Flow cytometry: illuminating microbiology

By means of flow cytometry, a technique whereby a hydrodynamically directed stream of cells is passed through a focus of exciting light, one can measure cell size and the macromolecular content of individual bacteria. The sensitivity and versatility of the flow cytometer make it a powerful tool in studies of the bacterial cell cycle, in identifying and characterizing bacterial infections, and in selecting bacteria of desired qualities. We review some of these applications of flow cytometry and conclude that this method holds great promise in many other areas of microbiology.     

Analysis of radiation-induced apoptosis in human lymphocytes: flow cytometry using Annexin V and propidium iodide versus the neutral comet assay

BACKGROUND: The neutral comet assay was devised to measure double-stranded DNA breaks, but it has also been used to measure apoptosis based on its characteristic DNA fragmentation patterns. There is still uncertainty about the reliability of this method. By comparing the comet assay with a flow cytometry method that uses Annexin V binding to apoptotic cells, we have provided further evidence for evaluating the usefulness of the comet assay for detecting apoptosis. METHODS: Apoptosis was induced in human peripheral blood mononuclear cells (PBMC) by ionizing radiation and measured using the comet assay and a flow cytometry method that measures Annexin V and propidium iodide (PI) staining. RESULTS: The Annexin V flow cytometry assay distinguished among early apoptosis, late apoptosis, and an apoptotic or necrotic phase in which the cells were labeled with both Annexin V and PI. The comet assay detected only the latter two phases of apoptosis. CONCLUSIONS: The comet assay is a useful tool for measuring the late stages of apoptosis whereas the Annexin V assay measures higher amounts of apoptosis because it can detect cells in an earlier stage of the apoptotic pathway.     

FLOW CYTOMETRY AND THE SINGLE CELL IN PHYCOLOGY

Flow cytometers measure light scattering and fluorescence characteristics from individual particles in a fluid stream as they cross one or more light beams at rates of up to thousands of events per second. Flow cytometrically detectable optical signals may arise naturally from algae, reflecting cell size, structure, and endogenous pigmentation, or may be generated by fluorescent stains that report the presence of otherwise undetected cellular constituents. Some flow cytometers can physically sort particles with desired optical characteristics out of the flow stream and collect them for subsequent culture or other analyses. The statistically rigorous, cell‐level perspective provided by flow cytometry has been advantageous in experimental investigations of phycological problems, such as the regulation of cell cycle progression. The capacity of flow cytometry to measure large numbers of cells in large numbers of samples rapidly and quantitatively has been used extensively by biological oceanographers to define the distributions and dynamics of marine picophytoplankton. Recent work has shown that flow cytometry can be used to elucidate relationships between the optical properties of individual cells and the bulk optical properties of the water they live in, and thereby may provide an explicit link between algal physiology and global biogeochemistry. Unfortunately, commercially available flow cytometers that are optimized for biomedical applications have a limited capacity to analyze larger phytoplankton. To circumvent these limitations, many investigators are developing flow cytometers specifically designed for analyzing the broad range of sizes, shapes, and pigments found among algae. These new instruments can perform some novel measurements, including simple fluorescence excitation spectra, detailed angular scattering measurements, and in‐flow digital imaging. The growing accessibility and power of flow cytometers may allow the technology to be applied to a wider array of problems in phycology, including investigations of nonplanktonic and multicellular algae, but also presents new challenges for effectively analyzing the large quantity of multiparameter data produced. Ultimately, the detection of molecular probes by flow cytometry may allow single‐cell taxonomic and physiological information to be garnered for a variety of algae, both in culture and in nature.     

Rapid detection of viable yeasts and bacteria in wine by flow cytometry

The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.     

