Does the High Nucleic Acid Content of Individual Bacterial Cells Allow Us To Discriminate between Active Cells and Inactive Cells in Aquatic Systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Does the high nucleic acid content of individual bacterial cells allow us to discriminate between active cells and inactive cells in aquatic systems?

The nucleic acid contents of individual bacterial cells as determined with three different nucleic acid-specific fluorescent dyes (SYBR I, SYBR II, and SYTO 13) and flow cytometry were compared for different seawater samples. Similar fluorescence patterns were observed, and bacteria with high apparent nucleic acid contents (HNA) could be discriminated from bacteria with low nucleic acid contents (LNA). The best discrimination between HNA and LNA cells was found when cells were stained with SYBR II. Bacteria in different water samples collected from seven freshwater, brackish water, and seawater ecosystems were prelabeled with tritiated leucine and then stained with SYBR II. After labeling and staining, HNA, LNA, and total cells were sorted by flow cytometry, and the specific activity of each cellular category was determined from leucine incorporation rates. The HNA cells were responsible for most of the total bacterial production, and the specific activities of cells in the HNA population varied between samples by a factor of seven. We suggest that nucleic acid content alone can be a better indicator of the fraction of growing cells than total counts and that this approach should be combined with other fluorescent physiological probes to improve detection of the most active cells in aquatic systems.     

Spatio-Temporal Variations of High and Low Nucleic Acid Content Bacteria in an Exorheic River

Bacteria with high nucleic acid (HNA) and low nucleic acid (LNA) content are commonly observed in aquatic environments. To date, limited knowledge is available on their temporal and spatial variations in freshwater environments. Here an investigation of HNA and LNA bacterial abundance and their flow cytometric characteristics was conducted in an exorheic river (Haihe River, Northern China) over a one year period covering September (autumn) 2011, December (winter) 2011, April (spring) 2012, and July (summer) 2012. The results showed that LNA and HNA bacteria contributed similarly to the total bacterial abundance on both the spatial and temporal scale. The variability of HNA on abundance, fluorescence intensity (FL1) and side scatter (SSC) were more sensitive to environmental factors than that of LNA bacteria. Meanwhile, the relative distance of SSC between HNA and LNA was more variable than that of FL1. Multivariate analysis further demonstrated that the influence of geographical distance (reflected by the salinity gradient along river to ocean) and temporal changes (as temperature variation due to seasonal succession) on the patterns of LNA and HNA were stronger than the effects of nutrient conditions. Furthermore, the results demonstrated that the distribution of LNA and HNA bacteria, including the abundance, FL1 and SSC, was controlled by different variables. The results suggested that LNA and HNA bacteria might play different ecological roles in the exorheic river.     

Community analysis of high- and low-nucleic acid-containing bacteria in NW Mediterranean coastal waters using 16S rDNA pyrosequencing

The ecological significance of the marine bacterial populations distinguishable by flow cytometry on the basis of the fluorescence (FL) of their nucleic acid (NA) content and proxies of cell size (such as side scatter, SSC) remains largely unknown. Some studies have suggested that cells with high NA (HNA) content and high SSC (HS) represent the active members of the community, while the low NA (LNA) cells are inactive members of the same phylogenetic groups. But group-specific activity measurements and phylogenetic assignment after cell sorting have suggested this is not be the case, particularly in open-ocean communities. To test the extent to which the different NA subgroups are similar, and consequently the extent to which they likely have similar ecological and biogeochemical roles in the environment, we analysed the phylogenetic composition of three populations after cell sorting [high NA-high SC (HNA-HS), high NA-low SC (HNA-LS), low NA (LNA)] by 454 pyrosequencing in two contrasting periods of the year in NW Mediterranean coastal waters (BBMO, Blanes Bay Microbial Observatory) where these three populations have recurrent seasonal patterns. Statistical analyses showed that summer and winter samples were significantly different and, importantly, the sorted populations within a sample were composed of different taxa. The majority of taxa were associated with one NA fraction only, and the degree of overlap (i.e. OTUs present simultaneously in 2 fractions) between HNA and LNA and between summer and winter communities was very small. Rhodobacterales, SAR116 and Bacteroidetes contributed primarily to the HNA fraction, whereas other groups such as SAR11 and SAR86 contributed largely to the LNA fractions. Gammaproteobacteria other than SAR86 showed less preference for one particular NA fraction. An increase in diversity was observed from the LNA to the HNA-HS fraction for both sample dates. Our results suggest that, in Blanes Bay, flow cytometric signatures of natural communities track their phylogenetic composition.     

