Flow cytometry advanced during the decade of the 1980s. Portability, as well as technological advances in the instruments, now allows the simultaneous study of chemical and biological parameters within intact cells both at sea and in the laboratory. Here we report on photoadaptation, fluorescence quenching, metabolic activity, and photosynthetic products (lipids). Endocytosis can also be monitored over time. Flow cytometry enables observation of many and various parameters of individual particles that cannot be obtained from bulk measurements alone.

Light scattering properties of biogenic CaCO3 particles [particulate inorganic carbon (PIC)] were determined on cultured calcifying algae and field‐derived CaCO3 particles. The particles were separated from particulate organic carbon (POC) with a flow cytometer, volume‐scattering functions were measured with a laser light‐scattering photometer, and particle composition was measured using atomic absorption spectrometry. Small calcite coccoliths were best sorted by gating on the ratio of horizontally polarized forward light scattering and vertically polarized forward light scattering; plated coccolithophores could be sorted by gating on side scattering and forward angle light scattering. Normalized volume‐scattering functions for the culture‐derived calcite particles varied by a factor of 2 for the different species. Backscattering cross sections (m2 particle−1) for calcite particles varied by ∼35 times and were generally a function of size. Backscattering efficiencies were ∼2–4 times higher for cells with CaCO3 than without it. CaCO3‐specific backscattering showed much less variability across various species; the calcite‐specific backscattering coefficient varied by only ∼38% for both cultured coccolithophores and field‐derived CaCO3 particles. Organic carbon‐specific backscattering of “naked” coccolithophores was highly consistent within all coccolithophores used in our experiments, as well as with values in the literature. Our results suggest that both POC and PIC can be optically estimated, the former by measuring backscattering of decalcified phytoplankton as well as their size distribution, and the latter is proportional to acid‐labile backscattering. These results show the feasibility of a rapid optical technique for measuring two biogeochemically important carbon fractions in the sea.

Benthic particle feeders are exposed to a food supply varying in both quantity and quality. Previous studies have shown that bivalve molluscs deal with such fluctuating particle regimes in a variety of ways, including adjustments in pumping and ingestion rates, and selective rejection of non-nutritive particles as pseudofeces. The actual site of particle selection within the pallial cavity, however, has remained a topic of speculation. During August 1995 and January and August 1996, we exposed the oysters Crassostrea virginica (Gmelin) and C. gigas (Thunberg), and the mussel Mytilus trossulus Gould to a mixture of ground, aged Spartina alterniflora Loisel and similar-sized phytoplankton at three concentrations (103, 104, 105 particles ml−1). We then examined the ctenidia and labial palps by means of endoscopy and sampled, in vivo, the particulate material from various ciliated tracts, and analyzed the samples with a flow cytometer. We found that in oysters, the ctenidia are responsible for particle sorting, whereas the labial palps play an accessory role in particle selection, or function to control the volume of material to be ingested. In mussels, however, the ctenidia play little role in particle selection and simply transport particulate matter to the palps for further processing. We suggest that selection by the ctenidia of oysters is a function of their architecture (plicate, heterorhabdic).

Bivalve molluscs form dense populations that exert profound effects on the particle loads and phytoplankton composition of coastal waters 1 . It has long been known that bivalves can select among different particle types, including selecting against those of poor nutritional value 2 , 3 , 4 , 5 , but because of difficulties in observing particle transport processes in the pallial cavity in vivo , the mechanism of selection was not known. We now use a combination of video endoscopy 6 and flow cytometry 7 to show that oysters can select living particles from non-living detritus on the gills. Our methods could aid the study of suspension feeding in many animal groups.

Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.