A method for analysing phosphatase activity in aquatic bacteria at the single cell level using flow cytometry

It has been demonstrated that ELF97-phosphate (ELF-P) is a useful tool to detect and quantify phosphatase activity of phytoplankton populations at a single cell level. Recently, it has been successfully applied to marine heterotrophic bacteria in culture samples, the cells exhibiting phosphatase activity being detected using epifluorescence microscopy. Here, we describe a new protocol that enables the detection of ELF alcohol (ELFA), the product of ELF-P hydrolysis, allowing the detection of phosphatase positive bacteria, using flow cytometry. Bacteria from natural samples must be disaggregated and, in oligotrophic waters, concentrated before they can be analyzed by flow cytometry. The best efficiency for disaggregating/separating bacterial cell clumps was obtained by incubating the sample for 30 min with Tween 80 (10 mg l(-1), final concentration). A centrifugation step (20,000 g; 30 min) was required in order to recover all the cells in the pellet (only 7+/-2% of the cells were recovered from the supernatant). The cells and the ELFA precipitates were resistant to these treatments. ELFA-labelled samples were stored in liquid nitrogen for up to four months before counting without any significant loss in total or ELFA-labelled bacterial cell abundance or in the ELFA fluorescence intensity. We describe a new flow cytometry protocol for detecting and discriminating the signals from both ELFA and different counterstains (4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)) necessary to distinguish between ELFA-labelled and non ELFA-labelled heterotrophic bacteria. The method has been successfully applied in both freshwater and marine samples. This method promises to improve our understanding of the physiological response of heterotrophic bacteria to P limitation.     

Current strategies to induce secondary metabolites from microbial biosynthetic cryptic gene clusters

The genome of actinomycetes and several other microorganisms are endowed with many cryptic gene clusters that can code for previously undetected, a plethora of complex secondary metabolites. Under standard laboratory controlled conditions, the genes regulating these biosynthetic clusters are expressed at very low levels or remain phenotypically cryptic (silent). Over the past several decades, multi-drug-resistant bacteria have been observed with increased frequency, posing a significant threat to human health worldwide. The present alarming situation urgently calls for concerted global efforts for the discovery of new antimicrobials. The present situation, if not controlled, will take us again to the pre-antibiotic era. Today, in the post-genomic era, various new strategies such as the activation of cryptic gene clusters in microorganisms rejuvenate a new conviction in the field of natural product research that may lead to the identification of yet-unidentified novel secondary metabolites of therapeutic and other use. Decryptification of this versatile endogenous genetic reservoir may provide in the near future the more concrete rationale for antibiotic discovery. The present review is an attempt to provide a comprehensive detail, outlining current strategies that have been shown successful to activate cryptic biosynthetic gene clusters in microorganisms.     

Profiling of bacterial adhesin--host receptor recognition by soluble immunoglobulin superfamily domains

Several gram-negative human pathogens recognize members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family. Pathogenic Neisseriae employ distinct isoforms of the colony opacity-associated proteins (Opa(CEA) proteins) to bind to the amino-terminal domains of CEACAMs. Here we present a novel approach to rapidly determine the CEACAM-binding properties of single bacteria. Expression of the isolated amino-terminal domains of various CEACAMs in eukaryotic cells yields soluble probes that selectively recognize Opa(CEA)-expressing bacteria in a pull-down assay format. Furthermore, by expressing soluble CEACAMs as fusions to green-fluorescent protein (CEACAM-N-GFP), CEACAM-binding bacteria can be decorated with a fluorescent label and analysed by flow cytometry allowing the specific detection of receptor binding events on the level of single bacteria. Besides its potential for rapid and quantitative analysis of pathogen-receptor interactions, this novel approach allows the detection of receptor recognition in heterogeneous bacterial populations and might represent a valuable tool for profiling the host binding capabilities of various microorganisms.     

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.     

Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc. Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109. The expression of the fusion protein was successfully demonstrated in the cloned cells. The fusion proteins had no effect on the viability of the host cells. Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.     

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.     

Hyperchromatic cytometry principles for cytomics using slide based cytometry.

BACKGROUND: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. METHODS AND RESULTS: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. CONCLUSION: With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance.     

