0 1990 Wiley-Liss, Inc

Cytometry 11:875-882 (1990)

Flow Cytometric Measurement of Rates of Particle Uptake From Dilute Suspensions by a Ciliated Protozoan' Daniel P. Lavin', A. G. Fredrickson, and Friedrich Srienc3 Department of Chemical Engineering and Materials Science and Institute for Advanced Studies in Biological Process Technology, University of Minnesota, Minneapolis 55455 and St. Paul 55108, Minnesota Received April 16, 1990; accepted July 27, 1990

Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The particles used are polystyrene microspheres containing a fluorescent dye. Measurements were made directly, that is, by determining the fluorescence intensities from microspheres ingested by cells in samples collected from the experimental feeding apparatus. The fact that fluorescence intensities from individual cells can be grouped into discrete classes based on the numbers of fluorescent particles associated with the cells makes it possible to calibrate the flow cytometer and convert fluorescence measurements into numbers of particles ingested by average

Feeding activity of a filter-feeding organism on a population of particles is usefully reported in terms of the organism's clearance rate. This is the rate of particle ingestion by the organism divided by the concentration of particles in the water processed by it (9). Clearance rate measurements can be carried out in a direct or indirect way. In the indirect procedure, one determines the rate at which particles disappear from the medium in which the feeding organisms are suspended, and this is equated to the total rate of particle uptake by these organisms. In a direct measurement, however, one determines the rate at which particles appear inside individual feeding organisms. Various phenomena that occur in the medium, like multiplication of (living) food particles or loss of particles from it by processes other than ingestion by the feeding population, usually complicate indirect determination of feeding rate; but if such effects do not occur or can be corrected for, indirect measurements should give the same results as direct ones. However, the direct method will give correct results even when such com-

cells. At low particle concentration or high ciliate concentration, ingestion data must be corrected for depletion of particles during the assay, and a method for doing this is described. Experiments at various ciliate concentrations show that ingestion rates are not affected by this concentration. The methods developed should allow measurements of rates of ingestion of particles from concentrated and polydisperse suspensions. For such measurements, nonfluorescent particles together with a fraction of fluorescent tracer particles would be used. Key terms: Tetrahymena pyriformis, fluorescent microspheres, fluorescence calibration, phagocytosis, clearance rates

plications occur. Moreover, and even more importantly, the indirect method can give only a n uptake rate per individual organism that is a n average over the whole population; the direct method is required to determine how individual organism particle uptake rate differs between the different classes, like size classes, into which individual organisms of a suspension feeding population may be grouped. Flow cytometry previously has been applied to make indirect measurements of nutrient uptake. This ap-

'This work was supported by the National Science Foundation, Grant No. NSF/BCS-8619399-02. We acknowledge with thanks the assistance of Pamela Sweeney and Christos Hatzis in the preparation of the manuscript. "Present address: Genetics Institute, 1 Burtt Road, Andover, MA 01810 .'Address reprint requests to F. Srienc, University of Minnesota, BPTI, 240 Gortner, St. Paul, MN 55108.

