Analysis of the distribution of ingested bacteria in nanoflagellates and estimation of grazing rates with flow cytometry

Bratvold, Delma; Srienc, Friedrich; Taub, Stephan R.

doi:10.3354/ame021001

Cited by 20 publications

(33 citation statements)

References 25 publications

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“…7) and comparable investigations (Bratvold et al 2000 and references therein) reflects specimens that did not feed during the period of incubation or others that do permanently select against FLB. Both possibilites may include experimental artifacts.…”

Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 80%

“…To minimize the handling effect, we reduced subsampling to 3 occasions (0, 20, 30 min) during each experiment. The physiological conditions of the grazers such as their nutritional status (Hatzis et al 1993, Jürgens & DeMott 1995 or the timing of their cell division and mouth-part formation may also lead to temporary non-feeding (Bratvold et al 2000). Prolongation of the experimental period does not overcome this problem, because incubation times in excess of digestion time yield unrealistic results (e.g.…”

Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 99%

“…Prolongation of the experimental period does not overcome this problem, because incubation times in excess of digestion time yield unrealistic results (e.g. Dolan & 2imek 1998, Bratvold et al 2000. Bacterial digestion time of small HNF species has been reported to range from < 30 min to 1.5 h (Sherr et al 1988, Gonzalez et al 1990b, 1993.…”

Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 99%

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Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance

Cleven

Weisse²

2001

Aquat. Microb. Ecol.

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We investigated the taxonomic composition of the heterotrophic nanoflagellate (HNF) assemblage and its taxon-specific bacterial grazing rates in Lake Constance (Germany) over the course of 1 yr. Bacterial grazing rates were measured using natural fluorescently labelled bacteria (FLB) and compared to bacterial production estimated by the uptake of 14 C-leucine incorporation. Glutaraldehyde-fixed, DAPI-stained flagellates were counted using epifluorescence microscopy. Based on annual averages, small species such as Spumella sp. (2 to 6 µm) were the most numerous HNF and the dominant bacterivores. Larger flagellates such as Kathablepharis sp. contributed significantly to total HNF biomass, in particular during spring, but were relatively unimportant as bacterial grazers. The HNF community structure changed during the transition from the phytoplankton spring bloom to the clearwater phase, with small flagellates such as heterokonts, kinetoplastids and choanoflagellates becoming increasingly abundant. The flagellate community composition was more diverse during summer and autumn than in spring. Per capita ingestion rates ranged from 0 to 31 bacteria HNF -1 h -1 and changed seasonally up to 10-fold within a given taxon. Mixotrophic species contributed little to total bacterivory. We provide evidence that the relative significance of bacterial ingestion by a given flagellate taxon may change seasonally. Based upon our experimental results, we discuss potential shortcomings inherent in the FLB technique.KEY WORDS: Heterotrophic nanoflagellates (HNF) · Seasonal succession · FLB · Grazing rate · Negative binomial distribution · Lake ConstanceResale or republication not permitted without written consent of the publisher

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Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 80%

Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 99%

Section: Potential Shortcomings Of the Flb Approachmentioning

confidence: 99%

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Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance

Cleven

Weisse²

2001

Aquat. Microb. Ecol.

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“…NB distributions are often found among bacterial counts in various scales, including repeated measurements and spatial or temporal distribution of specific bacteria (14,19,26,47), which correspond to distributions of ␣ i or ␥ ijk in the model of equation 1. A published case of NB distribution of bacterial cells in a microscope scale, i.e., the scale of ε ijkl in equation 1, can be found in counts of ingested bacteria within nanoflagellate cells (11). The authors of the latter study interpreted that the mechanism of NB distribution was the summation of different grazing rates of subpopulations of the protozoa.…”

