Comparison of fluorescence microscopy and solid-phase cytometry methods for counting bacteria in water

Total direct counts of bacterial abundance are central in assessing the biomass and bacteriological quality of water in ecological and industrial applications. Several factors have been identified that contribute to the variability in bacterial abundance counts when using fluorescent microscopy, the most significant of which is retaining an adequate number of cells per filter to ensure an acceptable level of statistical confidence in the resulting data. Previous studies that have assessed the components of total-direct-count methods that contribute to this variance have attempted to maintain a bacterial cell abundance value per filter of approximately 10(6) cells filter(-1). In this study we have established the lower limit for the number of bacterial cells per filter at which the statistical reliability of the abundance estimate is no longer acceptable. Our results indicate that when the numbers of bacterial cells per filter were progressively reduced below 10(5), the microscopic methods increasingly overestimated the true bacterial abundance (range, 15.0 to 99.3%). The solid-phase cytometer only slightly overestimated the true bacterial abundances and was more consistent over the same range of bacterial abundances per filter (range, 8.9 to 12.5%). The solid-phase cytometer method for conducting total direct counts of bacteria was less biased and performed significantly better than any of the microscope methods. It was also found that microscopic count data from counting 5 fields on three separate filters were statistically equivalent to data from counting 20 fields on a single filter.     

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.     

Rapid Quantification of the Toxic Alga Prymnesium parvum in Natural Samples by Use of a Specific Monoclonal Antibody and Solid-Phase Cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Internal Porosity of Mineral Coating Supports Microbial Activity in Rapid Sand Filters for Groundwater Treatment

A mineral coating develops on the filter grain surface when groundwater is treated via rapid sand filtration in drinking water production. The coating changes the physical and chemical properties of the filter material, but little is known about its effect on the activity, colonization, diversity, and abundance of microbiota. This study reveals that a mineral coating can positively affect the colonization and activity of microbial communities in rapid sand filters. To understand this effect, we investigated the abundance, spatial distribution, colonization, and diversity of all and of nitrifying prokaryotes in filter material with various degrees of mineral coating. We also examined the physical and chemical characteristics of the mineral coating. The amount of mineral coating correlated positively with the internal porosity, the packed bulk density, and the biologically available surface area of the filter material. The volumetric NH₄⁺ removal rate also increased with the degree of mineral coating. Consistently, bacterial 16S rRNA and amoA abundances positively correlated with increased mineral coating levels. Microbial colonization could be visualized mainly within the outer periphery (60.6 ± 35.6 μm) of the mineral coating, which had a thickness of up to 600 ± 51 μm. Environmental scanning electron microscopic (E-SEM) observations suggested an extracellular polymeric substance-rich matrix and submicron-sized bacterial cells. Nitrifier diversity profiles were similar irrespective of the degree of mineral coating, as indicated by pyrosequencing analysis. Overall, our results demonstrate that mineral coating positively affects microbial colonization and activity in rapid sand filters, most likely due to increased volumetric cell abundances facilitated by the large surface area of internal mineral porosity accessible for microbial colonization.     

Removal of Prymnesium parvum (Haptophyceae) cells under different nutrient conditions by clay

In this study, we have shown that the ichthyotoxic Prymnesium parvum (haptophyte) can be successfully removed by spraying the surface with a phosphatic clay/polyaluminium chloride (PAC) mix (final concentration, 4 g l⁻¹ and 5 ppm, respectively). The alga was grown in non-axenic batch cultures with nitrogen deficient, phosphorus deficient and nutrient sufficient media. Sub-samples of the nutrient sufficient culture were diluted to obtain cell abundances equal to those in the nutrient deficient cultures. Clay/PAC removed up to 100% of the cells in the low cell nutrient sufficient treatment after 72 h, but removal in the other treatments was lower (up to 84%). The nitrogen deficient cells were found to be the most toxic, measured as haemolytic activity of the cells (HE₅₀), just prior to the start of the experiment. However, the toxicity of the cells in all treatments was found to fluctuate during the incubation time with a general increase in toxicity towards the end, suggesting that the cells became stressed during sedimentation and/or when trapped in the clay. But the amount of released toxins was always below the detection limit of the haemolytic assay, and the abundance of free-living bacteria, derived from flow cytometer counts, increased throughout the experiment. This suggests that released toxins were either trapped by the clay particles or effectively degraded by the bacterial community. The study shows that the clay method can be efficient in mitigation of P. parvum blooms, but further studies have to be conducted where optimum clay concentrations are determined to ensure that the efficiency is high against nutrient deficient cells and at high cell abundances, i.e. conditions found during blooms in eutrophic coastal waters, and to determine the fate of the cells and their toxins during and after sedimentation.     

Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.     

Automated detection and enumeration for toxic algae by solid-phase cytometry and the introduction of a new probe for Prymnesium parvum (Haptophyta: Prymnesiophyceae)

Harmful algal blooms have a severe impact on aquaculture andfishery and can be caused by toxic haptophytes and dinoflagellates.Different toxic species, which are not easy to distinguish fromtheir morphologically similar and non-toxic relatives, occurin both groups. Sequencing of the large subunit ribosomal RNAof different strains and taxonomic relatives allowed the designof a probe specific to the toxic Prymnesium parvum spp. Forthe rapid detection and enumeration of Prymnesium and Alexandriumcells in cultures and environmental samples, respectively, protocolsfor fluorescence in situ hybridization were adapted for automateddetection by a solid-phase cytometer, the ChemScan. This cytometerenables the automated counting of fluorescently labelled cellson a membrane filter and subsequently a microscopic verificationof these results by the user, because the motorized stage ofthe microscope is driven to each positive signal by the computersoftware to localize that cell on the filter. With this fastdetection method, it was possible to detect, enumerate and verifymicroalgal cells on a filter, with a detection limit of onecell per membrane filter.     

Resistance to Co-Occurring Phages Enables Marine Synechococcus Communities To Coexist with Cyanophages Abundant in Seawater

Recent reports documenting very high viral abundances in seawater have led to increased interest in the role of viruses in aquatic environments and a resurgence of the hypothesis that viruses are significant agents of bacterial mortality. Synechococcus spp., small unicellular cyanobacteria that are important primary producers at the base of the marine food web, were used to assess this hypothesis. We isolated a diverse group of Synechococcus phages that at times reach titers of between 10³ and 10⁴ cyanophages per ml in both inshore and offshore waters. However, despite their diversity and abundance, we present evidence in support of the hypothesis that lytic phages have a negligible effect in regulating the densities of marine Synechococcus populations. Our results indicate that these bacterial communities are dominated by cells resistant to their co-occurring phages and that these viruses are maintained by scavenging on the relatively rare sensitive cells in these communities.     

Automated cerebrospinal fluid cytology: limitations and reasonable applications

OBJECTIVE: To evaluate the precision and clinical applicability of the Bayer ADVIA 120 cytometer (Bayer Healthcare, Fernwald, Germany) for cerebrospinal fluid (CSF) cell count and differentiation. STUDY DESIGN: One hundred six analyses of CSF from 98 patients by the ADVIA 120 were compared with routine cell count and microscopic differentiation. Correlation coefficients were calculated. RESULTS: In general, the total cell counts of both methods correlated well. The best correlations were seen at higher cell counts, > or = 100 cells per microliter with < 100 erythrocytes per microliter. The best correlations of cell differentiation were seen for lymphocytes and neutrophils, while the results for monocytes and eosinophils were less precise. In some cases, considerable differences between automated and microscopic cell counts and differentiation were seen that were relevant to clinical decision making. The detection of pathologic cell types, such as hemosiderophages, mitoses and neoplastic cells, was not provided by automated cytometry. CONCLUSION: When experienced personnel are not available, a preliminary cell count and differentiation between neutrophilic and lymphocytic reactions by automated cytometry may be valuable in allowing initial therapeutic decision making. Since the detection of pathologic cell types is not provided and the precision at low cell counts is only moderate, a personal microscopic evaluation of each sample is still indispensable to avoid misdiagnoses.     

