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Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

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Analysis of Blastocystis based on granularity.Blastocystis populations were plotted according to mean pixel intensities of both side scatter channel and PI staining to determine the cells' granularity and viability, respectively (A). Cells with higher PI staining have more granularity compared to cells with lower PI staining. This observation is common to all subtypes used in this study as shown in a graph (B); *, p < 0.05. Sample images showing cells with high granularity among viable and non-viable populations (C). These cells may represent true granular cells and degenerating cells, respectively.
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pone.0143974.g005: Analysis of Blastocystis based on granularity.Blastocystis populations were plotted according to mean pixel intensities of both side scatter channel and PI staining to determine the cells' granularity and viability, respectively (A). Cells with higher PI staining have more granularity compared to cells with lower PI staining. This observation is common to all subtypes used in this study as shown in a graph (B); *, p < 0.05. Sample images showing cells with high granularity among viable and non-viable populations (C). These cells may represent true granular cells and degenerating cells, respectively.

Mentions: Aside from the round vacuolar forms, round granular forms are also commonly observed in clinical samples and in cultures [6,16]. Vdovenko [23] observed that these forms can naturally arise from vacuolar forms possibly by environmental exposure or fixation artefact. A more recent study has also mentioned this phenomenon [16]. In this study, we used the side-scatter channel to measure granularity of the cells. The average intensity coming from the channel in all STs in non-viable cells are higher compared to viable cells. Cells that are more granular can be found mostly among non-viable cells (Fig 5A). Non-viable cells have wider range in terms of granularity in contrast to viable cells where these tend to have lower side-scatter channel intensity. These findings are consistent among the 3 STs studied (Fig 5B). Nevertheless, cells that are more granular can also be found among viable cells. Our data suggests that there exists two populations of granular cells: true granular cells and those that are degenerating (Fig 5C) as reported previously [1,23]. We also add that granularity of these dying cells may be due more to environmental exposure rather than caused by fixation since we have observed the same proportion of granular forms in both fixed and unfixed samples (data not shown). We also used the Hoechst staining intensity of viable granular cells to find their biological significance. We have observe that these cells have higher DNA content compared to granular ones (Fig 6) These cells may therefore be in a more active reproductive state compared to less granular ones. We also analysed the granularity of multinucleated cells.


Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Analysis of Blastocystis based on granularity.Blastocystis populations were plotted according to mean pixel intensities of both side scatter channel and PI staining to determine the cells' granularity and viability, respectively (A). Cells with higher PI staining have more granularity compared to cells with lower PI staining. This observation is common to all subtypes used in this study as shown in a graph (B); *, p < 0.05. Sample images showing cells with high granularity among viable and non-viable populations (C). These cells may represent true granular cells and degenerating cells, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4664278&req=5

pone.0143974.g005: Analysis of Blastocystis based on granularity.Blastocystis populations were plotted according to mean pixel intensities of both side scatter channel and PI staining to determine the cells' granularity and viability, respectively (A). Cells with higher PI staining have more granularity compared to cells with lower PI staining. This observation is common to all subtypes used in this study as shown in a graph (B); *, p < 0.05. Sample images showing cells with high granularity among viable and non-viable populations (C). These cells may represent true granular cells and degenerating cells, respectively.
Mentions: Aside from the round vacuolar forms, round granular forms are also commonly observed in clinical samples and in cultures [6,16]. Vdovenko [23] observed that these forms can naturally arise from vacuolar forms possibly by environmental exposure or fixation artefact. A more recent study has also mentioned this phenomenon [16]. In this study, we used the side-scatter channel to measure granularity of the cells. The average intensity coming from the channel in all STs in non-viable cells are higher compared to viable cells. Cells that are more granular can be found mostly among non-viable cells (Fig 5A). Non-viable cells have wider range in terms of granularity in contrast to viable cells where these tend to have lower side-scatter channel intensity. These findings are consistent among the 3 STs studied (Fig 5B). Nevertheless, cells that are more granular can also be found among viable cells. Our data suggests that there exists two populations of granular cells: true granular cells and those that are degenerating (Fig 5C) as reported previously [1,23]. We also add that granularity of these dying cells may be due more to environmental exposure rather than caused by fixation since we have observed the same proportion of granular forms in both fixed and unfixed samples (data not shown). We also used the Hoechst staining intensity of viable granular cells to find their biological significance. We have observe that these cells have higher DNA content compared to granular ones (Fig 6) These cells may therefore be in a more active reproductive state compared to less granular ones. We also analysed the granularity of multinucleated cells.

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

Show MeSH
Related in: MedlinePlus