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proach has been used to study food uptake by zooplankton and the trophic structure of aquatic ecosystems (23, 24). Also, it has been demonstrated with this measuring technology that algal food particles, even when they are similar in size, are processed selectively by the mussel Mytilus edulis or other filter-feeding bivalves (5, 20). Direct measurements of particle uptake by filter-feeding organisms have been made using radioactive labelling techniques (3, 16, 17) a s well as by employing direct microscopic observations (1, 8-10, 13, 15, 18, 19). Direct microscopic observations have the disadvantage that measurements can be made on only a few cells within a reasonable period of time. Confidence limits on estimates of uptake rate per cell are therefore likely to be far apart. In addition, the laborintensive nature of this technique prevents carrying out the number of experiments necessary to evaluate in detail single-cell uptake rates under all meaningful conditions. Use of electronic image analysis equipment could improve the microscopic method greatly, but to our knowledge such use has not yet been made. The present work is based on a direct measurement technique employing flow cytometry, which uses fluorescence from labelled particles to determine the number of such particles that have been ingested by individual feeding organisms. This technique has already been used to make direct measurements of the uptake of fluorescent microspheres by mammalian phagocytes (2, 11, 22). In addition, it has been used to measure particle uptake by ciliates also (12, 21). In the present work, it is applied to feeding of the small ciliate Tetrahymena on model bacterial particles suspended in water. Flow cytometry circumvents most of the disadvantages of microscopic and radioactive tracer direct measurements a s well a s those of indirect measurements. The rate of data acquisition is orders of magnitude faster than that of visual microscopic observation, thus permitting tens of thousands of organisms to be analyzed in a matter of minutes. Moreover, several characteristics of individual organisms, like light scattering from the cell and fluorescence from its DNA or protein, provided that these have been stained with appropriate dyes, can be measured simultaneously with fluorescence from ingested food particles. Thus, many subpopulations or classes of organisms characterized by a defined set of single organism parameters can be examined for class-to-class variation of singlecell ingestion rate. This paper describes use of flow cytometry to make direct, short-term measurements of particle uptake rates by ciliate cells. Because the measurement is direct, a n internal calibration procedure can be used to transform fluorescence data into average rates of particle uptake. A procedure is described that permits correction of the data for depletion of particles during the assay. The accurate flow cytometry assay has been used to test a previous report (6) that clearance rates are affected by ciliate concentrations where the latter are above 1,000 m1-l.

METHODS Organisms and Growth Media The organism used was Tetrahymena pyriformis GL, which will be called simply Tetrahymena pyriformis (14). This organism was originally obtained from Dr. M.A. Gorovsky, Department of Biology, University of Rochester, Rochester, New York. The ciliates were maintained a t 25°C in tube cultures containing PPYE medium (20 g 1-1 proteose peptone, 1g1-l yeast extract adjusted to pH 7.0 with 10 M NaOH). The liquid medium in which T. pyriformis cells for particle uptake experiments were grown was the foregoing PPYE medium supplemented with 5 g1-l of glucose (PPYEG medium). Before its use, medium was filtered twice through Whatman number 2 filter paper and sterilized by autoclaving. Glucose was sterilized separately. Determination of Ciliate Population Density and Size Distribution The population densities and size distributions of T . pyriformis cultures were measured with a n electronic particle counter and size distribution analyzer (model 8OXY Electrozone-Celloscope, Particle Data, Inc., Elmhurst, Illinois). A 95 pm aperture was used along with a sample volume of 501.9 p1. Preparation of Ciliate Inocula We found that clearance rates of different batches of T. pyriformis inocula varied considerably, depending on methods used to prepare the inocula. Therefore, a standard stepwise procedure for preparing inocula was developed and used in all of the work reported. The initial step of the procedure was to preculture ciliates in 180 x 25 mm culture tubes maintained a t 25°C using PPYEG medium. Cells from a 1-day-old culture tube were inoculated into 300 ml Erlenmeyer flasks containing 30 ml of fresh medium, and these were shaken at 200 cyclesimin on a n incubator shaker (LabLine Corp. Chicago, Illinois) maintained a t 25°C. After several days of growing in this well-aerated environment with daily transfers to fresh medium, the cells were inoculated into 2 liter Erlenmeyer flasks containing 120 ml of fresh medium. The timing of the transfer from a 300 ml flask to a 2 liter flask was based on the mean cell volume of the culture that decreased over the period of extended cultivation in exponential growth. The cells used in the particle uptake experiments were taken from these 2 liter flasks after 18 to 24 h of exponential growth. The specific growth rate during this exponential growth phase was approximately 0.14 h-l. The population densities a t harvest were typically about 30,000 m1-l. The population density and mean cell volume of a culture were monitored with the Electrozone-Celloscope a t least once daily from the time the culture was started in a 300 ml Erlenmeyer flask. Fluorescent Microspheres Carboxylated polystyrene microspheres containing a yellow-green fluorescent dye with excitation maxi-

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GREEN FLUORESCENCE CHANNEL FIG.1. Histogram of fluorescence intensities produced by flow cytometric analysis of a sample that had fed on a dilute, monodisperse suspension of 4.05 pm microspheres for 180 s. Each peak represents cells that have ingested a defined number of fluorescent microspheres.

