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 10⁶ 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 10⁵, 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.     

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.     

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.     

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.     

Detection of mRNA in flow-sorted cells

Cells were sorted onto nitrocellulose filters which were saturated with a lysing cocktail designed to preferentially immobilize cellular mRNA. After washing, these filters were incubated with 32P-labeled specific DNA probes. We used the phorbol ester/lipopolysaccharide (PMA + LPS) co-induction of IL-1 mRNA and CD13 expression in U937 cells to demonstrate the specificity of the technique. In addition we used the abundant expression of c-fos in U937 to demonstrate linearity. IL-1 beta mRNA is readily discernable autoradiographically from as few as 5,000 PMA + LPS-induced cells sorted onto a filter. With liquid scintillation counting we demonstrate good linearity of the c-fos quantitation over the range of 1,000 cells to 60,000 cells per filter target. The technique is easily adapted to any sorting flow cytometer and should prove useful to help correlate any flow cytometric cell phenotype with specific mRNA abundance.     

Are we missing half of the viruses in the ocean?

Viruses are abundant in the ocean and a major driving force in plankton ecology and evolution. It has been assumed that most of the viruses in seawater contain DNA and infect bacteria, but RNA-containing viruses in the ocean, which almost exclusively infect eukaryotes, have never been quantified. We compared the total mass of RNA and DNA in the viral fraction harvested from seawater and using data on the mass of nucleic acid per RNA- or DNA-containing virion, estimated the abundances of each. Our data suggest that the abundance of RNA viruses rivaled or exceeded that of DNA viruses in samples of coastal seawater. The dominant RNA viruses in the samples were marine picorna-like viruses, which have small genomes and are at or below the detection limit of common fluorescence-based counting methods. If our results are typical, this means that counts of viruses and the rate measurements that depend on them, such as viral production, are significantly underestimated by current practices. As these RNA viruses infect eukaryotes, our data imply that protists contribute more to marine viral dynamics than one might expect based on their relatively low abundance. This conclusion is a departure from the prevailing view of viruses in the ocean, but is consistent with earlier theoretical predictions.     

Comparative study of the abundance of various bacterial morphotypes in an eutrophic freshwater environment determined by AODC and TEM

Transmission electron microscopy (TEM) and epifluorescence microscopy were used to obtain comparative measurements of total bacterial counts, and to enumerate abundances of various bacterial morphotypes in an eutrophic freshwater habitat. Although particulate matter would have been expected to interfere with counting by obscuring large areas of the electron microscope grids, estimates of total bacterial abundance made by TEM were on average 1.2 times greater than those obtained using the acridine orange direct counting method (AODC). However, the precision of the AODC method was greater than that for TEM, with a coefficient of variation (C.V.) of 4.0% versus 8.8%, respectively. The total bacterial abundance ranged from 1.1 to 3.2 x 10(6) ml(-1). As was the case for total bacterial density, the numbers of rod- and vibrio-shaped cells were lower when counted in the epifluorescence microscope, indicating the presence of potential starvation forms or ultramicrobacteria. Greatest variations in counts made by TEM and AODC were found for filamentous and coccoid bacteria. Counts of filamentous bacteria made by AODC were only about half of those detected by TEM. In contrast, cocci were on average 1.5 times greater when counted by AODC compared to TEM estimates. Both counting differences were probably caused by the morphology and low density of filamentous and coccoid bacteria (1.7 and 1.4 x 10(5) ml(-1), respectively), which led to an uneven distribution on polycarbonate filters as well as on electron microscope grids. Besides, cocci might easily be mistaken for large viral particles when counted by AODC. Hence, the study supports the use of TEM over AODC for obtaining accurate estimates of total bacterial abundance and especially bacterial morphotypes in natural waters.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy.

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Detection, enumeration, and sizing of planktonic bacteria by image-analyzed epifluorescence microscopy

Epifluorescence microscopy is now being widely used to characterize planktonic procaryote populations. The tedium and subjectivity of visual enumeration and sizing have been largely alleviated by our use of an image analysis system consisting of a modified Artek 810 image analyzer and an Olympus BHT-F epifluorescence microscope. This system digitizes the video image of autofluorescing or fluorochrome-stained cells in a microscope field. The digitized image can then be stored, edited, and analyzed for total count or individual cell size and shape parameters. Results can be printed as raw data, statistical summaries, or histograms. By using a stain concentration of 5 micrograms of 4'6-diamidino-2-phenylindole per ml of sample and the optimal sensitivity level and mode, counts by image analysis of natural bacterial populations from a variety of habitats were found to be statistically equal to standard visual counts. Although the time required to prepare slides, focus, and change fields is the same for visual and image analysis methods, the time and effort required for counting is eliminated since image analysis is instantaneous. The system has been satisfactorily tested at sea. Histograms of cell silhouette areas indicate that rapid and accurate estimates of bacterial biovolume and biomass will be possible with this system.     

Automated enumeration of groups of marine picoplankton after fluorescence in situ hybridization

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.     

Automated Enumeration of Groups of Marine Picoplankton after Fluorescence In Situ Hybridization

We describe here an automated system for the counting of multiple samples of double-stained microbial cells on sections of membrane filters. The application integrates an epifluorescence microscope equipped with motorized z-axis drive, shutters, and filter wheels with a scanning stage, a digital camera, and image analysis software. The relative abundances of specific microbial taxa are quantified in samples of marine picoplankton, as detected by fluorescence in situ hybridization (FISH) and catalyzed reporter deposition. Pairs of microscopic images are automatically acquired from numerous positions at two wavelengths, and microbial cells with both general DNA and FISH staining are counted after object edge detection and signal-to-background ratio thresholding. Microscopic fields that are inappropriate for cell counting are automatically excluded prior to measurements. Two nested walk paths guide the device across a series of triangular preparations until a user-defined number of total cells has been analyzed per sample. A backup autofocusing routine at incident light allows automated refocusing between individual samples and can reestablish the focal plane after fatal focusing errors at epifluorescence illumination. The system was calibrated to produce relative abundances of FISH-stained cells in North Sea samples that were comparable to results obtained by manual evaluation. Up to 28 preparations could be analyzed within 4 h without operator interference. The device was subsequently applied for the counting of different microbial populations in incubation series of North Sea waters. Automated digital microscopy greatly facilitates the processing of numerous FISH-stained samples and might thus open new perspectives for bacterioplankton population ecology.     

