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Background: Light scattering is an essential parameter in flow cytometry, facilitating functions such as size measurement, discrimination of cell types on the basis of shape and morphology, detection of fluorescence-negative cells, and gating of fluorescence measurements. Light scattering measurement of viruses is generally not feasible with current flow cytometers due to their small size. The problem is aggravated by the fact that the light scattering of particles in this size range (< 200 nm) falls off with roughly the sixth power of their linear dimensions., Methods: A new optical layout using darkfield illumination and detection has been developed. A 532-nm laser was used for excitation, and scattered light was collected with large aperture optics., Results: Light scattering histograms of polymer particles with diameters of 70-300 nm were recorded without gating by other parameters. By extrapolation, a detection limit of about 50 nm was obtained. Different species of virus with sizes of approximately 100 nm also were recorded., Conclusions: Flow cytometric light scattering measurement of submicroscopic particles, in a size range that includes many viral species, is now feasible. The results indicate that it may be practically impossible to measure by flow cytometry the light scattering of particles smaller than 40 nm., (Copyright 2004 Wiley-Liss, Inc.)

Background: The sample injection systems of flow cytometers employ either a pressure differential between the sample vial and the sheath fluid reservoir or volumetric injection of the sample from a syringe. The pressure differential method facilitates rapid and efficient flushing to eliminate carryover between samples, but does not allow accurate determination of the rate of sample flow and cell concentration. Volumetric injection, which comprises a valve for switching the sample flow, facilitates highly accurate measurement of the cell concentration, but requires a less efficient and more time-consuming flushing procedure., Methods: Applying a removable syringe, which connects to the inlet of the sample tubing via a tight sealing, we eliminate the valve and obtain efficient flushing while maintaining the advantage of volumetric sample injection., Results: This device gives highly constant sample flow rates strictly proportional to syringe velocity over the range 0.2-50 microl/min with a settling time of about 2 sec., Conclusion: This device has the same precision as the conventional sample injection system, whereas the speed and efficiency of flushing are improved greatly., (Copyright 2002 Wiley-Liss, Inc.)

Background: Cells exclude their own volume of dye solution in the sample flow which carries them through the flow chamber of the flow cytometer, thereby affecting the otherwise constant signal arising from the fluorescence of this solution. Under certain conditions, this phenomenon may significantly influence the fluorescence signal of the cells., Materials and Methods: Using the slit scan technique, we studied this phenomenon as observed for monodisperse polystyrene particles in fluorescein solution., Results: The measurements show that dye solution accumulates just in front of the particle and just behind it, with a relative void in between. This phenomenon is most likely caused by the rapid constriction of the flow as it enters the orifice of the nozzle or flow chamber, giving rise to a pulse of fluorescence which adds to that of the particle or cell itself. The magnitude of this artifact depends on the design and dimensions of the nozzle/flow chamber as well as on the rate of sample flow., Conclusions: The dye exclusion artifact may affect measurements of cells when they are in a dye solution having a fluorescence per unit volume which is significant compared to that of the cells, especially at low sample flow rates., (Copyright 2002 Wiley-Liss, Inc.)

The measurement of microbes is an increasingly important application in flow cytometry. The small size of these organisms can create special difficulties for instruments designed to analyze much larger eukaryotic cells. This unit describes the problems encountered in making microbiological measurements, the instrument requirements, and some approaches that can be taken to optimize the instrument. This material is essential background for the protocols found in chapter 11, Microbiological Applications.

Measurement of bacteria and other microorganisms at the level of single cells has progressed enormously over the last couple of decades. Up to the late 1970s, there were no other means than microscopy for observation of single microorganisms, making any type of measurement very cumbersome and tedious, at best. Today, we measure several parameters simultaneously with a precision of a few per cent, and at a rate of 1000 cells per second. The first papers on the use of flow cytometry to measure bacteria appeared only in 1977, although the method had proved highly successful in studies of mammalian cells for almost a decade. There were several reasons for this relatively late introduction, including technical limitations, problems with adequate staining, and, not least, the human factor. Today, flow cytometry has a wide range of microbiological applications, ranging from studies of the bacterial cell cycle and many other cellular characteristics to assessment of antibiotic susceptibility of clinical samples, and monitoring of bacteria and other microorganisms in anything from sewage to sea water. Still, the potential of flow cytometry in microbiology is far from fully utilised. Better instruments and new stains will provide new opportunities to understand, control and exploit this vital part of the biosphere.

