Hematology reference values for peripheral blood of laboratory rats

Reference values for peripheral red and white blood cells, platelets and coagulation parameters from 11 and 18-week-old Charles River Sprague Dawley rats [Cr1:COBS CD(SD)] were obtained using automated methods. The Coulter Model S Senior, the Clay Adams Ultra-Flo Platelet Counter and the Sherwood Lancer Coagulyzer, were used to measure these parameters on 25 rats/sex. Detailed information on instrumentation or assay methods, and the animals, their source, environment and blood collection method was given. Platelet counts were in the range of 694 X 10(9)/1 to 1412 X 10(9)/1 which was higher than previously reported. In general, the remaining ranges were similar to previously published reference ranges.     

A new red cell discriminant incorporating volume dispersion for differentiating iron deficiency anemia from thalassemia minor

A novel red cell discriminant function [MCV2 x RDW/(Hgb x 100)] was compared to six other discriminants in 102 patients with established mild iron deficiency anemia and 33 patients with beta-thalassemia minor. The discriminant incorporates the two key measurements of erythrocyte cell volume distribution, namely the mean (MCV) and standard deviation (RDW), which are known to be helpful for distinguishing between these two frequent causes of microcytic hypochromic anemia. Data used for the learning set to develop the new discriminant were obtained using an electrical impedance automated whole blood analyzer (Coulter S + IV) and were applied as a validation set for six other discriminants. The discriminant was also tested on smaller subsets of the patients groups using data obtained on either an alternate electrical impedance instrument (Sysmex E-5000) or a laser light scattering based system (Technicon H*1). From the comparison it was concluded that use of a discriminant function that incorporates a measurement of red cell volume dispersion results in enhanced accuracy for distinguishing iron deficiency anemia from thalassemia minor.     

Flow cytometric enumeration of absolute lymphocyte number in peripheral blood using two parameters of light scatter

A method was developed to measure the absolute lymphocyte count (ALC) of whole blood using the Spectrum III automated flow cytometer. Ninety-nine samples of human peripheral blood were analysed on the Spectrum and the Coulter Counter S Plus II, to allow for comparison of the two machines. Regression analysis was used to test the extent of agreement between the sets of measurements on the two machines. The results demonstrated that the slope of the regression line was not significantly different from one, indicating a high level of correlation between Spectrum and Coulter ALC's. However, the mean difference between Coulter and Spectrum ALC's was not equal to zero, with the Spectrum giving counts approximately 10% lower than those of the Coulter machine. This is attributed to the different ways by which the two machines define a lymphocyte, the Spectrum III by two parameters of light scatter and the Coulter S Plus II by the single parameter of cell volume.     

Flow cytometric determination of basophils in whole blood with n-propyl Astra Blue iodide

Within the past year, it has become apparent, in connection with its use on automatic flow cytometers, that the quality of commercially available Alcian Blue has significantly declined. A homologous series of alkylated (C1-C7) Astra Blue quaternary ammonium halides was prepared, characterized, and evaluated for the detection of basophils in whole blood. On the Technicon H6000 flow cytometer, the resolution of the basophil cluster from the main population of unstained white blood cells was found to depend on the chain length of the quaternizing alkyl group. Optimal basophil resolution was observed for the n-propyl derivative. Correlation of the new method vs Alcian Blue as the reference on the H6000 was expressed as follows: %Baso (Astra Blue) = 0.89% Baso (Alcian Blue) + 0.12% for 180 fresh whole blood samples. Within-run precision at a basophil differential count of 0.73% was characterized by SD = 0.11, identical to that obtained for Alcian Blue. Aqueous solutions of n-propyl Astra Blue iodide, in contrast to Alcian Blue, are thermally stable. Heating the reagent for 1 h at 100 degrees C did not alter solubility or cytochemical behavior. In contrast, parallel treatment of Alcian Blue yielded insoluble material by hydrolysis of the isothiouronium groups. The reagent for basophil detection comprises n-propyl Astra Blue iodide, lanthanum chloride, sodium chloride, Tween 20, and cetylpyridinium chloride. The Astra Blue derivatives were characterized by uv-vis, ir, percentage halide, paper chromatography, and 13C NMR.     