ABSTRACTLaboratory experiments were performed with the prymnesiophyte Emiliania huxleyi (Lohm.) Hay and Mohler, strain 88E, to quantify calcification per cell, coccolith detachment, and effects of coccolith production on optical scattering of individual cells. 14C incorporation into attached and detached coccoliths was measured using a bulk filtration technique. 14C‐labeled cells also were sorted using a flow cytometer and analyzed for carbon incorporation into attached coccoliths. The difference between the bulk and flow cytometer analyses provided a 14C‐based estimate of the rate of production of detached coccoliths. Coccolith production and detachment were separated in time in batch cultures, with most detachment happening well after calcification had stopped. Accumulation of coccoliths was maximum at the end of logarithmic growth with 50–80 coccoliths per cell (three to five complete layers of coccoliths around the cells). Net accretion rates of coccoliths were on the order of 7 coccoliths· cell−1·d−1 while net detachment rates were as high as 15 coccoliths· cell−1·d−1 for stationary phase cells. Equal numbers of coccoliths were attached and detached early in logarithmic growth, and as cells aged, the numbers of detached coccoliths exceeded the attached ones by a factor of 6. Our results demonstrate pronounced charges of forward angle light scatter and 90° light scatter of cells as they grow logarithmically and enter stationary phase. Counts of loose coccoliths in batch cultures are consistent with the detachment of coccoliths in layers rather than individual coccoliths.

The clearance rate of natural planktonic assemblages was measured for the blue mussel Mytilus edulis (L.) and a co-occurring fouling community from mussel rope cultures using flow cytometry. Blue mussels had significantly higher clearance rates for all particle types and size classes. In addition, blue mussels showed selective feeding in favor of small phytoplankton (3–5 μm), whereas the solitary ascidian Ciona intestinalis (L.) and the suspension-feeding gastropod Crepidula fornicata (L.) showed preferential selection for large phytoplankton (> 16 μm). Clearance rates for large phytoplankton by these members of the fouling community were, however, always lower than blue mussels. Under conditions where food is not a limiting factor, interspecific competition for food by the associated fouling community should not significantly limit the yield of mussels.

Particles from both natural populations and culture mixtures are categorized into microautotroph and detritus/microheterotroph components based on size and chlorophyll autofluorescence. Subcomponents ofmicroautotroph pigment groups are possible by the addition of phycoerythrin autofluorescence measurements. Subcomponents of microautotrophs as "live" and "dead" cells are determined by the addition of the metabolic activity stain fluorescein diacetate (FDA). The detritus/microheterotroph fraction includes green-fluorescing cells, abundant in the Gulf of California, detritus, and bacteria. The relative contributions of icroautotrophs, microheterotrophs and detritus mustbe discerned in order to fully interpret beam attenuation and changes in beam attenuation.

Flow cytometry and sorting are now an important technology in aquatic research. Simultaneous measurements of individual particle cell size, fluorescence, and light scatter properties are directly applicable to current topics in aquatic research. Flow sorting may be employed to obtain subsets of cells for analysis by conventional methods. The manner in which rapid, precise measurements of single cells are made is complex, and the application of this technology to aquatic samples is subject to many analytical constraints. Flow cytometric measurements of algal cell size and pigment autofluorescence are relative and are therefore dependent on the optical configuration and variability of the instrument. Specific types of reference materials are used to establish the validity of analyses: 1) instrument standards, 2) fluorescence controls, and 3) internal stain standards. The selection and application of standards and controls are discussed in the context of allometric (cell size versus pigment fluorescence) and ataxonomic (pigment color groups) methods. The widespread acceptance of particular reference materials among research groups will result in comparable data sets describing aquatic particle distributions.

Phytoplankton can, through their autofluorescent characteristics, be thought of as tracer particles in much the same way as fluorescent microspheres when used in particle uptake experiments. Flow cytometric techniques can be used to differentiate phytoplankton from other suspended particles by the two primary autofluorescing photosynthetic pigments, chlorophyll and phycoerythrin. Based on these characteristics, phytoplankton assemblages have been used to assess grazing rates, particle selectivity, and endocytotic abilities in various marine species, from single-celled organisms to higher invertebrates.