For flow cytometry-based detection as well as susceptibility testing and counting, staining of the bacterial cells is essential. In an attempt to develop rapid preparatory procedures for nucleic acid staining of wild type Gram positive bacteria, the uptake of fluorescent dyes in viable S. aureus, E. faecalis, and B. cereus cells was studied by flow cytometry under conditions intended to block probe efflux and increase cell wall permeability. The aim of the study was to develop procedures which allow rapid nucleic acid staining independent of fixation, since ethanol fixation is time-consuming and may mask phenomena associated with viability and lead to uncontrolled loss and aggregation of cells. The dye uptake was measured repeatedly after treating cells with metabolic inhibitors in order to block probe efflux, or cold shock (0 degree C) to increase permeability. The probes used were mithramycin (Mi), ethidium bromide (EB), DAPI, Hoechst 33342 and Hoechst 33258. None of the procedures facilitated uptake of the dyes to a level similar to that obtained in fixed control cells in all of the species. After metabolic inhibition of B. cereus cells, DAPI and Hoechst fluorescence increased to a level similar to or above that found in fixed cells, indicating that the uptake of these dyes is limited by energy-dependent efflux. A similar increase of DAPI fluorescence was observed after cold shock suggesting the uptake of this dye to be limited also by permeability in B. cereus. The Mi and EB fluorescence increased to the level of the fixed control cells under all conditions tested, suggesting free probe influx in this species. Generally, probe uptake in S. aureus and E. faecalis was lower than in B. cereus cells, and no permeabilizing effect of cold shock was observed. In some experiments the fluorescence exceeded that of ethanol fixed control cells, indicating that the fixation may cause conformational changes in DNA.

An in vitro model for flow cytometric detection of heterogeneous drug response in exponentially growing Escherichia coli and Klebsiella pneumoniae was studied to evaluate the potential of this technology for rapid antibiotic susceptibility testing in polymicrobial samples. The cells, which exhibited 80-fold difference in in ampicillin susceptibility, were cultivated in the presence of ampicillin at a concentration equaling 1 MIC of the low-susceptibility strain (E. coli). Prior to flow cytometric analysis, the cells were fixed in 70% ethanol and stained with a DNA-specific dye. After 1 h of ampicillin incubation, the light scattering and fluorescence intensities of the susceptible cells increased 4.3-fold and 5-fold, respectively, but remained about constant for the resistant cells. The corresponding cell number increase for the resistant and the susceptible cells was 9.4 and 1.7, respectively. The two strains could be distinguished in the histograms on the basis of their light scattering and fluorescence intensities and by cell number. With an incubation of up to 3 h, the light scattering and fluorescence intensities of the susceptible cells grew stronger, at the expense of cell number. By combination of histograms, the discrimination of different cell populations could be improved. The results demonstrate the ability of flow cytometry to discriminate between species in heterogeneous cultures and suggest the potential of the technique for rapid assessment of bacterial susceptibility. However, the present results are preliminary, and the application to biological liquids and clinical samples has to be demonstrated further.

Exponentially growing E. coli cells were cultivated in the presence of ceftazidime, ciprofloxacin, and gentamicin in concentrations ranging from 0.5-8 minimal inhibitory concentration (MIC), permeabilized by means of cold shock in EDTA/azide, and stained with the DNA-specific dye combination of ethidium bromide and mithramycin before the fluorescence, light scattering, and cell number were measured flow-cytometrically. In order to evaluate the applicability of the cold-shock procedure, cells were also permeabillized by 70% ethanol. Permeabilization by cold shock, which eliminates washing of the cells, reduced the preparation time to <5 min. A statistically significant increase in light scattering and fluorescence, i.e., cell size and DNA content, could be detected already after 30 min of ceftazidime and ciprofloxacin exposure, even at sub-MIC concentrations. The results obtained with these drugs with cold-shock permeabilization were similar to those seen with ethanol fixation. For gentamicin-treated cells, however, a majority of the cells lost their fluorescence after cold shock. In gentamicin-treated cells fixed in ethanol, there was no consistent effect on either light scattering or fluorescence; however, we observed a substantial fragmentation and leakage of DNA in such cells. The cell proliferation was completely inhibited within 30 min of gentamicin incubation. For all three drugs, loss of light scattering and DNA were associated with cellular disintegration, i.e., reduced viability. The present results demonstrate that effects of ceftazidime, ciprofloxacin, and gentamicin on E. coli can be detected by flow cytometry within 1 h from the beginning of drug exposure to the completed measurement.