Cell analysis in cerebrospinal fluid (CSF) using Sysmex? hematology analyzers XT-4000i and XE-5000: evaluation with CSF controls of the Joint German Society for Clinical Chemistry and Laboratory Medicine (DGKL)

In cerebrospinal fluid (CSF) analysis, hematology analyzers (HAs) Sysmex? XT-4000i and XE-5000, equipped with flow cytometry (FCM), were used to count cells and differentiate leukocytes into mononuclear and polymorphonuclear cells (MNCs, PMCs) applying body fluid mode. FCM was evaluated with 20 DGKL CSF controls containing viable human leukocytes and erythrocytes. HA values were compared with reference values by Passing/Bablok regression analysis to reveal conformity. Conformity of white blood cells (WBCs) was obtained with native leukocytes, counted in calibrated Fuchs-Rosenthal chamber as reference; red blood cell counts proved inaccurate. CV <40% with WBC counts <20 per μL impairs accuracy. Reference WBC differentiation was assayed using FACS Canto II? and FC-500 SN with anti-CD45, anti-CD14, anti-CD16, anti-CD16/56 [Becton Dickinson (BD); Beckman Coulter (BC)]. BD FACS lysing solution?-no-wash-procedure was applied. BC pretreatment with Versalyse lysing solution was not recommended. MNCs (lymphocytes + monocytes) were significantly lower (～14%) on both HAs; PMCs (granulocytes or sum of neutrophils + eosinophils + basophils: range 1-86 M/L) were significantly higher (～2.2-fold). WBC HA differentiation is not reliable because MNC/PMC differentiation yielded lower and higher values than FACS-FCM references, respectively. This is attributed to incorrect discrimination of leukocytes with rounded/nonrounded nuclei; adding leukocytes with nonrounded nuclei to too low HA MNCs (about 40% not-activated) yielded P/B conformity; subtraction of leukocytes with nonrounded nuclei from elevated HA PMCs showed conformity (about 85% activated). Nucleus/activation state of leukocytes was assessed using microhistology. Sysmex XT-4000i and XE-5000 HAs systems are inappropriate for complete CSF cell analysis.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane.

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10-3-10-4 V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strength it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yeilds a mean value of about 1.6 V. for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 degrees C. as shown by incorporation of 131I-labeled albumin and repeated dielctric breakdown.     

Preparation of erythrocyte ghosts by dielectric breakdown of the cell membrane

Dielectric breakdown of membranes of red blood cells was observed in high electric fields (approx. 10³–10⁴ V/cm) using an improved Coulter Counter with hydrodynamic focussing. In making measurements of the size distributions of red blood cells as a function of increasing electric field strenght it was found that a sharp discontinuity occurred in the otherwise linear relation between the pulse heights in the Coulter Counter and the electric field strength due to dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated at breakdown in the cell membranes yields a mean value of about 1.6 V for the membrane potential of red blood cells. Due to the dielectric break-down, release of hemoglobin occurred. Mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter or thermal rupture could be excluded as hemolysing mechanisms. The leaky ghost cells resealed at 37 °C as shown by incorporation of ¹³¹I-labeled albumin and repeated dielectric breakdown.     

An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry.

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.     

The mean red cell volume in long distance runners

Red cell indices were determined in 6 well trained runners before and after a 100 km race, and Coulter Counter (CC) determinations compared with calculated values derived from centrifuged hematocrit (ctrf), red cell count (CC) and hemoglobin measurements. The following changes were observed immediately after the race, as compared to values 3 days before: MCV(ctrf) decreased by 4.9% (p less than 0.001), MCV(CC) increased by 1.9% (p less than 0.05), MCHC(ctrf) increased by 4% and MCHC(CC) decreased by 3%. The increase in MCV(CC) suggests that intraerythrocyte osmolality was increased, this probably leading to swelling of the cells induced by a shift of water from the diluting Coulter Counter solution into the red cells prior to the MCV measurement. The decrease in MCV(ctrf) immediately after the race was not correlated with the increase in plasma osmolality. This suggests that plasma osmolality alone was not the key factor for regulation of red cell volume. The changes in MCV(ctrf), which contributed to a surprising stability of the hematocrit value and plasma volume, might represent a physiological principle for the maintenance of a favourable blood viscosity.     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.     