Flow cytometry was found to be useful for describing the distribution of adsorption sites for yellow substance on mineral particles. Fewer than 0.1% of the particles measured in clay samples adsorbed enough fluorescing sorbate at 80 mg liter−1 to be detected with 150 mW of JV excitation energy. All of the particles would have been observed if the coverage had been uniform. Gravimetric measurements made on subsamples showed that clay components adsorb up to 2% of their weight of carbon as organic compounds and could provide a significant sink for the fluorescent products of decomposed vegetation.

A theoretical framework for interpreting flow cytometric histograms from homogeneous phytoplankton populations was developed in part I of this series of articles and applied to chlorophyll fluorescence histograms from clonal cultures in part II. In this paper, we demonstrate the application of this framework to the analysis of cell volume distributions found in a natural assemblage of phytoplankton from the Gulf of California. Flow cytometric analyses of a surface water sample incubated for a period of 61 h revealed the sequential growth and decline of three distinct subpopulations. Cell volume distributions for each subpopulation measured at different times were analyzed, and the theoretical density function described in parts I and II was fitted to these distributions. The range of cell volumes within each subpopulation was similar to that predicted for asynchronous populations.

Centrifugal elutriation is a technique for separating particles on the basis of their sedimentation velocity, an expression of size, shape, and specific gravity. Unialgal cultures, mixtures of two phytoplankton cultures, and natural seawater samples were elutriated to determine the feasibility of this technique for collecting fractions of different cell cycle phases, separating two phytoplankton species, and concentrating cells from dilute samples. Elutriation resulted in the separation of a culture of Dunaliella tertiolecta and Phaeodactylum tricornutum into homogeneous fractions of each species. Cells in the natural seawater sample were concentrated by nearly 2 orders of magnitude. Centrifugal elutriation provides an alternative cell separation and concentration technique when large numbers of cells are required.

Bulk fluorescence measurements have been popular in algal culture studies and in oceanographic and limnological applications. Usually, fluorescence is interpreted as an indicator of chlorophyll concentration or phytoplankton biomass, but sometimes measurements of fluorescence can be related to physiological properties of phytoplankton, such as responses to light. Now that in situ fluorometers are being deployed routinely with optical packages, there is active interest in interpreting the relationships between fluorescence, beam transmission, diffuse attenuation, and the physiological characteristics of phytoplankton. Flow cytometry offers the potential to extend these interpretations to the scale of individual cells. It may be difficult to compare measurements of fluorescence, however, because instruments differ greatly in excitation irradiance and time scale of measurement. With this in mind, we examined the short-term responses of a marine diatom to bright light, comparing different instruments (SeaTech in situ fluorometer, Turner Designs fluorometer, EPICS flow cytometer, FACS Analyzer, SeaTech beam transmissometer) while making concurrent measurements of photosynthesis vs irradiance and absorption spectra. Each fluorometer yielded somewhat different information, yet all showed a similar pattern of inhibition after exposure. One instrument, the in situ pulsed fluorometer, could show rapid changes of fluorescence immediately after large shifts of irradiance. Beam attenuation did not decline with the bright light treatment, nor did the specific absorption of chlorophyll. Photosynthetic efficiency was reduced after exposure to bright light, but the capacity for photosynthesis in high irradiance increased at the same time. These results are preliminary: nonetheless they support some interpretations of fluorescence/beam attenuation ratios, clarify some aspects of photosynthetic response to bright light, and suggest that flow cytometry may be useful for assessing physiological heterogeneity in phytoplankton assemblages.

We assessed damage done to especially delicate marine phytoplankton cells by passage through a Coulter Epics V flow cytometer. The cells did not distort or lyse after exposure to fluidics or to laser light to 1,000 mW. The cells did sustain damage evidenced by temporary growth rate depressions. The four clones tested eventually resumed control growth rates after growth lags to 48 h.