The effects of antibiotics on Escherichia coli during exponential growth in liquid medium were monitored by flow cytometry measuring cellular DNA content and cell size of individual cells in large numbers as well as cell counts. A statistically significant increase in cellular DNA as well as size was recorded after 20 and 40 min of incubation with the MIC of ampicillin and mecillinam, respectively. The optical density (OD600nm) of treated cultures continued to increase for at least 80 minutes, showing that the increase in cellular DNA and size reflected continued growth and elongation of cells coupled with inhibition of cell division, the latter being confirmed by the cell counting. Since fixation, staining and flow-cytometric analysis can be carried out within a few minutes, the present results suggest that flow cytometry may have potential as a rapid and quantitative technique that can be automated for clinical and experimental susceptibility testing of antibacterial drugs.

It has been found that the type of short arc high-pressure lamps used in some flow cytometers can be pulsed to reach intensities many times higher than that measured when they are operated on constant power. A 75 W mercury-xenon lamp was fed 20 microseconds current pulses super-imposed on its rated current of 5.4 A. Pulses of 50 A produced a 75-fold increase of the emission intensity at the excitation wavelength of FITC, which means that the excitation intensity in the flow chamber at this wavelength exceeded 100 mW. At the major emission lines of mercury, i.e., 366, 436, and 546 nm, the increase was about 25-fold, corresponding to intensities of the order of 300 mW. The light pulses were found to be reproducible to within 2% and the intensity was independent of the pulse frequency up to at least 250 pulses/s. Operated in this mode the lamp produced more than 1 x 10(8) 30 A pulses, which is sufficient to measure some 10,000 typical size samples. The rise time of the light pulses was about 5 microseconds. This was sufficiently short that the leading edge of the light scattering signal from a cell entering the excitation focus of the flow cytometer could be used to trigger the current pulse so that the cells were exposed to the full light pulse intensity while they were still within the focus.(ABSTRACT TRUNCATED AT 250 WORDS)

The sensitivity and resolution of flow cytometers are functions of the signal produced by a given particle as well as by the noise in the presence of which the signal is detected. The noise is primarily due to the fact that emission of light as well as its detection by photoelectric devises are stochastic processes. This fact leads to equations describing how resolution and sensitivity are limited by the magnitude of the signal, the background, and the photoelectron quantum yield of the detector. The equations are pointing to a method by which the signal and noise of a flow cytometer can be measured in absolute terms, as well as a way to determine fluorescence sensitivity without having to extrapolate to the noise level. The equations appear to be validated when applied to measuring data obtained with two different flow cytometers.

7-aminoactinomycin D (7-AMD) efficiently discriminates between cells in the G0 and G1 phases of the cell cycle (Stokke et al., Cancer Res. 48:6708, 1988). The fluorescence and light scatter of cells stained with 7-AMD, Hoechst 33258 (H33258), and fluorescein isothiocyanate (FITC)-labeled antibodies were measured by dual wavelength excitation flow cytometry (488 nm, ultraviolet). The H33258 fluorescence was found to reflect DNA content in the presence of 7-AMD, although energy transfer caused an approximately 50% reduction in H33258 fluorescence intensity. However, energy transfer was more pronounced in dead cells, permitting exclusion of such cells during analysis. The G0, G1, S, and G2 phases of the cell cycle could be identified in the 7-AMD versus H33258 fluorescence histograms, as was demonstrated with mitogen-stimulated B lymphocytes and a mixture of unstimulated B lymphocytes and a proliferating B-cell line. One hour fixation with paraformaldehyde was compatible with prefixation labeling of surface antigens with indirectly FITC-labeled antibodies as well as postfixation labeling of intracellular antigens. Studies of expression of some surface and nuclear activation-associated antigens confirmed that cell cycle-resolved antigen expression and the time course of appearance of such antigens could be assessed accurately. Phycoerythrin could be used to label a second antigen.