Encephalitozoon cuniculi: Quantitation of Parasites and Evaluation of Viability1

Two methods (manual and automated) for quantitation of viable versus dead Encephalitozoon cuniculi are reported. The manual method uses ethidium bromide and acridine orange to stain dead and viable organisms, respectively. The stained organisms are visually differentiated with the aid of a fluorescence microscope. The automated method uses propidium iodide to stain dead parasites, which are differentiated from viable unstained parasites with the aid of a flow cytometer. An automated cell counter (Coulter Counter) was used to count rapidly large numbers of samples and to improve the sensitivity of counting low concentrations of parasites. These methods will enhance investigators' abilities to conduct quantitative experiments on host defense mechanisms against E. cuniculi.     

Flow cytochemical patterns of white blood cells in human hematopoietic malignancies: III. Miscellaneous hemopoietic diseases

Peripheral blood samples from 48 untreated and 20 treated patients with disease entities that directly or indirectly affect hematopoiesis [dys-myelopoietic syndrome (DMS), refractory anemia with excess blasts (RAEB) or in transformation (RAEBIT), lymphoma, myeloma, acquired immunodeficiency syndrome (AIDS), and solid tumors with uninvolved bone marrow] were measured with the Technicon H-6000 automated hematology analyzer; this instrument provides a differential count on 10(4) white blood cells (WBC) effected by means of flow cytochemistry (peroxidase content) and volume (light scatter) discrimination. Cases with DMS and RAEB showed statistically significantly lower WBC counts than normal, whereas cases with lymphoma showed significantly higher values. No disease entity demonstrated changes in mean peroxidase activity (MPA) that were significantly different from normal, although all disease entities, including cases with solid tumors, showed significantly higher (two to severalfold) proportions of cells with high peroxidase (HPX) content, probably as a reflection of a disturbance of normal hemopoiesis with the emergence of younger granulocytic forms. All cases with paraleukemia (DMS, RAEB, and RAEBIT) showed significantly higher values of large unstained cells (LUC), whereas cases with lymphoma showed significantly lower LUC values. There were no statistically significant differences for any parameter (WBC counts, MPA, HPX, or LUC) among the paraleukemia subtypes. However, based on the displayed trends, a case presenting with dyserythropoiesis, relatively low WBC counts, abnormal HPX values, and LUC below 10% should be suspected for RAEB, whereas the presence of greater than 10% LUC and almost normal or even slightly elevated WBC counts should suggest a more accelerated phase of RAEB. Unless complicated by a leukemic phase, cases of lymphoma or myeloma did not display changes in any of the parameters analyzed by the H-6000. Similarly, patients with AIDS had no overt changes other than a trend to lower WBC counts with occasionally higher or lower absolute lymphocyte counts than normal. The peripheral blood of patients with solid tumors displayed a slight increase in HPX, suggesting an indirect effect on hemopoiesis since careful workup failed to demonstrate bone marrow involvement. Our data demonstrates that an H-6000 analysis has a role in the evaluation and follow-up of all these entities particularly to document leukemic transformation of either lymphoma, myeloma, or RAEB.     

Rapid,Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer,and a Comparison to Other Methods

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument''s automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.     

