells are not prevented from entering the critical S phase in the absence of phosphorus.
Porter J, Deere D, Pickup R, Edwards C (1996) Fluorescent probes and flow cytometry : New insights into environmental bacteriology. Cytometry 23 :91-96
Recent trends in now cytometry have established new techniques in bacteriology, Advances in fluorescent dye technology complement these improvements, offering probes for a variety of cellular functions. Bacterial ecology requires the application of new techniques to help answer questions unanswerable by traditional methods alone, Here we review some aspects of how coupling the two technologies has enabled researchers to directly study individual bacterial cells, and revealed the complexity and heterogeneity present in both laboratory cultures and in environmental samples. Results are discussed with respect to viability analysis, stress induced changes, specific cell detection and cell sorting. (C) 1996 Wiley-Liss, Inc.
Vaquer A, Troussellier M, Courties C, Bibent B (1996) Standing stock and dynamics of picophytoplankton in the Thau Lagoon (northwest Mediterranean coast). Limnology and Oceanography 41 :1821-1828
Eucaryotic picophytoplankton (PEUC), picocyanobacteria, and larger phytoplanktonic cells from the Thau Lagoon (northwest Mediterranean coast) were numbered by flow cytometry from November 1991 to February 1994. PEUC cells dominated the phytoplanktonic assemblage and exhibited seasonal dynamics. Monthly mean abundances of larger phytoplanktonic cell and PEUC were significantly correlated with temperature and solar irradiance values, with the highest correlations having been obtained for PEUC abundances. The mean abundance of total picophytoplankton (3.5 x 10(4) cells ml(-1)) was among the highest recorded in marine waters. The dominance of picophytoplankton over larger phytoplanktonic cells (average abundance, 5 x 10(3) cells ml(-1)) in the nutrient-rich waters of the lagoon was related to grazing from large-scale shellfish breeding (oyster’s biomass approximate to 35,000 t), which seems to act preferentially on the largest cells. Several hypotheses, including the potential selective effect of copper, were proposed to explain the dominance of the PEUC form (average abundance, 3.4 x 10(4) cells ml(-1)).
Watabe S, Wada S, Saito S, Matsunaga S, Fusetani N, Ozaki H, Karaki H (1996) Cellular changes of rat embryonic fibroblasts by an actin-polymerization inhibitor, bistheonellide A, from a marine sponge. Cell Struct Funct 21 :199-212
Bistheonellide A, an inhibitor of actin polymerization from the marine sponge Theonella sp., was introduced at a concentration of 100 nM into rat fibroblast of 2.4 x 10(4) cells/ml. Within 1 h, it disrupted stress fibers, accompanied by a marked change of the cell morphology, resulting in the formation of processes from the cell surface. Further incubation for 24 h in the presence of 100 nM bistheonellide A led to binucleation in most cells and subsequent inhibition of cell cycle progression. When bistheonellide A was withdrawn from the culture medium, binuclear cells began to grow again within 20 h and reverted to mononuclear morphology. Flow cytometric analysis fluorescence-activated cell sorting showed that 2C diploid DNA content in G1 phase was changed into 4C content of tetraploid for the bistheonellide A treated-cells in G1 phase and into 8C content during G2 and M phase. Therefore, we suggested that the bistheonellide A treatment inhibited cytokinesis, but not mitosis in M phase, and that treated cells were arrested at the early G1 phase. These effects of bistheonellide A on the cell cycle progression of 3Y1 fibroblast were also observed more prominently in cells synchronized in S phase with hydroxyurea. Cells in G0 phase were then activated by the addition of fetal calf serum in the presence of 100 nM bistheonellide A. Cell cycle progression of the bistheonellide A-treated cells was obviously slowed down or completely inhibited during G1 phase. These results reveal that actin filaments are not only essential to cytokinesis but also for promoting the progression of cell cycle G1 to S phase.
