Estimating microbial populations by flow cytometry: Comparison between instruments (original) (raw)
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Applied and Environmental Microbiology, 2007
The relationship between flow cytometry data and epifluorescence microscopy measurements was assessed in bacterioplankton samples from 80 lakes to estimate bacterial biovolume and cell size distribution. The total counts of 4,6-diamidino-2-phenylindole-stained cells estimated by both methods were significantly related, and the slope of their linear regression was not significantly different from 1, indicating that both methods produce very similar estimates of bacterial abundance. The relationships between side scatter (SSC) and 4,6-diamidino-2-phenylindole fluorescence and cell volume (microscopy values) were improved by binning of the data in three frequency classes for each, but further increases in the number of classes did not improve these relationships. Side scatter was the best cell volume predictor, and significant relationships were observed between the SSC classes and the smallest (R 2 ؍ 0.545, P < 0.001, n ؍ 80) and the largest (R 2 ؍ 0.544, P < 0.001, n ؍ 80) microscopy bacterial-size classes. Based on these relationships, a reliable bacterial biomass estimation was obtained from the SSC frequency classes. Our study indicates that flow cytometry can be used to properly estimate bacterioplankton biovolume, with an accuracy similar to those of more time-consuming microscopy methods.
Journal of Plankton Research, 2007
Phytoplankton observation is the product of a number of trade-offs related to sampling processes, required level of diversity and size spectrum analysis capabilities of the techniques involved. Instruments combining the morphological and high-frequency analysis for phytoplankton cells are now available. This paper presents an application of the automated high-resolution flow cytometer Cytosub as a tool for analysing phytoplanktonic cells in their natural environment. High resolution data from a temporal study in the Bay of Marseille (analysis every 30 min over 1 month) and a spatial study in the Southern Indian Ocean (analysis every 5 min at 10 knots over 5 days) are presented to illustrate the capabilities and limitations of the instrument. Automated high-frequency flow cytometry revealed the spatial and temporal variability of phytoplankton in the size range 12$50 mm that could not be resolved otherwise. Due to some limitations (instrumental memory, volume analysed per sample), recorded counts could be statistically too low. By combining high-frequency consecutive samples, it is possible to decrease the counting error, following Poisson's law, and to retain the main features of phytoplankton variability. With this technique, the analysis of phytoplankton variability combines adequate sampling frequency and effective monitoring of community changes.
Cytometry, 2001
Background: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. Methods: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements.
Flow cytometry: A powerful tool in analysis of biomass distributions in phytoplankton
Water Science and Technology, 1995
The large range in cOllcentraJions and cell-sizes of algal cells and colonies and the large variety of cell types are the main reasons for developing a dedicated cytometer for the analysis of phytoplankton. A ElD'Opean Community funded cOllsonium has developed the EUlQPA cytometer. which is easily transponed and can be operated at sea. With the EuJOPA. both small smgle cells and large colonies of cyanobacteria can be analyzed In OIIe run. ThIs provides correlated mformaUon 011 optical characteristics. pigments contents and taxonomy. The resulting dlstnbution of (chlorophyll) biomass over taxonomic groups can be inter-caJjbrated With standard spectrometnc analysIs teChniques. The EmQPA can be used successfully for analysis of field samples and phytoplankton cultures. It is well SUited for phytoplankton monitoring and grazing studies.
Flow cytometry assessment of bacterioplankton in tropical marine environments
Journal of Microbiological Methods, 2003
Flow cytometry was used to characterize bacterioplankton from two tropical environments in Brazil: the eutrophic Guanabara Bay and the oligotrophic southwest Atlantic Ocean. Bacterial abundance was evaluated by flow cytometry, and cells were stained with SYTO 13, allowing demonstration of differences in nucleic acid content. Bacterial production was also evaluated by means of 3 H-leucine incorporation. Bacterial numbers were different for both sites. In Atlantic Ocean samples, we found a maximum of 5.50 Â 10 5 cells ml À 1 , and low nucleic acid content organisms predominated. In Guanabara Bay, bacterial numbers were one order of magnitude higher than in the ocean, and they varied from outer bay (1.01 Â 10 6 cells ml À 1) to inner bay (6.90 Â 10 6 cells ml À 1). Bacterial activity in ocean samples varied from 4.6 to 126 ng C l À 1 h À 1 , while in the bay, mean values ranged from 1.95 Ag C l À 1 h À 1 (outer bay) to 7.35 Ag C l À 1 h À 1 (inner bay). Values found for both parameters are characteristic of different trophic situations. These results illustrate the utility of cytometric analyses of bacterioplankton populations in characterizing their large spatial and temporal scales of distribution in aquatic ecosystems.