Standardized flow cytometric method for the accurate determination of platelet counts in patients with severe thrombocytopenia

BACKGROUND: The therapeutic option of prophylactic platelet (PLT) transfusion in cases of severe thrombocytopenia critically depends on the availability of accurate and precise counts because clinical decisions are widely based on decision or trigger points. Although often applied in current practice at a level of 20 Gpt/L, there is increasing evidence that the trigger points could safely be reduced to 10 or even 5 Gpt/L. In order to facilitate this downward revision, it is necessary to have PLT counting methods that are able to provide reliable results in the appropriate decision range. METHODS: Postchemotherapy-induced pancytopenia PLT counting was performed in patients with hematological malignant disorders. This study describes a novel flow cytometric method that utilizes a PLT-specific monoclonal antibody (CD41a) in conjunction with fluorescent reference beads in order to derive absolute platelet numbers. RESULTS: Applying a mathematical model, this flow cytometric method was shown to have a detection limit of 0.24 Gpt/L and a lower limit of quantification (coefficient of variation [CV] = 10%) of 1.1 Gpt/L. These values are a substantial improvement on previously reported results for the Technicon H1 automated instrument or manual hemocytometry. Moreover, although the flow cytometry and Technicon H3 methods were found by supplementary analyses to show a reasonably good correlation, the hematology instrument showed a distinct tendency to overestimate PLT counts at low levels. CONCLUSION: It is proposed that this standardized immunoplatelet method offers the best approach in evaluating, at the clinical level, the possibility of lower PLT transfusion triggers. It can be used to evaluate the performance limitations of automated hematology analyzers that are widely used at the present time.     

Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements.

BACKGROUND: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements. METHODS: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and beta2-microglobulin with antibodies conjugated with fluorescein- and indocarbocyanine-like fluorophores and analyzed in FCET measurements on a cell-by-cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray. RESULTS: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye- and instrument-dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor-acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap. CONCLUSIONS: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546-Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.     

Comparison of Coulter Counter and 14C measurements of Acartia tonsa (Copepoda,Calanoida) grazing on Chlamydomonas sp. (Volvocales)

Both the Coulter Counter and ¹⁴C method were used to measure the grazing (clearance rates) of the marine calanoid copepod Acartia tonsa on different concentrations of a Chlamydomonas sp. culture. In most cases, clearance rates measured by the Coulter Counter method were higher than those measured by the ¹⁴C method by factors of 2 to 3. We explore several possibilities for the differences obtained between the two methods. We suggest that loss of radioactivity through grazer egestion might be the main reason for the discrepancy between methods. Food concentration did not affect the comparability of both methods' measurements.     

Dielectric Breakdown of Cell Membranes

With human and bovine red blood cells and Escherichia coli B, dielectric breakdown of cell membranes could be demonstrated using a Coulter Counter (AEG-Telefunken, Ulm, West Germany) with a hydrodynamic focusing orifice. In making measurements of the size distributions of red blood cells and bacteria versus increasing electric field strength and plotting the pulse heights versus the electric field strength, a sharp bend in the otherwise linear curve is observed due to the dielectric breakdown of the membranes. Solution of Laplace's equation for the electric field generated yields a value of about 1.6 V for the membrane potential at which dielectric breakdown occurs with modal volumes of red blood cells and bacteria. The same value is also calculated for red blood cells by applying the capacitor spring model of Crowley (1973. Biophys. J. 13:711). The corresponding electric field strength generated in the membrane at breakdown is of the order of 4 · 10⁶ V/cm and, therefore, comparable with the breakdown voltages for bilayers of most oils. The critical detector voltage for breakdown depends on the volume of the cells. The volume-dependence predicted by Laplace theory with the assumption that the potential generated across the membrane is independent of volume, could be verified experimentally. Due to dielectric breakdown the red blood cells lose hemoglobin completely. This phenomenon was used to study dielectric breakdown of red blood cells in a homogeneous electric field between two flat platinum electrodes. The electric field was applied by discharging a high voltage storage capacitor via a spark gap. The calculated value of the membrane potential generated to produce dielectric breakdown in the homogeneous field is of the same order as found by means of the Coulter Counter. This indicates that mechanical rupture of the red blood cells by the hydrodynamic forces in the orifice of the Coulter Counter could also be excluded as a hemolysing mechanism. The detector voltage (or the electric field strength in the orifice) depends on the membrane composition (or the intrinsic membrane potential) as revealed by measuring the critical voltage in E. coli B harvested from the logarithmic and stationary growth phases. The critical detector voltage increased by about 30% for a given volume on reaching the stationary growth phase.     