Sampling Design and Enumeration Statistics for Bacteria Extracted from Marine Sediments

The spatial and temporal distributions of marine bacteria were studied at both a muddy and a sandy subtidal site in North Inlet, S.C. The sampling design was hierarchical, since subsampling (by a dilution series) of the sediments was necessary to count bacterial cells using acridine orange epifluoresence microscopy. The cell count data fit a log-normal distribution. The abundance of bacteria was 10¹¹ g⁻¹ (dry weight) of mud and 10⁹ g⁻¹ (dry weight) of sand. Variance component analyses demonstrated that variation due to the subsampling procedures was always statistically significant. Thus the common practice of counting 20 fields from one filter preparation is inadequate for estimating the true bacterial population variance in marine sediments. It is recommended that replication of the subsampling level be performed. Standardization of data (by dry weight of sediment) decreased sampling variance at the mud site but not at the sand site, implying that bacteria are more homogeneously distributed in sand than in mud.     

Rapid quantification of the toxic alga Prymnesium parvum in natural samples by use of a specific monoclonal antibody and solid-phase cytometry

The increasing incidence of harmful algal blooms around the world and their associated health and economic effects require the development of methods to rapidly and accurately detect and enumerate the target species. Here we describe use of a solid-phase cytometer to detect and enumerate the toxic alga Prymnesium parvum in natural samples, using a specific monoclonal antibody and indirect immunofluorescence. The immunoglobulin G antibody 16E4 exhibited narrow specificity in that it recognized several P. parvum strains and a Prymnesium nemamethecum strain but it did not cross-react with P. parvum strains from Scandinavia or any other algal strains, including species of the closely related genus Chrysochromulina. Prymnesium sp. cells labeled with 16E4 were readily detected by the solid-phase cytometer because of the large fluorescence signal and the signal/noise ratio. Immunofluorescence detection and enumeration of cultured P. parvum cells preserved with different fixatives showed that the highest cell counts were obtained when cells were fixed with either glutaraldehyde or formaldehyde plus the cell protectant Pluronic F-68, whereas the use of formaldehyde alone resulted in significantly lower counts. Immunofluorescence labeling and analysis with the solid-phase cytometer of fixed natural samples from a bloom of P. parvum occurring in Lake Colorado in Texas gave cell counts that were close to those obtained by the traditional method of counting using light microscopy. These results show that a solid-phase cytometer can be used to rapidly enumerate natural P. parvum cells and that it could be used to detect other toxic algae, with an appropriate antibody or DNA probe.     

Seasonal Changes of Bacterioplankton in a Reservoir Related to Algae. I. Numbers and Biomass

A reservoir for drinking water supply was studied at 10 day intervals for one season. Changes in bacterial microscopic counts and chlorophyll and algal biomass showed a similar pattern. Bacterial peaks usually lagged slightly behind the algal maxima. Average volumes of bacterial cells, corrected for shrinkage, were mostly lower than 0.2 μm³. The average concentration of bacterial C in water was estimated to be 18 μg μg 1 ⁻¹ (from a volume biomass of 0.09 mm³ per litre). Several years' fluctuations in bacterial numbers were compared with hydrological data and phyto- and zooplankton changes.     

Detection of bacterial contamination in starch and resin-based papermaking chemicals using fluorescence techniques

Rapid fluorescence techniques were evaluated for the detection of bacterial contaminants in papermaking chemicals including starch and the resin-based sizes and starch slurries used in the paper industry. Viable and non-viable bacterial cells were visualised by fluorescent probes and detected by epifluorescence microscopy and flow cytometry. The best discrimination ability was obtained with the fluorescent probes LIVE/DEAD and SYBR Green, based on the staining of cellular nucleic acid, and ChemChrome V3, which demonstrated cellular enzymatic activity. The process samples had to be diluted and filtered before fluorescence staining and analysis because they were viscous and contained solid particles. Fluorescence microscopic counts of bacteria in highly contaminated process samples were similar to plate counts, but flow cytometric enumeration of bacterial cells in process samples yielded 2- to 10-fold lower counts compared with plate counts, depending on the consistency of the sample. The detection limits in flow cytometric analysis and in epifluorescence microscopy were 10³–10⁶ cells ml⁻¹ and 10⁵–10⁶ cells ml⁻¹, respectively. Intrinsic bacterial contamination was detectable with fluorescence techniques and highly contaminated process samples could be analysed with fluorescence methods. Electronic Publication     