mum a t 458 nm and emission maximum a t 540 nm (fluoresbrite, Polysciences, Inc., Warrington, Pennsylvania) were used to simulate nutrient particles in the feeding studies. For each size bead used, the standard deviation of the bead diameter was a t most 5% of the mean. The concentration of the fluorescent microspheres in a suspension was microscopically determined using a Neubauer hemacytometer counting chamber (Cambridge Instruments, Inc., Buffalo, New York). Microspheres were always microscopically examined for agglomeration prior to use in uptake experiments. In those cases where agglomeration occurred, the microspheres were gently sonicated for 10 to 25 min using a Branson 1200 ultrasonic bath (Branson Cleaning Equipment Company, Shelton, Connecticut) to break up the clumps.

Particle Uptake Experiment The uptake experiments were carried out at 25°C in 250 ml beakers stirred with a 1.5 inch magnetic stir bar a t about 135 rpm. The appropriate volume of microspheres was mixed into about 10 ml of growth medium in the 250 ml beaker. Approximately 25 ml of cell suspension (the exact volume depended on the protozoan population density) from a 2-liter flask of exponential phase cells was added to the suspension at the start of the assay. In all cases the protozoan concentration in the suspension in the beaker during a n uptake experiment was 20,000 1,500 ml-' except for the experiments in which the effect of protozoan concentration on uptake was studied. Samples of 3 to 5 ml were withdrawn and quickly added to formalin to a final formaldehyde concentration of 1%(v/v). After fixation for 38-48 h at 4"C, the cells were washed three times with a saline-phosphate solution (5 gl-' NaC1, 0.4 gl-' KC1, 0.4 81-1 Na,HPO,, 0.15 gl- KH,PO,, pH = 7.0) to separate microspheres in suspension from the protozoa. In the washing steps, the protozoa were allowed to

*

settle by gravity for 6-8 h, and the supernatant was then carefully drawn off from each tube. In this procedure, the majority of microspheres remained in the supernatant.

Flow Cytometry The flow cytometer used (Cytofluorograf Model IIs, Ortho Instruments, Westwood, Massachusetts) was equipped with the standard nonsorting analytical flow cell and a Coherent Innova 90-5 argon-ion laser (Palo Alto, California) tuned to a wavelength of 488 nm and operated a t a light-stabilized beam power of 0.1 W. Three properties of individual cells were measured and stored in list mode: fluorescence intensity of ingested and attached microspheres, forward-angle light-scattering intensity, and right-angle light-scattering intensity. The 515-530 nm narrow band-pass filter (part number 300-0281-002) was used to measure the fluorescence intensity. For measurements of the forwardangle light-scattering intensity of the protozoan cells, a neutral density filter with a transmittance of 0.10 %, was used. Samples were analyzed a t a rate of 100-500 cells s-'. For data analysis, the signals were gated from a forward-angle, right-angle light-scattering cytogram that allowed discrimination of the ciliate cell population. Mean values of the distributions were evaluated with the built-in software routines of the data acquisition unit (Model 2151, Ortho Instruments, Westwood, MA). RESULTS Calibration of the Flow Cytometer A typical result from the flow cytometric analysis of a sample is shown in Figure 1. The histogram, which was obtained from a sample of cells that had fed on 4.05 pm microspheres for 180 s, shows the distribution of channel numbers into which the fluorescence intensi-