Wilkins MF, Boddy L, Morris CW, Jonker R (1996) A comparison of some neural and non-neural methods for identification of phytoplankton from flow cytometry data. Comput Appl Biosci 12 :9-18
Four artificial neural network paradigms (multilayer perceptron networks, learning vector quantization networks, and radial and asymmetric basis function networks) and two statistical methods (parametric statistical classification by modelling each class with Gaussian distributions, and non-parametric density estimation via the K-nearest neighbour method) were compared for their ability to identify seven freshwater and five marine phytoplankton species from flow cytometric data. Kohonen self-organizing maps were also used to examine similarities between species. Optimized networks and statistical methods performed similarly, correctly identifying between 86.8% and 90.1% of data from freshwater species, and between 81.3% and 84.1% of data from marine species. Choice of identification technique must therefore be made on the basis of other criteria. We highlight the way each method partitions the data space and thereby separates the data clusters, and discuss the relative merits of each with reference to complexity of data boundaries, training time, analysis time and behaviour when presented with ’novel’ data.
Wu D, Meydani SN, Meydani M, Hayek MG, Huth P, Nicolosi RJ (1996) Immunologic effects of marine- and plant-derived n-3 polyunsaturated fatty acids in nonhuman primates. Am J Clin Nutr 63 :273-280
The effect of marine- and plant-derived n-3 polyunsaturated fatty acids (PUFAs) on T cell-mediated immune response was studied in cynomolgus monkeys. Animals were first fed a 14-wk baseline diet ; 10 animals were then fed diets containing 1.3% or 3.3% of energy as eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) which the other 10 were fed diets containing 3.5% or 5.3% of energy as alpha-linolenic acid (ALA) for two consecutive 14-wk periods. Both diets significantly decreased the percentage of T cells (except 1.3% EPA + DHA), T helper cells (except 1.3% EPA + DHA and 3.5% ALA), and T suppressor cells. Proliferative response of lymphocytes to T cell mitogens significantly increased after the diet containing 3.3% EPA + DHA. Interleukin 2 production significantly increased after the diets containing 1.3% and 3.3% EPA + DHA. No significant changes in mitogenic response or interleukin 2 production were found after ALA diets. Feeding 1.3% or 3.3% EPA + DHA or 5.3% ALA significantly suppressed prostaglandin E2 production in response to T cell mitogens. Plasma tocopherol concentrations were decreased significantly only in monkeys fed ALA diets. We conclude that after adjustment for the tocopherol concentration, marine-derived n-3 PUFAs but not plant-derived n-3 PUFAs increased T cell-mediated mitogenic response and interleukin 2 production. This is most likely due to diet-induced quantitative differences in cellular fatty acid composition and, thus, in prostaglandin E2 production and tocopherol status.
Zettler ER, Olson RJ, Binder BJ, Chisholm SW, Fitzwater SE, Gordon RM (1996) Iron-enrichment bottle experiments in the equatorial Pacific : Responses of individual phytoplankton cells. Deep-Sea Research Part Ii-Topical Studies in Oceanography 43 :1017-1029
Iron-enrichment bottle experiments were monitored using how cytometry to investigate the hypothesis that phytoplankton in the equatorial Pacific are iron-limited. Iron-enriched Synechococcus, ultraphytoplankton, nanophytoplankton, pennate diatoms, and coccolithophorids had higher fluorescence and/or forward light scatter per cell than control cells ; for Prochlorococcus the trends were the same although the differences were not significant. This suggests that most phytoplankton cells were physiologically affected by the low iron concentrations in this region. However, only pennate diatoms showed significant increases in cell concentrations due to iron enrichment. The sum of chlorophyll fluorescences of individual cells measured by flow cytometry yielded patterns similar to those of extracted bulk chlorophyll, with increases of up to 10-fold in iron-enriched bottles but at most 3-fold in control bottles ; pennate diatoms accounted for most of the increase in chlorophyll in iron-enriched bottles. Copyright (C) 1996 Elsevier Science Ltd.