When Phase Contrast Fails: ChainTracer and NucTracer,Two ImageJ Methods for Semi-Automated Single Cell Analysis Using Membrane or DNA Staining

Within bacterial populations, genetically identical cells often behave differently. Single-cell measurement methods are required to observe this heterogeneity. Flow cytometry and fluorescence light microscopy are the primary methods to do this. However, flow cytometry requires reasonably strong fluorescence signals and is impractical when bacteria grow in cell chains. Therefore fluorescence light microscopy is often used to measure population heterogeneity in bacteria. Automatic microscopy image analysis programs typically use phase contrast images to identify cells. However, many bacteria divide by forming a cross-wall that is not detectable by phase contrast. We have developed ‘ChainTracer’, a method based on the ImageJ plugin ObjectJ. It can automatically identify individual cells stained by fluorescent membrane dyes, and measure fluorescence intensity, chain length, cell length, and cell diameter. As a complementary analysis method we developed ''NucTracer'', which uses DAPI stained nucleoids as a proxy for single cells. The latter method is especially useful when dealing with crowded images. The methods were tested with Bacillus subtilis and Lactococcus lactis cells expressing a GFP-reporter. In conclusion, ChainTracer and NucTracer are useful single cell measurement methods when bacterial cells are difficult to distinguish with phase contrast.     

Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high-resolution flow cytometry

The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.     

Evaluation of the interactions between strains of S. aureus and peripheral blood phagocytic cells

The aim of this study was to investigate the interactions occurring between peripheral blood phagocytes and strains of S. aureus isolated from different clinical specimens (blood, respiratory tract, pus). To evaluate the sensitivity of microorganisms to bactericidal activity of phagocytes, monocytes and granulocytes separated from peripheral blood by standard density gradient and by counter-current centrifugal evaluation these cells were incubated with suspensions of opsonized bacteria. In parallel, the viability of phagocytes was examined by flow cytometry, and the ability of bacteria to trigger reactive oxygen intermediates (ROI) production was evaluated by chemiluminescence measurement. To investigate the efficiency of phagocytosis, bacteria were labelled with fluorescein isothiocynate (FITC) and the percentage of cells containing FITC-labelled bacteria were analysed by flow cytometry. The data obtained show the strains of S. aureus derived from different clinical specimens, differ in their sensitivity to bactericidal activity of phagocytes--strains isolated from the blood show the highest, but strains isolated from the respiratory tract have the lowest sensitivity to killing. These strains differ too in their ability to trigger monocyte CL response. On the contrary, there was no difference in toxicity of bacteria against phagocytes. Strains isolated from peripheral blood showed a significant negative correlation between the ability to trigger CL response and toxicity against phagocytes.     

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuberculosis infection.     

In vivo plant flow cytometry: a first proof-of-concept

In vivo flow cytometry has facilitated advances in the ultrasensitive detection of tumor cells, bacteria, nanoparticles, dyes, and other normal and abnormal objects directly in blood and lymph circulatory systems. Here, we propose in vivo plant flow cytometry for the real-time noninvasive study of nanomaterial transport in xylem and phloem plant vascular systems. As a proof of this concept, we demonstrate in vivo real-time photoacoustic monitoring of quantum dot-carbon nanotube conjugates uptake by roots and spreading through stem to leaves in a tomato plant. In addition, in vivo scanning cytometry using multimodal photoacoustic, photothermal, and fluorescent detection schematics provided multiplex detection and identification of nanoparticles accumulated in plant leaves in the presence of intensive absorption, scattering, and autofluorescent backgrounds. The use of a portable fiber-based photoacoustic flow cytometer for studies of plant vasculature was demonstrated. These integrated cytometry modalities using both endogenous and exogenous contrast agents have a potential to open new avenues of in vivo study of the nutrients, products of photosynthesis and metabolism, nanoparticles, infectious agents, and other objects transported through plant vasculature.     