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ties from single cells fell. The peaks shown in the histogram represent cells that contain 1, 2, 3, and 4 and more microspheres. A peak representing cells with no ingested or attached beads is present in channel 1also. However, a s many cells are compacted into one or perhaps two channels, this peak is too high to be shown without making a n unacceptable expansion of the vertical scale of the graph. Such peaks were obtained in all experiments when fluorescent microspheres of diameter 1.7 pm and larger were used. These peaks, which correspond to a definite number of particles per cell, provide a convenient means of calibrating the flow cytometer and converting the measured quantity of average fluorescence channel number of a sample of cells into the desired quantity of number of particles associated with the average cell in the sample. To determine the average fluorescence channel number corresponding to a specific microsphere number, the fluorescence histogram (Fig. 1) was divided into regions that were assumed to represent cells that had 0, 1, 2, or 3 beads associated with them. The divisions were drawn a t the minima between the peaks of the histogram. The numbers of cells in each region a s well as the mean fluorescence channel numbers of each region were then calculated. No region contained fewer than 300 cells. The numbers of cells in the regions are estimates of the numbers of cells in the sample that have associated with them 0, 1,2, and 3 beads, and the mean fluorescence channel numbers of the regions are estimates of the channel numbers into which fall the fluorescence from average cells that have associated with them 0, 1, 2, and 3 beads. Evidently, these estimates are subject to some errors, as a few of the cells assigned to a given subpopulation really belong to the adjacent subpopulation and vice versa. However, when one does not attempt to apply the procedure to subpopulations of cells that have ingested more than three beads (in the case of the figure noted), the numbers of cells misassigned by the procedure will be small compared with the numbers correctly assigned; moreover, errors in assigning cells to subpopulations made a t its two boundary lines will tend to have opposite signs, thus partially cancelling one another. A more objective assignment of the cells into particular classes could be done based on statistical techniques (7). A plot of these averages vs. number of beads associated was prepared for each bead size; Figure 2 is the plot for feeding on 2.74 pm beads. This figure shows that the difference, S,, between the fluorescence channel numbers of average cells that have associated with them i and i + 1 beads is independent of i, for i 2 1. Therefore vi, the fluorescence channel number of the average cell that has i beads associated with it (i = 1, 2, 3, 4), is given by v L = (i6, + p), where p is the y-intercept of the plot shown in Figure 2. In general, the difference between the fluorescence channel numbers of average cells that have associated with them 0 beads and 1bead is not equal to 6,, and so the intercept p is not the same as v,,, the fluorescence channel num-

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1 2 3 4 NUMBER OF MICROSPHERES PER CELL

FIG. 2. Channel numbers of fluorescences from average cells that have ingested 0 , 1 , 2 , 3 beads ofdiameter 2.74 +m vs. number ofbeads ingested.

ber of the average cell that has 0 beads associated with it. Therefore, we have to write vo,

it?,

+ i3,

i i

=

=

0 1,2,3,. , .

Let f , (i = 0, 1 , 2 , . . .) be the fraction of cells in a sample that have i beads associated with them. For i no greater than two or three these fractions are readily obtained from the counts of the cells in the various regions of the histogram and the total number of cells in the sample. The sum of the f, over all values of i is one. The number of particles associated with the average cell in the sample is then defined by 7 .

i=

CiL r=n

(2)

Similarly, the fluorescence channel number of the fluorescence from the average cell in the sample is given by t

=

C"&

(3)

r-O

Substitution of equation (1)into equation ( 3 )and use of equation (2) then gives the relation between the channel number of the fluorescence from the average cell and the number of beads associated with it as t

=

i6,

+ p - (p - lJ",f,.

(4)

This is the equation used to convert the fluorescence data for a sample into the (population-average) number of particles associated with the average cell. To use it, one must know the fraction of cells in the sample that have no beads associated with them as well as the three calibration constants, S,, p, and uo. In analyzing the data from a sequence of samples taken during a n experiment, fo will be 1 at the beginning of the experiment and will decrease toward a small but nonzero asymptotic value as the experiment proceeds. Thus, the contribution of (6 - vo)fo to the fluorescence of the average cell decreases a s the experiment proceeds, whereas the contribution of (i 6, + p)

RATES O F PARTICLE UPTAKE CY 7'. p y n f o w ~ 7 a

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FIG.3. Test of the linearity of the fluorescence calibration when extrapolated to large numbers of particles ingested. The particle diameter was 1.67 pm. See text for explanation.

increases. It turns out that the calibration constants and feeding rates are such that the first contribution above can be ignored except when feeding is on very dilute suspensions. Figure 2 shows that equation (1)is valid up to i = 4, but of course one would like to extrapolate the calibration equation based on i t to values of i considerably larger than 4. Hence, a number of experiments were done to see if such extrapolation is valid. In these experiments, the organisms were allowed to feed on suspensions of various concentrations in order to vary the number of beads ingested by the average cell over a wide range. Samples of cells were analyzed flow cytometrically to determine the fluorescence channel number of the average cell. In addition, the samples were analyzed microscopically, and estimates of the numbers of beads associated with average cells were made from counts made on individual cells in the sample. Figure 3 shows results obtained when feeding was on beads of 1.67 Fm diameter. In these experiments, the heads associated with 30-40 cells from each sample were counted, and the means and confidence limits of the numbers of beads associated with the average cells in the various samples were calculated. The results show that the correlation remains linear up to a n average of 150 beads associated with each cell.

