Environmental health: flow cytometric methods to assess our water world.

Flow cytometry/cell sorting in aquatic sciences has been driven in two directions. The frontier directions are on shipboard and shore-based. On the one hand, the rapid analytical technique has been taken on shipboard to provide a real-time assessment of the particles and phytoplankton in water masses. These data also give information on the amount of vertical mixing and advection, and denote fronts between two or more water masses. There is an optical characterization (based on sizes, numbers, and pigment groups) of the individual primary producers, as well as detritus and suspended sediments. An optical-closure question is being addressed: "Does the total optical signal equal the sum of the parts?" Additionally, associations with chemical and physical oceanographic features are readily accomplished. A "census" of thousands of phytoplankton cells is obtained and can be mapped. Scientists are able to identify "who is where?" Such data are critical to understand the optical-feedback loop or the so-called photon-budget-in-the-sea, which in turn controls the rates at which growth processes occur in nature. On the other hand, an in-depth understanding is sought as to how particle size, shape, refractive index, nutritional status (nutrient and/or light limitation), growth dynamics, and cell cycle combine to control the optics (light scatter and fluorescence at the moment, and ideally absorption as well) or the photon-budget-of-the-cell. For this purpose, a shore-based facility associated with a diverse collection of phytoplankton is ideal. The development at Bigelow Laboratory of the Jane J. MacIsaac Facility is to provide services for the oceanographic community. Association and co-location with the Provasoli-Guillard Center for Culture of Marine Phytoplankton is key. Visitors are trained and given access to state-of-the-art instrumentation. Visiting investigators have available "the tropical, temperate, and polar seas" in concentrated form, as marine phytoplankton isolated worldwide and maintained as living clonal cultures. In this way, frontline cell biology questions can be addressed. The relentless exploration of standards and controls appropriate for the aquatic community must be continued. An intercalibration effort is a vital step. It is only with the widespread acceptance of particular reference materials and uniform optical filters among research groups utilizing FCM that comparable data sets describing aquatic particle distributions will be possible. For a global science, this strategy is imperative.

Variance within homogeneous phytoplankton populations, I: Theoretical framework for interpreting histograms.

A framework is presented for interpreting frequency distributions of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. The framework, based on both laboratory experience and theoretical concepts, is illustrated with the use of a simulation model. Asynchronous, synchronous, and phased populations were simulated, with constant and variable growth patterns over the cell cycle. Though simulations produced a wide variety of histogram shapes, including multimodal distributions, the primary difference between asynchronous and synchronous/phased distributions lies in their temporal variation. Histograms that are constant in time indicate asynchronous populations; when populations are not asynchronous, their histogram shapes vary with a periodicity on the same time scale as the cell cycle. A probability density function for the case of asynchronous populations with a constant growth rate is derived. When fitted to simulated histograms this two-parameter density function yields estimates of the two parameters: mean and variance of cell volume (or mass) at age 0.

Variance within homogeneous phytoplankton populations, II: Analysis of clonal cultures.

In part I of this series of articles, a framework was presented for interpreting histograms of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. In this paper, the analytical framework is applied to flow cytometric histograms from laboratory experiments involving clonal phytoplankton cultures. The density function derived in part I was modified to include a third parameter representing a linear shift of the origin. This modified density function was fitted to chlorophyll fluorescence histograms for populations believed to be asynchronous (grown in continuous light) and also to histograms from populations grown on a 14:10 (h:h) light/dark cycle. Near-synchronous subpopulations sorted from an asynchronous population were also analyzed. In populations in which underlying assumptions (asynchronous divisions, constant growth) are valid, curve fits provide estimates of the inherent variability among cells at age 0. The implication of fitting the density function to populations in which these assumptions are not valid is discussed.

Variance within homogeneous phytoplankton populations, III: Analysis of natural populations.

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.

Rapid analytical technique for the assessment of cell metabolic activity in marine microalgae.