Impact of Mount St. Helens Eruption on Bacteriology of Lakes in the Blast Zone

Lakes lying within the blast zone of Mount St. Helens showed dramatic increases in heterotrophic bacterial numbers after the eruption of 18 May 1980. The total microscopic counts of bacteria in some of the most severely affected lakes were more than 10⁷ cells per ml, an order of magnitude above the counts in outlying control lakes. Likewise, the numbers of viable bacteria reached levels of more than 10⁶ cells per ml, compated with fewer than 10⁴ cells per ml in control lakes. The CPS medium used for enumeration provided growth of up to 81.5% of the bacteria during sampling of one of the blast zone lakes. The high numbers of bacteria and the efficacy of the viable enumeration procedure are evidence that the lakes have been transformed rapidly from oligotrophy to eutrophy due to the eruption and its aftermath. Organic material leached from the devastated forest vegetation is thought to be responsible for the enrichment of heterotrophs. Total coliform bacteria were found in all of the blast zone lakes, and some lakes contained fecal coliform bacteria. Klebsiella pneumoniae was the predominant total coliform and was also identified as one of the fecal coliform bacteria, although Escherichia coli was the predominant species in that category. Our data indicate that bacterial populations peaked in the outer blast zone lakes in the summer of 1980 and in most of the inner lakes during the summer of 1981.     

Dominant marine heterotrophic flagellates are adapted to natural planktonic bacterial abundances

Grazing controls bacterial abundances and composition in many ecosystems. In marine systems, heterotrophic flagellates (HFs) are important predators. Assemblages of HFs are primarily formed by species still uncultured; therefore, many aspects of their trophic behaviour are poorly known. Here, we assessed the functional response of the whole assemblage and of four taxa grown in an unamended seawater incubation. We used fluorescently labelled bacteria to create a prey gradient of two orders of magnitude in abundance and estimated ingestion rates. Natural HFs had a half-saturation constant of 6.7 × 10⁵ prey ml⁻¹, a value lower than that of cultured flagellates and within the range of marine planktonic bacterial abundances. Minorisa minuta was well adapted to low prey abundances and very efficient in ingesting bacteria. MAST-4 and MAST-7 were also well adapted to the typical marine abundances but less voracious. In contrast, Paraphysomonas imperforata, a typical cultured species, did not achieve ingestion rate saturation even at the highest prey concentration assayed. Our study, beside to set the basis for the fundamental differences between cultured and uncultured bacterial grazers, indicate that the examined predator taxa have different functional responses, suggesting that they occupy distinct ecological niches according to their grazing strategies and prey preferences.     

Marine bacterial community structure resilience to changes in protist predation under phytoplankton bloom conditions

To test whether protist grazing selectively affects the composition of aquatic bacterial communities, we combined high-throughput sequencing to determine bacterial community composition with analyses of grazing rates, protist and bacterial abundances and bacterial cell sizes and physiological states in a mesocosm experiment in which nutrients were added to stimulate a phytoplankton bloom. A large variability was observed in the abundances of bacteria (from 0.7 to 2.4 × 10⁶ cells per ml), heterotrophic nanoflagellates (from 0.063 to 2.7 × 10⁴ cells per ml) and ciliates (from 100 to 3000 cells per l) during the experiment (∼3-, 45- and 30-fold, respectively), as well as in bulk grazing rates (from 1 to 13 × 10⁶ bacteria per ml per day) and bacterial production (from 3 to 379 μg per C l per day) (1 and 2 orders of magnitude, respectively). However, these strong changes in predation pressure did not induce comparable responses in bacterial community composition, indicating that bacterial community structure was resilient to changes in protist predation pressure. Overall, our results indicate that peaks in protist predation (at least those associated with phytoplankton blooms) do not necessarily trigger substantial changes in the composition of coastal marine bacterioplankton communities.     