Section: Vol 76 2010 Comparison Of Microscopic Direct Count Methodsmentioning

confidence: 92%

Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

Seo

Ahn

2010

Appl Environ Microbiol

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To assess interchangeability of estimates of bacterial abundance by different epifluorescence microscopy methods, total bacterial numbers (TBNs) determined by most widely accepted protocols were statistically compared. Bacteria in a set of distinctive samples were stained with acridine orange (AO), 4-6-diamidino-2-phenylindole (DAPI), and BacLight and enumerated by visual counting (VC) and supervised image analysis (IA). Model II regression and Bland-Altman analysis proved general agreements between IA and VC methods, although IA counts tended to be lower than VC counts by 7% on a logarithmic scale. Distributions of cells and latex beads on polycarbonate filters were best fitted to negative binomial models rather than to Poisson or log-normal models. The fitted models revealed higher precisions of TBNs by the IA method than those by the VC method. In pairwise comparisons of the staining methods, TBNs by AO and BacLight staining showed good agreement with each other, but DAPI staining had tendencies of underestimation. Although precisions of the three staining methods were comparable to one another (intraclass correlation coefficients, 0.97 to 0.98), accuracy of the DAPI staining method was rebutted by disproportionateness of TBNs between pairs of samples that carried 2-fold different volumes of identical cell suspensions. It was concluded that the TBN values estimated by AO and BacLight staining are relatively accurate and interchangeable for quantitative interpretation and that IA provides better precision than does VC. As a prudent measure, it is suggested to avoid use of DAPI staining for comparative studies investigating accuracy of novel cell-counting methods.Bacterial abundance is an instrumental parameter in assessing the roles of bacteria in the environments (18,27,30,45). While a variety of techniques are available (1,30,53,60), staining bacterial cells with acridine orange (AO) (29) or 4Ј,6-diamidino-2-phenylindole (DAPI) (48) and counting them on black polycarbonate (PC) filters by epifluorescence microscopy have become the standard procedure for direct counting (9,18,30). The Live/Dead BacLight staining kit, which is widely accepted as a rapid measure of viability of individual cells, also provides a total count of bacteria (10). Currently, most studies reporting total bacterial numbers (TBNs) use one of the three staining methods described above. However, the basic question of which fluorochrome to use for a given samples still presents challenges, as comparative studies using two or more of these fluorochromes have often yielded conflicting results (10,17,20,34,37,40,49,52,54,57,58).A more perplexing question is whether TBN values based on different fluorochromes are interchangeable for a quantitative interpretation incorporating TBN data from different methods. A large-scale intersystem study, an analysis of long-term collection of longitudinal data, or a collaborative study by multiple laboratories often requires an amalgamated use of TBN values from different fluorochromes. Apart from the interc...

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“…Bougrier et al (1997) used flow cytometry to study preingestive selection of different microalgal mixtures in Crassostrea gigas and Mytilus edulis. A rapid cytometric method assessing the distribution of ingested bacteria in nanoflagellates was presented by Bratvold et al (2000), investigating grazing behaviour of cultures of Rhynchomonas nasuta and Paraphysomonas vestita. Their results suggest a heterogeneous population composed of subgroups of flagellates with different grazing rates, although the specific biological implications of the statistical models used with regard to the number of flagellate subgroups remain to be proven.…”

Section: Size and Biomassmentioning

confidence: 99%

Flow cytometry as a tool for the study of phytoplankton

Dubelaar¹,

Jonker²

2000

Sci. Mar.

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SUMMARY: An overview is presented on flow cytometry as a tool for counting, analysis and identification of phytoplankton species and groups. The paper covers basics on the analysis technique and instrumentation such as the measuring principle, the type of instrument, limitations and pitfalls with phytoplankton samples and sample handling and preprocessing. Possibilities of the measured entities are discussed, roughly divided in light scatter and related parameters, the endogenous fluorescence and exogenous fluorescence, followed by a discussion on the actual applications such as phytoplankton abundance analysis, ecology and physiology research and monitoring of particle size and biomass. In addition to a limited literature review, we tried to assess how flow cytometry is used in routine laboratory practice and monitoring operations. Therefore, a questionnaire was sent out via email to 47 scientists at 43 institutes known to us as involved in flow cytometric analysis of phytoplankton. In total, 19 scientists responded. Specific survey results are included in italic print whereas some more general answers were integrated in the overview.

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Analysis of the distribution of ingested bacteria in nanoflagellates and estimation of grazing rates with flow cytometry

Cited by 20 publications

References 25 publications

Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance

Seasonal succession and taxon-specific bacterial grazing rates of heterotrophic nanoflagellates in Lake Constance

Agreement, Precision, and Accuracy of Epifluorescence Microscopy Methods for Enumeration of Total Bacterial Numbers

Flow cytometry as a tool for the study of phytoplankton

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