Phylogenetic characterisation of picoplanktonic populations with high and low nucleic acid content in the North Atlantic Ocean

In flow cytometric analyses of marine prokaryotic picoplankton often two populations with distinct differences in their apparent nucleic acid content are discernable, one with a high and one with a low nucleic acid content (HNA and LNA, respectively). In this study we determined the phylogenetic composition of flow cytometrically sorted HNA and LNA populations, collected at six stations along a transect across three oceanic provinces from Iceland to the Azores. Catalysed reporter deposition fluorescence in situ hybridisation (CARD-FISH) analysis of sorted cells revealed distinct differences in phylogenetic composition between the LNA and HNA populations with only little overlap. At all stations the LNA population was dominated by the alphaproteobacterial clade SAR11 (45–74%). Also, Betaproteobacteria were always present at 2–4%. While the LNA composition was rather stable, the HNA populations were composed of distinct phylogenetic clades in the different oceanic provinces of Arctic and Tropics. For example Cyanobacteria dominated the North Atlantic Gyre HNA population (29–44%) with Prochlorococcus as the major clade (34–44%), but were low in Arctic and Polar waters (1% and 5%, respectively). In contrast, Bacteroidetes accounted for the majority of HNA cells in the Polar and Arctic province (26% and 32%, respectively), but were low in the Gyre region (3–10%). The DNA content of the HNA population was about 3.5 times higher than that of the LNA populations. This reflects differences in the genome sizes of closely related cultured representatives of HNA clades (3–6 Mbp) and LNA clades (1.3–1.5 Mbp).     

Antarctic marine bacterioplankton subpopulations discriminated by their apparent content of nucleic acids differ in their response to ecological factors

Bacterial abundances determined in Drake Passage and Bransfield and Gerlache Straits (Antarctica) in the Austral summer ranged from 0.78 to 9.4×10⁵ cells ml⁻¹, and were positively correlated with standing stocks of Chl a. Two bacterial subpopulations were discriminated based in their different levels of green fluorescence and wide angle light scatter (SSC) per cell after SYTO-13 staining for the first time in Antarctic waters. High nucleic acid (HNA) and low nucleic acid (LNA) subpopulations differed considerably in their response to changes in environmental variables. The apparent content of nucleic acids per cell for the HNA subpopulation (FL1-HNA) showed vertical profiles similar to those of Chl a, including the presence of a maximum at the subsurface chlorophyll maximum. FL1-HNA was positively correlated with Chl a. No similar trends were observed for the LNA fraction. HNA and LNA subpopulations differed in the response of the wide angle light scatter signal to environmental factors as well. SSC-HNA decreased strongly with depth and was positively correlated with Chl a. Again, no similar trends were observed for the LNA subpopulation. The percentage of HNA cells (%HNA) ranged between 35.0 and 76.7% and showed a general tendency to increase with depth. This increase seemed to be larger when the stratification of the water column was higher. Differences in grazing pressure could be responsible of the unexpected vertical distribution of HNA cells. Our results shows that in situ LNA and HNA bacterioplankton subpopulations are under different ecological controls and likely to play different trophodynamic roles in Antarctic waters.     