Is relative abundance a good indicator of population size? Evidence from fragmented populations of a specialist butterfly (Lepidoptera: Lycaenidae)

A common task for conservation biologists and ecologists is to establish how many individuals there are in a population, usually within a defined area of habitat. Estimates of both absolute and relative population sizes are widely used in many aspects of population conservation and management. Mark–recapture studies are appropriate for estimating the absolute population sizes of a wide range of animals, in both open and closed populations, while relative abundances can be estimated from a variety of survey methods. Relative abundances are often used in a comparative way to compare both population size and fluctuations in abundance. Here, we used transect counts and capture–recapture studies to estimate the relative abundances and population sizes of a specialist butterfly, Theclinesthes albocincta (Lycaenidae) in three habitat fragments, over two consecutive years. The sizes of the three populations differed significantly between sites and were highly variable between years. One population was extremely small and is likely to become locally extinct. We found that estimates of relative abundance were highly correlated with estimates of population size (r ² = 0.88, P = 0.017) derived from the open population models. The combination of transect counts and capture–recapture studies used in this study appears to be a very informative tool for the conservation and management of this butterfly species and could be extended to other insects.     

Strong small-scale temporal bacterial changes associated with the migrations of bloom-forming dinoflagellates

Bacterial abundances in nearshore Mediterranean planktonic environments tend to change seasonally by 10-fold. Strong daily changes in bacterial abundance, at least as large as seasonal range, occurred in the presence of large dinoflagellate populations performing daily vertical migrations. The daily variability of heterotrophic bacteria was associated with the daily migrations of a bloom of Alexandrium taylori in La Fosca Bay, and Gymnodinium impudicum in Barcelona harbor. Bacterial abundance in surface waters can change daily as much as from 1 × 10⁶ to 5 × 10⁶ with apparent net change rates of 0.24 h⁻¹. We suggest that the migrating dinoflagellates create microstructures exploited by the bacteria, and that the large algal populations (>10⁶ cells l⁻¹) make this microstructure visible with conventional sampling protocols. We also show evidence of the link between dinoflagellate abundance and relative bacterial activity in these waters, as measured by the percentage of bacteria with high nucleic acid content.     

Flow Cytometric Analysis of Marine Bacteria with Hoechst 33342

We investigated the accuracy and precision of flow cytometric (FCM) estimates of bacterial abundances using 4′, 6-diamidino-2-phenylindole (DAPI) and Hoechst 33342 (HO342, a bisbenzamide derivative) on paraformaldehyde-fixed seawater samples collected from two stations near Oahu, Hawaii. The accuracy of FCM estimates was assessed against direct counts by using epifluorescence microscopy. DAPI and HO342 differ in two aspects of their chemistry that make HO342 better suited for staining marine heterotrophic bacteria for FCM analysis. These differences are most important in studies of open-ocean ecosystems that require dual-beam FCM analysis to clearly separate heterotrophic bacterial populations from populations of photosynthetic Prochlorococcus spp. Bacterial populations were easier to distinguish from background fluorescence when stained with HO342 than when stained with DAPI, because HO342 has a higher relative fluorescence quantum yield. A substantially higher coefficient of variation of blue fluorescence, which was probably due to fluorescent complexes formed by DAPI with double-stranded RNA, was observed for DAPI-stained populations. FCM estimates averaged 2.0 and 12% higher than corresponding epifluorescence microscopy direct counts for HO342 and DAPI-stained samples, respectively. A paired-sample t test between FCM estimates and direct counts found no significant difference for HO342-stained samples but a significant difference for DAPI-stained samples. Coefficients of variation of replicate FCM abundance estimates ranged from 0.63 to 2.9% (average, 1.5%) for natural bacterial concentrations of 6 × 10⁵ to 15 × 10⁵ cells ml^-1.     

Bacterial population dynamics in a meromictic lake.

Polyclonal antibodies against nine different bacteria isolated from Lake Saelenvannet in western Norway were produced, and the population dynamics of these strains in the lake were monitored through two spring seasons by immunofluorescence staining. The total counts of bacteria varied over time and space from 1.5 x 10(6) to 1.5 x 10(7) cells ml-1. The counts of specific bacteria were in the range of 10(3) to 10(4) cells ml-1 or less; in sum, they generally made up less than 1% of the bacterial community. Some populations showed significant changes in abundance, with blooms lasting 1 to 3 weeks. The rate of change (increase and decrease) in abundance during blooms was estimated to be 0.2 to 0.6 day-1. The average virus-to-bacteria ratio was 50, and there was a significant correlation between the abundances of virus and bacteria. Both protozoan grazing and lytic virus infection were assessed as possible mechanisms driving the variations in bacterial population density.