Flow cytometric identification of microorganisms by dual staining with FITC and PI.

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.     

RAPID DETECTION of STAPHYLOCOCCUS AUREUS IN FOOD BY FLOW CYTOMETRY

This report describes the detection of Staphylococcus aureus in buffer and in several kinds of food by flow cytometry. Fluorescein isothiocyanate conjugated anti-protein A antibodies were used in a 4-h procedure to label cells, 10⁵-10⁶ cells/mL are needed. the use of single parameter, green fluorescence, enabled specific differentiation of S. aureus from other bacteria including 11 Staphylococcus species. the flow cytometric method can detect S. aureus in food samples after 48-h enrichment in trypticase soy broth with 10% NaCl. As low as 2 S. aureus cells present in 10 mL enrichment broth could grow to a population density detectable by the flow cytometric method after enrichment. This method was faster and less laborious than the conventional BAM (Bacteriological Analytical Manual) or AOAC (Association of Official Analytical Chemists) methods, and could be automated for analysis of S. aureus in food.     

Development of a flow cytometric assay to study glucocorticoid receptor-mediated gene activation in living cells

A flow cytometry-based reporter gene assay was developed and utilized to measure glucocorticoid receptor (GR)-mediated gene activation at the single cell level in living cells. A reporter gene was generated that contains two copies of the glucocorticoid response element and an E1b TATA box upstream of a destabilized enhanced green fluorescent protein. Glucocorticoid activation of the reporter gene in Cos-1 and HTC cell lines was measured in vivo by flow cytometry and was shown to be dose dependent, leading to an increase in total fluorescence of the cell population. Flow cytometric analysis indicated this increase in total fluorescence per sample resulted from an increase in the number of cells expressing the activated green fluorescent protein (GFP) reporter as well as an overall increase in the mean GFP fluorescence within cells. Activation of reporter gene activity was time dependent occurring as early as 1-2h after dexamethasone addition. Activation of the reporter gene was specific as it exhibited different sensitivities to a range of glucocorticoids and activation could be blocked with glucocorticoid receptor antagonists. Coexpression of the coactivator SRC-1a or P65 subunit of NF-kappa B with GR led to enhancement or repression, respectively. Taken together, these data suggest the reporter-based flow cytometry assay is an effective method for analyzing glucocorticoid receptor-mediated gene expression at the single cell level in living cells.     

Accurate flow cytometric membrane potential measurement in bacteria using diethyloxacarbocyanine and a ratiometric technique.

BACKGROUND: Membrane potential (MP) plays a critical role in bacterial physiology. Existing methods for MP estimation by flow cytometry are neither accurate nor precise, due in part to the heterogeneity of size of the particles analyzed. The ratio of a size- and MP-sensitive measurement, and an MP-independent, size-sensitive measurement, should provide a better estimate of MP. METHODS: Flow cytometry and spectrofluorometry were used to detect red (488 --> 600 nm) fluorescence associated with aggregates of diethyloxacarbocyanine (DiOC2(3)), which, in the monomeric state, is normally green (488 --> 530 nm) fluorescent. RESULTS: In bacteria incubated with 30 microM dye, aggregate formation increases with the magnitude of the interior-negative membrane potential. Green fluorescence from stained bacteria predominantly reflects particle size, and is relatively independent of MP, whereas red fluorescence is highly dependent on both MP and size. The ratio of red to green fluorescence provides a measure of MP that is largely independent of cell size, with a low coefficient of variation (CV). Calibration with valinomycin and potassium demonstrates that the method is accurate over the range from -50 mV through -120 mV; it also accurately tracks reversible reductions in MP produced by incubation at 4 degrees C and washing in glucose-free medium. CONCLUSIONS: The ratiometric technique for MP estimation using DiOC2(3) is substantially more accurate and precise than those previously available, and may be useful in studies of bacterial physiology and in investigations of the effects of antibiotics and other agents on microorganisms.