The epi-illumination optics employed in most arc lamp-based flow cytometers may be modified so as to produce a dark-field configuration which facilitates highly sensitive detection of both forward and large angle light scattering in an instrument with a "jet on open surface" flow chamber. Forward scattering is detected at angles upwards from about 2 degrees, while large angle scattering includes angles above 18 degrees. Theoretical considerations suggest that large angle scattering measured around 20 degrees may be as efficient as that measured at 90 degrees for the purpose of distinguishing cells on the basis of intracellular structure. This was supported by the finding that dual parameter light scattering histograms of leukocyte suspensions obtained with the arc lamp-based instrument were closely similar to such histograms recorded with a laser-based instrument with the large angle detector at 90 degrees. Different species of bacteria could be distinguished by means of the dual parameter light scattering device, as could different species of sea algae. The sensitivity of the device is sufficient to measure 0.2 microns polystyrene particles in both forward and large angle scattering.

By means of a new flow chamber, a standard fluorescence microscope with Epi illumination and 100 W mercury arc excitation has been turned into a flow cytophotometer combining high resolution and sensitivity with simplicity of operation. In the flow chamber, cells are passed in a narrow stream through the microscope focus carried by a laminar flow of water running on the open surface of a cover glass which is coupled to the oil immersion microscope objective. Two spectral components of the fluorescence, for example, resulting from specific staining of two different cellular constituents with different dyes, can be measured simultaneously in separate channels so as to produce three-dimensional histograms. The scattered light of the cells is detected in dark field by a second microscope situated opposite the primary objective. Scattered light detection is integrating with regard to scattering angle from 0 degree to 90 degrees. Hence, diffraction pattern effects are eliminated and the light scatter signal is approximately proportional to cell dry weight. The Epi illumination, which implies that excitation and fluorescence collection are parfocal, greatly simplifies instrument adjustment, which is further facilitated by the fact that the cell stream can be viewed at high magnification. Cell measuring time is about 3 microseconds which implies a measuring rate of 3 x 10(3) cells/s at 1% coincidence rate. Sensitivity is sufficient for measuring the DNA content of bacteria (that is, approximately 5 x 10(-15) g/cell) with a coefficient of variance (CV) of about 6%. CV less than 1% is achieved for DNA histograms of mammalian cells. A 5 W argon laser as excitation source facilitates slit scan analysis and increases the sensitivity and measuring rate by one to two orders of magnitude.

A device is described for simultaneous separate detection of the light scattering of cells at low and large scattering angles in an arc lamp-based flow cytometer with epi-illumination through an oil immersion microscope objective. Light scattering was measured in a dark field configuration that allows separate detection of light scattering greater than 2 degrees and 15 degrees, respectively. Dual parameter light scattering histograms of a blood cell suspension containing various types of leukocytes were closely similar to that obtained with a commercial laser-based instrument with light scattering detection at forward and right angles. The sensitivity of the device was sufficient to measure polystyrene particles with 0.25-micron diameter. A potential application may be differentiation of bacteria.

The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).

The feasibility of flow cytometry for measurements on bacteria has been demonstrated by measurements of DNA-associated fluorescence of Escherichia coli K-12 in various phases of cell growth. Bacteria were stained with a combination of ethidium bromide and mithramycin after fixation in 70% ethanol. Cultures grown to stationary phase accumulated in two peaks representing cells with two and four chromosomes. Qualitatively similar histograms were obtained with cells grown in the presence of chloramphenicol, whereas cells of the temperature sensitive strain E 177 (dnaA) ended up with only one chromosome per cell at the restrictive temperature. The fluorescence intensity of cells with one chromosome was about 10(3) times smaller than that of human diploid cells. Instrumental resolution at this level of intensity was CV = 5%, whereas peak widths corresponded to CV = 7-8%. Dyes bound to RNA did not appear to contribute significantly to the fluorescence.