A Model-Based Approach for Species Abundance Quantification Based on Shotgun Metagenomic Data

The human microbiome, which includes the collective microbes residing in or on the human body, has a profound influence on the human health. DNA sequencing technology has made the large-scale human microbiome studies possible by using shotgun metagenomic sequencing. One important aspect of data analysis of such metagenomic data is to quantify the bacterial abundances based on the metagenomic sequencing data. Existing methods almost always quantify such abundances one sample at a time, which ignore certain systematic differences in read coverage along the genomes due to GC contents, copy number variation and the bacterial origin of replication. In order to account for such differences in read counts, we propose a multi-sample Poisson model to quantify microbial abundances based on read counts that are assigned to species-specific taxonomic markers. Our model takes into account the marker-specific effects when normalizing the sequencing count data in order to obtain more accurate quantification of the species abundances. Compared to currently available methods on simulated data and real data sets, our method has demonstrated an improved accuracy in bacterial abundance quantification, which leads to more biologically interesting results from downstream data analysis.     

Rapid,Precise, and Accurate Counts of Symbiodinium Cells Using the Guava Flow Cytometer,and a Comparison to Other Methods

In studies of both the establishment and breakdown of cnidarian-dinoflagellate symbiosis, it is often necessary to determine the number of Symbiodinium cells relative to the quantity of host tissue. Ideally, the methods used should be rapid, precise, and accurate. In this study, we systematically evaluated methods for sample preparation and storage and the counting of algal cells using the hemocytometer, a custom image-analysis program for automated counting of the fluorescent algal cells, the Coulter Counter, or the Millipore Guava flow-cytometer. We found that although other methods may have value in particular applications, for most purposes, the Guava flow cytometer provided by far the best combination of precision, accuracy, and efficient use of investigator time (due to the instrument''s automated sample handling), while also allowing counts of algal numbers over a wide range and in small volumes of tissue homogenate. We also found that either of two assays of total homogenate protein provided a precise and seemingly accurate basis for normalization of algal counts to the total amount of holobiont tissue.     

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.     

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10(2) to 10(7) cfu/ml. Cytometric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.     

Toward Reliable Estimates of Abundance: Comparing Index Methods to Assess the Abundance of a Mammalian Predator

Due to time and financial constraints indices are often used to obtain landscape-scale estimates of relative species abundance. Using two different field methods and comparing the results can help to detect possible bias or a non monotonic relationship between the index and the true abundance, providing more reliable results. We used data obtained from camera traps and feces counts to independently estimate relative abundance of red foxes in the Black Forest, a forested landscape in southern Germany. Applying negative binomial regression models, we identified landscape parameters that influence red fox abundance, which we then used to predict relative red fox abundance. We compared the estimated regression coefficients of the landscape parameters and the predicted abundance of the two methods. Further, we compared the costs and the precision of the two field methods. The predicted relative abundances were similar between the two methods, suggesting that the two indices were closely related to the true abundance of red foxes. For both methods, landscape diversity and edge density best described differences in the indices and had positive estimated effects on the relative fox abundance. In our study the costs of each method were of similar magnitude, but the sample size obtained from the feces counts (262 transects) was larger than the camera trap sample size (88 camera locations). The precision of the camera traps was lower than the precision of the feces counts. The approach we applied can be used as a framework to compare and combine the results of two or more different field methods to estimate abundance and by this enhance the reliability of the result.     

A note on 'plotless' methods for estimating bacterial cell densities

R oser , D., N edwell , D.B. & G ordon , A. 1984. A note on "plotless" methods for estimating bacterial cell densities. Journal of Applied Bacteriology 56 , 343–347.
'Plotless' techniques for determining population densities have been developed for, and applied to, higher plant populations. They can often be carried out more rapidly than techniques involving total counts of individuals in plots, or quadrants, but such plotless techniques have not been generally applied to the estimation of densities of bacterial cells. Direct microscopical counting of cell numbers in a field of view, an example of a plot-related method, has been traditionally used for micro-bial cell counts. In this study 'plot' and 'plotless' methods on a variety of bacterial samples are compared. Estimates of bacterial cell density were obtained by measuring the distance of cells from a fixed point in a field of view. The values, which were more rapidly obtained, were directly correlated with total cell counts. Although there was some apparent deviation from a perfect 1:1 relationship with total counts, as indicated by a correlation coefficient less than 1.0, there were no significant differences between the replicated counts of bacteria on samples of tissue from the surface of Hypholoma basidiocarps ( P < 0.05). This indicated that the methods of enumeration were comparable. The distance-related estimates could readily be obtained from fields of view with cell densities varying over several orders of magnitude. It was more rapidly applied, particularly at high density, and the method was applicable not only to random cell distributions but also to the non-random distributions encountered when microbial cells aggregated into micro-colonies. The method appears to be particularly well-suited for automated, digitized, direct counting procedures, as well as to estimating bacterial numbers on membrane filters and natural substrates.     

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.     

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10² to 10⁷ cfu/ml. Cyto-metric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.