Corrections for Attached but not Ingested Beads The triple washing of fixed cells removed most of the attached but not ingested beads from the cells, but complete removal from every cell could not be attained. Therefore, it was necessary to correct for the presence of such beads. To do this, control samples were prepared by adding cells which had not fed on the mi-

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crospheres to the fixing solutions. These solutions contained beads a t the same concentrations as corresponding samples from the feeding experiment. Concentrations of cells and formalin were adjusted so that they were the same in the control samples a s they were in the noncontrol samples. After fixation and washing, the control samples were analyzed by flow cytometry and &, the number of beads attached to the average cell of the sample, was determined using the calibration described above (equation 4).In addition, fo,, the fraction of cells with no attached beads, was determined for each control sample. Largest values of & were observed a t the highest bead concentrations used and were about 0.26 per cell. Smallest values of fOc were observed under the same conditions and were about 0.85. The relation between i', the number of beads ingested by the average cell, i, the number of beads ingested by or attached to the average cell, and &, is given by l'=i-&

(5)

This equation was used to calculate i'. The relation between f o ' , the fraction of cells in a sample with no ingested beads, f o , the fraction of cells in a sample with no ingested or attached beads, and foc is f,, = t x o c

(6)

and this equation was used to calculate f;,'.

Direct Measurements of Particle Uptake by Flow Cytometry Typical data obtained from three series of such feeding experiments are shown in Figure 4.Here the population-average number of particles ingested per cell, i.e., the total number of ingested microspheres divided by the corresponding cell number, is plotted as a function of feeding time for series of experiments in which cells were offered five different concentrations of 2.74 km beads. Concentrations of microspheres ranged from 1 x 105m1-l to 1 x lo6 m1-l. All cell suspensions used in these experiments had a mean cell volume of 6,185 pm3 according to the electronic volume measurement. Several conclusions based on these data can be made. First, the linear increase with time of the populationaverage ingestion per cell indicates that the physiological state of the ciliates does not change enough to affect the feeding rate during the duration of the experiment. Second, the linear relationship, found even a t low microsphere concentrations, indicates t h a t depletion of microspheres is not significant enough to cause feeding rates to decrease during the experiment unless i t is quite long. Third, the linearity of the relation shows that microspheres ingested in the initial stages of the experiment are not defecated toward the end of the experiment. Fourth, the facts that the lines for each series of experiments are straight and pass through the origin show that feeding activity exhibits no lag period a t any of the bead concentrations used. Fifth, the fact t h a t the lines are straight and pass through the origin show that the method of adjusting the data for the presence of attached but not ingested

L A W N ET AL.

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FIG.4. Time courses of increase of population-average numbers of 2.74 IJ-mmicrospheres ingested per cell for feeding of T . pyrzformis on suspensions of concentrations (in ml - I ) : 1 x 10" (o), 3.2 x lo5 (@), and 1.0 x 10" (A).