The DNA and protein content of individual Escherichia coli cells were measured at a rate of 10(4) cells per second with a sensitive microscope-based flow cytometer. DNA and protein were quantified by measuring the fluorescence from cells stained with a combination of the DNA-binding drugs Mithramycin and ethidium bromide and by scattered light, respectively. Separate experiments demonstrated that the light scatter signal was proportional to protein content. Dual parameter histograms (fluorescence/scattered light) of bacterial cultures gave detailed pictures of changes dependent upon the growth conditions and of the cell cycle kinetics. Effects of antibiotics could be readily detected and characterized after a few hours. The results demonstrate that flow cytometry is a promising method for application in experimental and clinical microbiology.

Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.

The effect of various concentrations of rat-liver macrophages on the Con-A-induced blastogenesis of syngeneic spleen lymphocytes has been studied by means of flow-cytometric methods and by measurement of the uptake of 3H-thymidine. By measuring the distributions of cellular and nuclear volume and cellular DNA to characterize the progression of responding lymphocytes through the cell cycle, we have distinguished a promotive and an inhibitory effect of macrophages. The promotive effect is on the number of cells initiating blastogenesis and on their rate of progression through the cell cycle. The degree of promotion increased strongly with the concentration of macrophages even for concentrations that suppressed the incorporation of 3H-thymidine almost completely from about 30 h of culture. The inhibition observed for macrophage concentrations +/- 10% was a late effect causing stagnation of cell cycle and reduced viability only from about 24 h of culture.

Light-scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc-lamp-based flow cytometer (AL-FCM). The results were compared with those obtained with a conventional, laser-based flow cytometer (L-FCM) with forward scattering (2-20 degrees) and scattering at right angles. Measuring with the AL-FCM in the angle range upward from 13 degrees, the relative light-scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right-angle scattering in the L-FCM. With a range of scattering angles upward from 2 degrees the AL-FCM yielded histograms similar although not identical to that of the forward-scattering detector in the L-FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.

Flow cytometric determination of the DNA and protein content of E. coli has been carried out by means of a microscope-based flow cytophotometer with a high pressure arc lamp excitation ligh source. Fluorescence (DNA)/light scatter (total cell protein) dual parameter histograms with a resolution cv of 5% were obtained for cells labeled with a combination of mithramycin and ethidium bromide. Histograms of E. coli in rapid and slow exponential growth are presented to exemplify how the cell cycle kinetics of bacteria can be studied in much more detail than has been possible by other methods. Significant effects of chloramphenicol and penicillin on the cell cycle distribution and cell numbers of E. coli cultures were evident after one hour of culture. The data provided information on which parts of the cell cycle and what types of processes were affected by the drug. It appears that flow cytometry may become a valuable tool in studies of the cell cycle of bacteria as well as in clinical drug testing.

A microscope-based flow cytometer, which is based on a new flow system, has been extended to include light scatter detection, dual parameter fluorescence analysis, and a device which compensates for variations in excitation light intensity. A dichroic mirror splits the fluorescence into two spectral components that are further purified by additional filters situated in front of the photomultiplier tube detectors (PMT). Light scatter detection is obtained by a dark field configuration. An objective focuses the scattered light emitted within the dark field onto an adjustable slit in front of a PMT. Resolution of light scatter histograms of 1.5-micrometer latex spheres is about CV = 2.5%. Dual parameter analysis revealed a linear correlation between the light scatter signal and fluorescence of cells stained with fluorescein isothiocyanate, indicating that the light scatter signal is closely proportional to cell dry mass. The instrumental light scatter, which is proportional to the excitation light intensity, gives rise to a DC signal. This signal regulates the fluorescence signal amplifiers so that these signals are compensated for fluctuations in the excitation light intensity.

Knowledge of the sensitivity of a flow cytometer is important both in choosing an instrument for a particular application and in determining whether the resolution obtained on real samples is limited by instrumental factors or sample variability. The coefficient of variation (CV) obtained for measurement is given by CV2 = CVS2 + CVI2 + 1/n + K(delta VB2)/n2, where CVS is the variability due to the sample, CVI that due to variations in illumination of the sample particles, n the number of photoelectrons emitted from the photomultiplier photo cathode per sample particle (which is the signal intensity), delta VB2 the mean square background noise voltage (usually due to background illumination), and K a constant. Measurement of the CV of light flashes from a light emitting diode as a function of their intensity allows development of standard curves which can be used to directly determine the degree to which the resolution obtained on actual samples is limited by signal intensity and background light. The use of standard particles allows relative calibration of the curves for different instruments and which also ranks their sensitivities.