Linking Host Prokaryotic Physiology to Viral Lifestyle Dynamics in a Temperate Freshwater Lake (Lake Pavin,France)

In aquatic ecosystems, fluctuations in environmental conditions and prokaryotic host physiological states can strongly affect the dynamics of viral life strategies. The influence of prokaryote physiology and environmental factors on viral replication cycles (lytic and lysogeny) was investigated from April to September 2011 at three different strata (epi, meta, and hypolimnion) in the mixolimnion of deep volcanic temperate freshwater Lake Pavin (France). Overall, the euphotic region (epi and metalimnion) was more dynamic and showed significant variation in microbial standing stocks, prokaryotic physiological state, and viral life strategies compared to the aphotic hypolimnion which was stable within sampled months. The prokaryotic host physiology as inferred from the nucleic acid content of prokaryotic cells (high or low nucleic acid) was strongly regulated by the chlorophyll concentration. The predominance of the high nucleic acid (HNA) prokaryotes (cells) over low nucleic acid (LNA) prokaryotes (cells) in the spring (HNA/LNA?=?1.2) and vice versa in the summer period (HNA/LNA?=?0.4) suggest that the natural prokaryotic communities underwent major shifts in their physiological states during investigated time period. The increase in the percentage of inducible lysogenic prokaryotes in the summer period was associated with the switch in the dominance of LNA over HNA cells, which coincided with the periods of strong resource (nutrient) limitation. This supports the idea that lysogeny represents a maintenance strategy for viruses in unproductive or harsh nutrient/host conditions. A negative correlation of percentage of lysogenic prokaryotes with HNA cell abundance and chlorophyll suggest that lysogenic cycle is closely related to prokaryotic cells which are stressed or starved due to unavailability of resources for its growth and activity. Our results provide support to previous findings that changes in prokaryote physiology are critical for the promotion and establishment of lysogeny in aquatic ecosystems, which are prone to constant environmental fluctuations.     

A single-cell analysis of virioplankton adsorption, infection, and intracellular abundance in different bacterioplankton physiologic categories

Culture studies of phage–host systems have shown that phage proliferation strongly depends on the physiological state of the host, but it is still unclear to what extent this holds true within aquatic ecosystems. We used a combination of flow sorting and electron microscopy to explore how the frequency of bacterial cells with attached viruses (FCAV), of visibly infected cells, and the number of intracellular viruses are distributed within five physiologic categories: cells with high (HNA) and low (LNA) nucleic acid content, with a compromised membrane, in division, and with an intact-looking morphology. FCAV was not different between the cellular physiologic categories, suggesting low influence of host physiology on viral adsorption. Infected cells were found within all the physiologic categories, besides the dividing cells, but showed different levels of new virion production, with the abundance of intracellular viruses ranked as follows: HNA > intact-looking cells > LNA > compromised membrane cells. These results favor the physiological control hypothesis of viral progeny production. The calculation of viral production rate of the HNA and LNA cells show that viral infection of HNA cells likely accounts for the majority of viral production. It also show that cells considered as less active can still act as resources for phages, although they contain much less intracellular phage particles.     

Comparison of cellular and biomass specific activities of dominant bacterioplankton groups in stratified waters of the Celtic Sea.

A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [(35)S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of alpha-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the alpha-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of gamma-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production: different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.     

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [³H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.     

Human bone marrow CFU-GM and BFU-E localized by light scatter cell sorting

Flow cytometric separation was performed on the normal human bone marrow (BM) by using the low-angle (0 degrees) or high-angle (90 degrees) light scatter. Four distinct subpopulations of cells can be enriched from normal human BM and these fractions were subsequently evaluated for their morphological properties as well as their clonogenic capacity in various progenitor cell assays. Our results indicate that human erythroid and granulocyte-macrophage progenitor cells can be separated from BM low-density cells by cell sorting, and these cells show similar 0 degrees and 90 degrees light scatter properties to those observed with murine bone marrow studies. Flow cytometric analysis also suggests that the majority of sorted BFU-E and CFU-GM resides in the blast cell subset of human BM mononuclear cells.     

Flow cytometry and phytoplankton

Flow cytometry and sorting are now an important technology in aquatic research. Simultaneous measurements of individual particle cell size, fluorescence, and light scatter properties are directly applicable to current topics in aquatic research. Flow sorting may be employed to obtain subsets of cells for analysis by conventional methods. The manner in which rapid, precise measurements of single cells are made is complex, and the application of this technology to aquatic samples is subject to many analytical constraints. Flow cytometric measurements of algal cell size and pigment autofluorescence are relative and are therefore dependent on the optical configuration and variability of the instrument. Specific types of reference materials are used to establish the validity of analyses: 1) instrument standards, 2) fluorescence controls, and 3) internal stain standards. The selection and application of standards and controls are discussed in the context of allometric (cell size versus pigment fluorescence) and ataxonomic (pigment color groups) methods. The widespread acceptance of particular reference materials among research groups will result in comparable data sets describing aquatic particle distributions.     