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particles neither overcorrects nor undercorrects by significant amounts. Finally, the reproducibility of the experimental conditions during sampling seems to be excellent, as individual data points fall very closely on five different straight lines. Correction for depletion of microspheres during a feeding experiment. The linearity of the graphs of population-average number of particles ingested per cell in Figure 4 shows that depletion of the microspheres was negligible during the course of the experiments. However, ciliate concentrations in these experiments were all approximately 20,000 ml-', and a t higher ciliate concentrations (or lower particle concentrations or longer feeding times), depletion might well become a factor that cannot be neglected. Figure 5a shows plots of population-average number of microspheres ingested per cell vs. time for two experiments in which ciliates a t a concentration of 19,500 ml-' fed on suspensions of 2.74 pm fluorescent microspheres having a concentration of 5 x lo5 ml-' and 3 x lo4 m1-l. In contrast to the previous data, one can clearly see that the plots are nonlinear. The most likely explanation of the nonlinearity is that it is the result of depletion of the microsphere population. The simplest way to make corrections for depletion is to evaluate the population-average ingestion rate a t the beginning of the experiment, when the microsphere concentration has its initial, known value. One simply finds the limiting slope of the plot of population-average number of particles ingested vs. time as time approaches zero; this slope is the population-average ingestion rate a t the initial microsphere concentration. A slight difficulty with this procedure is that it is not clear how objective estimates of the uncertainty in the ingestion rate can be made. Hence a n alternate procedure, which avoids this difficulty, is needed and is as follows. The rate of decrease of the concentration of particles in suspension is equal to the average rate of ingestion of a cell times the cell concentration. From the defini-

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FIG. 5. a: Time courses of population-average number of microspheres ingested per cell from experiments in which microsphere size (2.74 km) and ciliate concentration (1.95 x lo4 ml-9 were the same, but initial concentration of the microspheres (in m1-l) was 1.0 x lo5 (@), and 3.0 x lo4 (0). b: Replot of the data of (a) based on equation ( 8 )of the text. For data points taken at a given ciliate concentration, the slope of the best straight line passing through the origin and the points is the population-average clearance rate at the ciliate concentration used. Symbols used are as in (a).

tion of clearance rate, the rate in the decrease of particle concentration in a homogeneous suspension is therefore given by db dt

-

C'bp,

(7)

where b and p are the microsphere and protozoan concentrations, respectively, C' is the population-average clearance rate of a cell, and t is time. When the microsphere suspension is dilute, C' is independent of b, by definition. In addition, in a short-term experiment, p does not change by a significant amount. Therefore, C' and p may be treated as constants for short-term feeding experiments on dilute suspensions, and integration of equation (7) with C' and p constant leads to b

=

b,e

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(8)

where b, is the initial microsphere concentration. Let i' be the population-average number of microspheres ingested a t some arbitrary stage of the experiment, when the concentration of the microspheres is 6. Then clearly (9)

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RATES OF PARTICLE UPTAKE BY 2'. pqnforncis

Elimination of b between equations (8) and (9) leads to the result

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(10)

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and this equation is useful for graphical evaluation of the population-average clearance rate per cell from the flow cytometric data: A plot of the quantity In [b,,/(b,, -E'p)llp vs. t for the data taken a t a given value of p should result in a straight line whose slope is C'. An estimate of C' and confidence limits for it can be found by straightfoward curve-fitting techniques. Figure 5b shows the data of Figure 5a replotted in the manner suggested. It can be seen that this procedure straightens out the plot of the data for both microsphere concentrations used. Moreover, both experiments yield virtually the same line. One should note that equation 10 takes advantage of the direct measuring approach, because the population average number of microspheres ingested is a direct result from the flow cytometry measurement, and the actual particle concentration in suspension does not have to be determined during the experiment. Equation 8 can be used for estimating feeding rates from data obtained from indirect measurements that require determination of the actual particle concentration in suspension during the experiment, a s suggested by Coughlan (4). Effects of ciliate cell concentration on population-average clearance rate. The effect of ciliate concentration on the population-average clearance rate was determined in experiments in which the concentration of ciliates feeding on 1.74 km microspheres varied from 2,500 ml-' to 47,000 m1-l. In the first experiment, the protozoa were cultured until the population density reached 50,000 ml-'; then they were diluted to produce concentrations of 2,470, 6,560, 18,900, and 44,800 ml-', and each dilution was offered a suspension with a concentration of 5 x lo6 microspheres m1-l. This experimental protocol was repeated with a different culture of cells. In this experiment the ciliate concentrations were 2,460, 6,510, 19,000, and 47,200 ml-'. The mean cell volume for the cell population in the first experiment was 6,840 km3; whereas i t was 6,913 pm3 in the second experiment. Estimates of population-average clearance rates per cell and confidence limits on these estimates were determined at all four cell concentrations for both cultures by the method based on equation (10) and described in the preceding paragraph. The results are plotted in Figure 6. Error bars indicating the 95% confidence level intervals for each of the estimates of the population-average clearance rate per cell suggest that there is no significant influence of cell concentration on clearance rate, except perhaps there is some falling-off of the clearance rate at the highest cell concentration in the case of the culture with mean cell volume of 6,913 km3.