A standard method for the assessment of cell viability has been developed for marine phytoplankton using an inexpensive stain, fluorescein diacetate (FDA), at .75 microM for 10 min. A flow cytometer was used as the fluorescence detector, providing an assessment of viability for each individual particle. Cell size and chlorophyll fluorescence per cell were assessed simultaneously, permitting an assignment of viability to specific subpopulations, thus increasing the power of the technique. A reasonable correspondence between FDA mean fluorescence intensity per cell and an independent metabolic indicator, photosynthetic capacity measured by 14C, was found. Both FDA mean fluorescence intensity and photosynthetic capacity vary as a function of cell volume. Recovery after extended periods of darkness indicate that cells that are FDA negative may not be dead, but merely quiescent or inactive.

Phytoplankton optical properties: flow cytometric examinations of dilution-induced effects.

Flow cytometry is used to measure dilution-induced changes in the optical properties of Dunaliella tertiolecta-light scatter, cell fluorescence, and refractive index. Observed changes in cell optical properties are compared to simultaneous measures of cell volume and count, extracted chlorophyll a concentration, and the (14)C uptake rate. Flow cytometric measurements reveal short-term dilution effects (within 1 h of dilution) that, are not evident in other morphological or physiological measurements such as cell volume, extracted chlorophyll a concentration, and (14)C uptake rate. Data are presented which suggest that these short-term changes in cell optical properties are the result of changes in the real component of refractive index, possibly due to a rapid and temporary rearrangement of the internal cellular structure. Long-term changes are observed in time series measurements of cell volume and count, extracted chlorophyll a concentration, and (14)C uptake rate.

Effects of flow cytometric analysis on morphology and viability of fragile phytoplankton.

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.

Observations on the intra- and interspecific single cell optical variability of marine phytoplankton.

Phytoplankton occur as a wide variety of sizes, shapes, textures, and conformational inclusions. Optical reseachers, however, to date represent phytoplankton in models as uniform or varying by only a small amount. We give evidence here of changes in the distribution of light scatter for single cells of individual species. This observation is noted even within one species. Optical characteristics show ranges of intraspecific variability (as functions of physiological or nutritional state) comparable in magnitude to interspecific variability. Such variability may be attributed to changes in the cellular refractive-index distribution and the cell size and shape.

Effects of flow cytometric analysis and cell sorting on photosynthetic carbon uptake by phytoplankton in cultures and from natural populations.

Photosynthetic rates of phytoplankton were significantly lower after analysis by flow cytometry (FCM) than before. Exposure to the laser beam during the sorting process caused significant physiological damage. The cellular content of a radiolabel accumulated prior to FCM was not affected by FCM. Although it may not be possible to use FCM to preconcentrate cells for further physiological studies, samples may be incubated with stable or radioactive isotopes and then analyzed by FCM.

Seasonal persistence of resting cyst toxicity in the dinoflagellate Gonyaulax tamarensis var. excavata.

Resting cysts collected in the autumn from bottom sediments were stored in the laboratory, in the dark at 4 degrees C, through the winter. Over a six month period, toxicity of the stored resting cysts was compared with toxicity of cysts from freshly-collected sediments. Resting cysts retained toxin over the winter months. Toxin levels show an apparent rise. Toxicity measurements of cysts from sediment samples kept in the laboratory reflect in situ levels.

Toxicity in Resting Cysts of the Red-Tide Dinoflagellate Gonyaulax excavata from Deeper Water Coastal Sediments.

For the first time, Gonyaulax excavata cysts have been shown to be toxic. Bottom sediments from a water depth of 90 meters off the Maine coast were extremely rich in cysts, which were approximately ten times more toxic than the corresponding motile stages. Cysts are probably ingested by shellfish, thereby causing shellfish toxicity in deeper waters offshore and contributing to shellfish toxicity in shallower coastal waters. A new approach to the problem of paralytic shellfish poisoning is therefore needed, one that takes into account benthic cysts and sedimentary factors affecting their distribution. The possible dangers of spreading poisoning through human activities must be considered.

Lack of secondary intoxification by red tide poison in the American lobster Homarus americanus.

Lobsters are able to feed on shellfish which are toxic with PSP (paralytic shellfish poisoning from Gonyaulax tamarensis) with no apparent harm to themselves, and no measurable assimilation of the poison into their tissues. Lobsters consumed food containing in excess of 1000 mug PSP. There was no impairment of respiration (oxygen consumption) measurable two to three hours after feeding, and no PSP measured in the meat of the claws and tail 48 to 120 hours after feeding. The only PSP was in the guts and contents which were measured 48 hours after feeding began.