Comparison of Cellular and Biomass Specific Activities of Dominant Bacterioplankton Groups in Stratified Waters of the Celtic Sea

A flow-sorting technique was developed to determine unperturbed metabolic activities of phylogenetically characterized bacterioplankton groups with incorporation rates of [³⁵S]methionine tracer. According to fluorescence in situ hybridization with rRNA targeted oligonucleotide probes, a clade of α-proteobacteria, related to Roseobacter spp., and a Cytophaga-Flavobacterium cluster dominated the different groups. Cytometric characterization revealed both these groups to have high DNA (HNA) content, while the α-proteobacteria exhibited high light scatter (hs) and the Cytophaga-Flavobacterium cluster exhibited low light scatter (ls). A third abundant group with low DNA (LNA) content contained cells from a SAR86 cluster of γ-proteobacteria. Cellular specific activities of the HNA-hs group were 4- and 1.7-fold higher than the activities in the HNA-ls and LNA groups, respectively. However, the higher cellular protein synthesis by the HNA-hs could simply be explained by their maintenance of a larger cellular protein biomass. Similar biomass specific activities of the different groups strongly support the main assumption that underlies the determination of bacterial production: different bacteria in a complex community incorporate amino acids at a rate proportional to their protein synthesis. The fact that the highest growth-specific rates were determined for the smallest cells of the LNA group can explain the dominance of this group in nutrient-limited waters. The metabolic activities of the three groups accounted for almost the total bacterioplankton activity, indicating their key biogeochemical role in the planktonic ecosystem of the Celtic Sea.     

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.     

Identification of inflammatory cells in bovine milk by flow cytometry

Cells recovered from normal or mastitic bovine milk were examined by flow cytometry. All milk samples contained particulate material that was heterogeneous in size and that produced a right-angle light-scatter signal equal to or greater than that produced by human or bovine neutrophils. Although this material labeled with Hoechst 33342, it produced fluorescence intensities below that of intact bovine cells, suggesting that it consisted of cell fragments. Mastitic milk additionally contained other populations of cells that were poorly resolved from the normal particulate material by size (electronic volume sensor) and right-angle light scatter. In order to improve this resolution, the milk cells were incubated with carboxydimethylfluorescein diacetate (CMFDA) to label intact cells. When milk samples labeled with CMFDA were examined by dual-parameter analysis using green fluorescence and right-angle light scatter, five or more populations of cells could be identified in mastitic milk. These populations included intact and degenerate neutrophils, lymphocytes, including both small and activated cells, monocytes, and large activated macrophages containing many vacuoles and phagocytosed particles. Using this procedure, all the animals in the University of Nevada-Reno Holstein dairy herd were tested once a month for 6 months. In addition, individual animals with mastitis were examined one or more times each day during the course of the inflammatory process. In the routine screening, the flow cytometric examination detected mastitis before overt symptoms developed. In cows identified to have mastitis, the flow cytometric examination provided prognostic information regarding the success of treatments.     

Effects of mismatched refractive indices in aquatic flow cytometry.

BACKGROUND: Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural waters. The refractive index of water samples from aquatic environments can differ because of a variety of factors such as dissolved organic content, aldehyde preservative, sample salinity, and temperature. In flow cytometric analyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We measured the effect of this mismatch on cell size measurements. METHODS: We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity ratio. The effects were characterized for standard microspheres, cultured phytoplankton cells of different sizes, and natural populations from an estuarine river. RESULTS: We found that the distorted scatter signals resulted in an increase in the apparent size of small cells (1--2 microm) by a factor of 4.5 times. Cells in the size range of 3--5 microm were less affected by the salinity differences, and cells larger than 5 microm were not affected. Chlorophyll and phycoerythrin fluorescences and 90 degrees light scatter signals were not changed by sheath and sample salinity differences. CONCLUSIONS: Care must be taken to ensure that the sheath and sample refractive index are matched when using forward light scatter to measure cell size spectra, especially in estuarine studies, where salinity can vary greatly. Of the factors considered that can change the sample refractive index, salinity gradients in an estuary cause the largest index mismatch and, consequently, the largest error in scatter.     