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CELL CONCENTRATION [rnl-']

FIG.6. Variation of population-average clearance rate with ciliate concentration for cultures with mean cell volume 6,840 pmq (0) and 6,913 +ma ( 0 ) The . clearance rates and their error bars were obtained by curve-fitting the data of the type shown in Figure 5b.

DISCUSSION In this work we have used flow cytometry instrumentation to make direct measurements of uptake of fluorescent microspheres from dilute suspensions by T. pyriformis. About 20,000 cells were analyzed in each sample taken during a feeding experiment so that population-average quantities are not much affected by the randomness of the feeding of single cells. Interpretation of the data in terms of population average quantities requires, however, that the exact correlation between fluorescence values and cellular microsphere content is established. Such correlation can then be used to convert mean fluorescence values into average numbers of microspheres per cell. In the presented work, the calibration process can easily be carried out using the measured fluorescent microspheres as a n internal standard, a s cells exhibit discrete levels of fluorescence intensities depending on the number of beads ingested. The data confirm that a linear correlation between fluorescence intensity and number of microspheres is maintained over a wide range. This correlation does not hold, however, for the range of microsphere contents between 0 and 1, presumably because of imprecise zero level adjustments of the analog-todigital converter. One should note that this nonlinearity must be taken into account in the conversion of the population average data, especially as the fraction of cells without beads changes during the kinetic experiment. Failure to do so would introduce a significant error in samples with a large fraction of cells without beads; i.e., the error would most significantly affect initial data of the feeding experiment and data on cell populations with very low ingestion rates. Furthermore, a simple procedure has been presented that permits correction of the data for depletion of beads during the experiment. Such depletion becomes important during conditions of either low microsphere concentration or high ciliate concentration. The developed method permits precise determination of clear-

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ance rates of ciliate populations, and our data show that clearance rates are not influenced by the concentration of ciliates up to 50,000 cells per ml-', which is in contrast to previous reports in the literature (22). This difference is most likely because depletion of nutrient particles during the uptake experiment has not been sufficiently accounted for in the previously reported studies or because other complicating effects have not been considered since live bacterial cells have been used a s nutrient particles. The direct measurement on single cells permits application of the presented internal calibration procedure. The measured clearance rates have been expressed in this work a s population average data, which could be obtained, in principle, by indirect measurements of the ingestion rates also. One should note, however, that the analysis can be extended to subgroups of cells because of the direct measurement technique employed, and for this purpose the developed direct measuring procedure will be very useful. Although the methods described above have been applied only to measure uptake from dilute suspensions of particles, they should be applicable also to measure uptake from concentrated suspensions of particles. For this purpose, only a fraction of the particles would be fluorescently labelled; the fraction of tracer particles would be adjusted so that histograms like that of Figure 1 would still be obtained even for feeding on concentrated suspensions. One would have to make sure, however, that the relevant properties of the tracer particles are the same to be able to exclude any selective uptake of the particles used.

LITERATURE CITED 1. Beirsheim KY: Clearance Rates of bacteria-sized particles by freshwater ciliates measured with monodisperse fluorescent latex beads. Oecologia 63:286-288, 1984. 2. Blair OC, Carbone R, Sartorelli AC: Differentiation of HL-60 promyelocytic leukemia cells: Simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry. Cytometry 7:171-177, 1986. 3. Bogdan KG, Gilbert JJ, Starkweather PL: In situ clearance rates of planktonic rotifers. Hydrobiologia 73:73-77, 1980. 4. Coughlan J: The estimation of filtering rate from the clearance of suspensions. Marine Biol. 2:356-358, 1969. 5. Cucci TL, Shumway SE, Newell RC, Selvin R, Guillard RRL, Yentsch CM: Flow cytometry: A new method for characterization of differential ingestion, digestion, and egestion by suspension feeders Mar Ecol Progr Ser 24:201-204, 1985.

