Effects of mismatched refractive indices in aquatic flow cytometry.

BACKGROUND: Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural waters. The refractive index of water samples from aquatic environments can differ because of a variety of factors such as dissolved organic content, aldehyde preservative, sample salinity, and temperature. In flow cytometric analyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We measured the effect of this mismatch on cell size measurements. METHODS: We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity ratio. The effects were characterized for standard microspheres, cultured phytoplankton cells of different sizes, and natural populations from an estuarine river. RESULTS: We found that the distorted scatter signals resulted in an increase in the apparent size of small cells (1--2 microm) by a factor of 4.5 times. Cells in the size range of 3--5 microm were less affected by the salinity differences, and cells larger than 5 microm were not affected. Chlorophyll and phycoerythrin fluorescences and 90 degrees light scatter signals were not changed by sheath and sample salinity differences. CONCLUSIONS: Care must be taken to ensure that the sheath and sample refractive index are matched when using forward light scatter to measure cell size spectra, especially in estuarine studies, where salinity can vary greatly. Of the factors considered that can change the sample refractive index, salinity gradients in an estuary cause the largest index mismatch and, consequently, the largest error in scatter.

Flow cytometric analysis of 5-cyano-2,3-ditolyl tetrazolium chloride activity of marine bacterioplankton in dilution cultures.

The respiratory activity of marine bacteria is an important indication of the ecological functioning of these organisms in marine ecosystems. The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) is reduced intracellularly in respiring cells to an insoluble, fluorescent precipitate. This product is detectable and quantifiable by flow cytometry in individual cells. We describe here an evaluation of flow cytometry for measuring CTC activity in natural assemblages of marine bacteria growing in dilution cultures. We found that more CTC-positive cells are detected by flow cytometry than by visual epifluorescence microscopy. Samples can be stored refrigerated or frozen in liquid nitrogen for at least 4 weeks without a significant loss of total cells, CTC-positive cells, or CTC fluorescence. Cytometry still may not detect all active cells, however, since the dimmest fluorescing cells are not clearly separated from background noise. Reduction of CTC is very fast in most active cells, and the number of active cells reaches 80% of the maximum number within 2 to 10 min. The proportion of active cells is correlated with the growth rate, while the amount of fluorescence per cell varies inversely with the growth rate. The CTC reduction kinetics in assemblages bubbled with nitrogen and in assemblages bubbled with air to vary the oxygen availability were the same, suggesting that CTC can effectively compete with oxygen for reducing power. A nonbubbled control, however, contained more CTC-positive cells, and the amount of fluorescence per cell was greater. Activity may have been reduced by bubble-induced turbulence. Addition of an artificial reducing agent, sodium dithionite, after CTC incubation and fixation resulted in a greater number of positive cells but did not "activate" a majority of the cells. This indicated that some of the negative cells actually transported CTC across their cell membranes but did not reduce it to a detectable level. Automated analysis by flow cytometry allows workers to study single-cell variability in marine bacterioplankton activity and changes in activity on a small temporal or spatial scale.

Phagotrophy of fluorescently labeled bacteria by an oceanic phytoplankter.

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.

Flow cytometry and phytoplankton.

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.

Separation and concentration of phytoplankton populations using centrifugal elutriation.

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.

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.

A flow cytometric approach to assessing the environmental and physiological status of phytoplankton.

Individual particle analysis using a flow cytometer (FCM) was made on natural phytoplankton communities in oligotrophic waters. Our objective was to develop an assay to yield information on the nutrient history of individual cells using FCM. Results from nutrient assays showed that both biovolume and total red fluorescence are affected by the nutrient conditions in the incubator. The light effect was measured by changes in the chlorophyll content of the cells, and after the 12 h incubation the cells seemed well adapted to the light conditions. The estimated kinetic constant for the chlorophyll synthesis averaged 1.5 x 1.0(-2) h-1, whereas the growth rate calculated from the changes in the cell numbers changed from 0.14 to greater than 2.5 doubling per day. The smallest size fraction presented the highest growth rate (greater than 2.5 doublings per day). The relationship between the total red fluorescence as estimated with the FCM and the biovolume revealed that the cells from the 2 m samples at the beginning of the experiment were probably nutrient limited. Important changes in the size of the cells under nutrient limitation were also observed. The FCM data suggest that the FCM is a valuable tool for estimating the relative growth response and nutritional state of natural phytoplankton populations.

Using phytoplankton and flow cytometry to analyze grazing by marine organisms.

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.

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.