Correlated analysis of cellular DNA, membrane antigens and light scatter of human lymphoid cells

Flow cytometric correlated analysis of membrane antigens, DNA, and light scatter was performed on human lymphoid cells using fluorescein (FITC)-conjugated antibodies to label B- and T-cell antigens and propidium iodide (PI) to stain DNA after ethanol fixation and RNase treatment. A FACS II flow cytometer was modified to obtain digitized measurements of two color fluorescence and light scatter emissions, simultaneously. Software was written to allow single parameter analysis or correlated analysis of any two of the three parameters acquired. Ethanol fixation preserved FITC surface labeling for at least 15 weeks, but produced marked changes in light scatter. No changes in FITC distributions were observed after RNase treatment and PI staining, and the presence of FITC labeling did not affect DNA distributions. Within heterogeneous cell populations, the DNA distribution of cell subpopulations identified by a membrane antigen was clearly demonstrated.     

Multivariate analysis and list mode processing of murine hemopoietic subpopulations for cytokinetic studies

Multivariate analyses and list mode data processing were used to obtain cytokinetic information on flow cytometrically distinct hemopoietic subpopulations. In one application, viable, unfixed hemopoietic subpopulations were discriminated on the basis of cyanine dye fluorescence and orthogonal light scatter; Hoechst dye fluorescence was measured to determine the proliferative status of the subpopulations. In another application, ethanol-fixed mouse bone marrow cells were triply stained with Hoechst dye, rhodamine-conjugated wheat germ agglutinin (WGA), and a fluorescein-labeled monoclonal antibody against bromodeoxyuridine. In both of these studies, flow cytometric data for all three variables were acquired in list mode fashion, stored on magnetic tape, and processed by list mode software on a computer-based multivariable pulse-height analyzer. In the first application, subpopulations distinguished by cyanine dye intensity and light scatter appeared to be more related to cell lineage and cell size than proliferative status. In the second application, WGA affinity discriminated two subpopulations in mouse bone marrow S-phase cells in each subpopulation that actively incorporated bromodeoxyuridine (BrdUrd). List mode data processing obviates the need for routine electronic sorting of cells and thus facilitates characterization of discriminated subpopulations. In this regard, it is particularly useful for the study of flow cytometrically distinct, low frequency subpopulations.     

深圳近海表层浮游细菌分布特征及其环境影响因素

于2015年3月、5月、8月和10月在深圳市近岸海域(珠江口、深圳湾和大亚湾)采集表层水样,利用流式细胞仪测定总浮游细菌、高DNA含量亚群细菌(HNA)、低DNA含量亚群细菌(LNA)的丰度,分析它们的时空分布特点,阐释环境因子对浮游细菌时空分布格局的影响。结果表明,珠江口、深圳湾和大亚湾海域表层浮游细菌的平均丰度依次降低,分别为3.82×10~6个/mL、7.67×10~6个/mL和3.38×10~6个/mL。珠江口海域浮游细菌丰度由远岸到近岸递增,深圳湾海域湾内各站位浮游细菌丰度差异较小,大亚湾海域浮游细菌丰度空间差异不显著(P0.05)。浮游细菌丰度时间差异主要受温度影响,空间差异主要受营养盐和叶绿素a影响。HNA亚群丰度时空差异性比LNA亚群的大,HNA亚群受温度影响显著(P0.01),而LNA亚群与温度相关性不显著(P0.05)。环境对HNA和LNA亚群丰度的影响有许多相似之处,但两者对某些环境因子有着不同的响应,说明它们在近海表层生态系统中可能扮演着部分重叠但略有不同的角色。