6. Curds CR, Cockburn A: Studies on the growth and feeding of Tetruhymena pyrzformis in axenic and monoxenic culture. J Gen Microbiol 54:343-358, 1968. 7. Everitt BS, Hand DJ: Finite Mixture Distributions. Chapman and Hall, London, New York, 1981. 8. Fenchel T: Suspension Feeding in ciliated protozoa: Feeding rates and their ecological significance. Microbiol Ecol 6:13-25, 1980b. 9. Fenchel T: Suspension feeding in ciliated protozoa: Functional response and particle size selection. Microbiol Ecol6:1-11, 1980a. 10. Fenchel T: Protozoan filter feeding. Prog Protist01 1:65-113, 1986. 11. Fujikawa-Yamamoto K, Yokoe H, Odashima S: Evaluation of the phagocytosis of microspheres in V79 cells by flow cytometry. Cell Struct Funct 12:83-91, 1987. 12. Gerritsen J, Sanders RW, Bradley SW, Porter KG: Individual feeding variability of protozoan and crustacean zooplankton analyzed with flow cytometry. Limnol Oceanogr 32:691-699, 1987. 13. McManus GB, Fuhrman JA: Bacterivory in seawater studied with the use of inert fluorescent particles. Limnol Oceanogr 31: 420-426, 1986. 14. Nanney DL, McCoy JW: Characterization of the species of the Tetruhymeraa pyrzformis complex. Trans Am Microsc SOC 95:664682, 1976. 15. Pace ML, Bailiff MD: Evaluation of a fluorescent microsphere technique for measuring grazing rates of phagotrophic microorganisms. Mar Ecol Progr Ser 40:185-193, 1987. 16. Porter KG, Orcutt Jr JD: Nutritional adequacy, manageability, and toxicity as factors that determine the food quality of green and blue-green algae for daphnia. In: ASLO Special Symposium 111: The Evolution and Ecology of Zooplankton Communities, Kerfoot WC (ed). University Press of New England, Hanover, New Hampshire, 1980. 17. Porter KG, Gerritsen J, Orcutt Jr JD: The Effect of food concentration on the swimming patterns, feeding behavior, ingestion, assimilation, and respiration by Daphnia. Limnol Oceanogr 27: 935-949, 1982. 18. Sherr BF, Sherr EB: High rates of consumption of bacteria by pelagic ciliates. Nature 325:710-711, 1987. 19. Sherr BF, Sherr EB, Fallon RD: Use of monodispersed, Fluorescently labeled bacteria to estimate in situ protozoan bacterivory. Appl Environ Microbiol 53:958-965, 1987. 20. Shumway SE, Cucci TL, Newell RC, Yentsch CM: Particle selection, ingestion, and absorption in filter-feeding bivalves. J Exp Mar Biol Ecol 91:77-92, 1985. 21. Srienc F, Fredrickson AG, Lavin DP: Feeding, growth, and reproduction of ciliate microorganisms. An engineering view. Ann NY Acad Sci 506:357-370, 1987. 22. Steinkamp JA, Wilson JS, Saunders GC, Stewart CC: Phagocytosis: Flow cytometric quantitation with fluorescent microspheres. Science 215: 64-66, 1982. 23. Yentsch CM, Horan PK, Muirhead K, Dortch Q, Haugen E, Lebendre L, Murphy LS, Perry MJ, Phinney DA, Pomponi SA: Flow cytometry and cell sorting: A technique for analysis and sorting of aquatic particles. Limnol Oceanogr 28:1275-1280, 1983. 24. Yentsch CM, Yentsch CS: Emergence of optical instrumentation for measuring biological properties. Oceanogr Mar Biol Annu Rev 2255-98, 1984.

Flow cytometric measurement of rates of particle uptake from dilute suspensions by a ciliated protozoan.

Flow cytometry is used to measure rates of ingestion of particles from dilute monodisperse suspensions by the ciliate Tetrahymena pyriformis. The part...
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