Flow cytometry (FCM) is used for analyzing and counting particles by suspending them in a flow stream passing through an excitation light source like a laser beam. Interaction between the light beam and the particle causes characteristic scattering of light and the excitation of fluorochromes if present in the particles. The scatter signal is divided in “forward scatter (FSC)”, or small angle signal, measuring light diverted at a low angle (0.5 – 5°), and “sideward scatter (SSC)”, or large angle signal, measuring light collected at angles greater than 15°. The angle of scattered light provides information on the nature of particles including surface and intracellular characteristics. The cell suspension may be stained using fluorophore dyes that can be design to attach to particular molecules such as DNA, RNA, protein, or antibodies or by using fluorescent oligonucleotides probe.
Histograms with two variables (e.g. light scattering versus fluorescence, fluorescence versus counts) or contour plots may be generated from scattered light, which provide information on structural properties of the analyzed cell populations (58,59).
1.3.1 Application of flow cytometry in aquatic microbiology
Already applied for more than 30 years in medical research and routine diagnosis/analysis, FCM has been increasingly used also in the field of aquatic microbiology during the last three decades.
Since less than 1% of microbial species present in the environments are cultivable on solid media, microscopy is the routine technique for detection and quantification of bacteria, especially in aquatic samples. FCM is a powerful tool for counting of microorganisms and in combination with various nucleic acid stains (SYTO 9, SYTO 13, SYBR Green) it has been used for quantification of microbial communities in various aquatic environments (60). FCM procedures have been extensively applied and are now an established and accepted approach for the quantification of total bacterial abundance in drinking water distribution systems and
wastewater (61–64). Moreover, it has been demonstrated that the total cell number is a good indicator of water quality and that it may change during treatment of waste water usings sand filtration, granular activated carbon, and ozonation. Disinfection treatments of waters using chemical oxidants may differ in selectivity and reactivity and thus the efficiency of bacterial disinfection processes might vary. FCM can be applied to evaluate both the cellular activity and the physiological state of microbial cells. For instance, it allows differentiating between living and dead cells. The combination of propidium iodide (PI) and SYBR Green stains is generally used to monitor membrane integrity and, consequently, cell viability (58,61). In combination with nucleic acid staining FCM can be applied to separately count bacteria from low nucleic acid (LNA) and high nucleic acid (HNA)- containing bacteria. The discrimination between these two groups is based on differences in fluorescence intensity that in turn is related to the nucleic acid content (60,65). Clusters of low-DNA and high-DNA bacteria are widespread in nearly all aquatic samples, from marine, brackish and freshwater environments.
1.3.2 Application flow cytometry for research on phototrophic organisms
The natural cellular pigmentation of phototrophic bacteria can be used as a signal to discriminate phototrophic and heterotrophic microorganisms. Chlorophyll (found naturally in all phytoplankton cells) and phycobilin (found in cyanobacteria) are natural fluorophores showing characteristic optical wavelength excitation and emission profiles. The fluorescence of chlorophyll can thus be used as the primary gating factor to discriminate phytoplankton from other particles. Chlorophyll a is the dominant photosynthetic pigment in most phytoplankton species and its excitation with blue laser light (488-nm) results in a measurable fluorescent signal in the long red wavelength (> 640 nm) (Fig. 5).
Fig. 5 Natural fluorescence of bacteriochlorophyll in the long red wavelengths (FL3, red filter > 640 nm).
Histogram without phototrophic sulfur microorganisms (left), and after addition of phototrophic sulfur bacteria (right). Threshold for bacteriochlorphyll autofluorescence signal corresponded to FL3 > 1’000. The vertical red line separates the non-auto-fluorescent cells (left, from 101 to 103) from the phototrophic auto-fluorescent cells (right, from 103 to 107). Note the large difference in y-axis range between the two panels.
Natural fluorophores provide the great advantage in that they allow detection, discrimination, morphological analysis and quantification of microorganisms using FCM without the addition of dyes such as SYBR Green (66). FCM can thus be used to analyze phytoplankton from freshly collected environmental water samples by measuring relative cell size and intrinsic fluorescence profiles (67,68). Other accessory pigments in phytoplankton are the phycobilins and the carotenoids. Phycocyanins are a class of phycobilins present in all species of phototrophic cyanobacteria and have been widely used for detection of cyanobacteria. The red FCM (640 nm) maximally excites phycocyanins near their absorption maximum producing strong fluorescence emissions detected at 675 nm (66).
Due to these developments, FCM has become an established method for phytoplankton studies (67–69). The main advantage of this technique lays in the processing of samples at a high rates, allowing the analysis of a large number of individuals. Moreover, each analyzed cell is
characterized by several parameters, such as size, light scatter, and fluorescence emission, which allows estimates of the relative abundance of cells belonging to several groups. These approaches were used, for instance, for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content and for estimating the relative activity and physiological state of each cell (68).
The potential of FCM as a fast tool for the characterization and counting of phototrophic sulfur bacteria has documented in the past. Purple and green sulfur bacteria from both laboratory strains and from environmental samples obtained from the stratified meromictic Lake Vilar (Spain) were detected and counted in unstained samples using a blue laser-based FCM (70).
Moreover, variations in cell-specific pigment content and the dynamics of sulfur accumulation were also quantified using FCM as sulfur accumulation changes the light scatter characteristic of the phototrophic cells. Therefore, the rapid identification and the physiological characterization provided by FCM could be applied in complex ecophysiological experiments in natural environments.
In detailed studies of bacterial populations analysis, FCM combined with flow cell sorting (also known as fluorescence-activated cell sorting – FACS) has been applied to concentrate and count subpopulations from complex bacterial communities (71,72). FCM has also been combined with radioactive or stable isotope incubations of the sorted sub-populations in order to measure the metabolic activity of specific functional groups (73). In a recent study, samples from Lake Cadagno incubated with 15N2 and 13CO2 were flow cell sorted using gating criteria based on the auto-fluorescence signature of the green sulfur bacterium Chlorobium phaeobacteroides. The sorted cells were subsequently transferred to a filter membrane for NanoSIMS analysis (54).