Hemopoietic stem and precursor cell analysis in umbilical cord blood using the Sysmex SE-9000 IMI channel

Circulating hematopoietic progenitor cells (HPCs) are routinely measured by flow cytometry using CD34 expression. As an alternative, the "immature information" (IMI) channel measurement of the automated hematology analyzer Sysmex SE machines was developed. We tested the IMI channel HPC method with umbilical cord blood specimens. The IMI-HPCs were compared with CD34 counts and numbers of colony-forming units (CFUs). The IMI-HPC data were reproducible and dilution experiments yielded a log-linear relationship. The mean percentage of CD34(+) cells in 50 umbilical cords was 0.43 versus 0.11 of HPCs in the IMI channel (correlation coefficient r = 0.67). Absolute numbers yielded 96.79 x 10(6)/L CD34(+), 33.17 x 10(6)/L IMI-HPC, and 35.04 x 10(6)/L CFU-HPC. Receiver operating characteristics curves were made at various cutoff levels for CD34(+) cells to visualize sensitivity and specificity profiles. With median values of 13.56 x 10(6)/L for IMI-HPC and 20 x 10(6)/L for CD34(+) as cutoff points (the levels used in the laboratory to start stem cell pheresis), the percentage of false-negative observations was 70.4%. To exclude the influence of storage time, tests were repeated until 72 h after umbilical cord collection. Total white blood cell count decreased in most cases, whereas absolute number of IMI-HPC tended to increase in time. In conclusion, if HPC measurements in the IMI channel are used to monitor circulating stem cells during mobilization, one has to be aware of a very low correlation between these results and those of other methods such as CD34(+) analysis and colony growth. False-negative results do occur, but if events are seen in the IMI channel, this simple monitoring technique is useful to predict the presence of circulating stem cells.     

Cell analysis system based on immunomagnetic cell selection and alignment followed by immunofluorescent analysis using compact disk technologies

BACKGROUND: Although the flow cytometer has become the standard in cell analysis, it has limitations. Recently, we introduced a new cell analysis method based on immunomagnetic selection and aligning of cells. No flow system is needed and cell analysis can be performed in whole blood. METHODS: Whole blood is incubated with fluorescent labels and immunomagnetic nanoparticles. The blood is injected into a capillary that is in a strong magnetic field. The immunomagnetic-labeled cells move upward and align themselves along ferromagnetic lines present on the upper surface of the capillary. An optical focus and tracking system analogous to that used in a conventional compact disk player focuses a 635-nm laser-diode on the magnetically aligned cells. The emitted fluorescence signals are projected on two photomultipliers. Allophycocyanin (APC)-labeled CD4 (CD4-APC) and Cyanin5.5 (Cy5.5)-labeled CD8 (CD8-Cy5.5) antibodies and Oxazine750, all red excited, are used as fluorescent labels. RESULTS: A differential white blood cell count performed in whole blood is obtained using the CD4-APC in combination with Oxazine750. The results are compared with the Technicon-H1 hematology analyzer. Correlation coefficients of 0.91 for neutrophilic granulocytes, 0.93 for lymphocytes, 0.93 for monocytes, and 0.96 for eosinophilic granulocytes were obtained. Immunofluorescence is demonstrated using CD4-APC and CD8-Cy5.5. The absolute counts obtained for CD4+ and CD8+ are compared with the Coulter Epics XL flow cytometer. Correlation coefficients of, respectively, 0.91 and 0.94 were obtained. CONCLUSION: We conclude that our system is as capable as a standard flow cytometer or hematology analyzer for a reliable routine white blood cell analysis, including immunophenotyping, and can be used as an easy-to-handle disposable white blood cell test.     

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and ¹⁴C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10³ cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.