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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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Round Blastocystis have higher DNA content.Blastocystis cells were plotted according to circularity using the software’s shape wizard. The population of cells was then divided into three groups: low, mid and high circularity (A). A dot-plot was generated to analyze the relationship between circularity and DNA content indicated by Hoechst staining of Blastocystis cells (B). The dot-plot shows that some highly circular cells have the highest DNA content while the least circular cells have the lowest DNA content. Bar graphs show the average Hoechst-staining of the three groups of Blastocystis cells based on circularity (C). Differences between groups based on circularity in each ST were found to be significant (p < 0.05) using ANOVA.
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pone.0143974.g004: Round Blastocystis have higher DNA content.Blastocystis cells were plotted according to circularity using the software’s shape wizard. The population of cells was then divided into three groups: low, mid and high circularity (A). A dot-plot was generated to analyze the relationship between circularity and DNA content indicated by Hoechst staining of Blastocystis cells (B). The dot-plot shows that some highly circular cells have the highest DNA content while the least circular cells have the lowest DNA content. Bar graphs show the average Hoechst-staining of the three groups of Blastocystis cells based on circularity (C). Differences between groups based on circularity in each ST were found to be significant (p < 0.05) using ANOVA.

Mentions: Analysis of single Hoechst-staining was also done to further correlate Blastocystis shape and reproductive status. Hoechst-staining is used for cell-cycle analysis. Actively dividing cells register higher fluorescence compared to inert cells. In all the isolates studied, round-shaped cells had higher average Hoechst-staining (Fig 4). This finding links well with the observation using PI-staining that most irregularly-shaped may not have biological or reproductive roles at all.


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

Yason JA, Tan KS - PLoS ONE (2015)

Round Blastocystis have higher DNA content.Blastocystis cells were plotted according to circularity using the software’s shape wizard. The population of cells was then divided into three groups: low, mid and high circularity (A). A dot-plot was generated to analyze the relationship between circularity and DNA content indicated by Hoechst staining of Blastocystis cells (B). The dot-plot shows that some highly circular cells have the highest DNA content while the least circular cells have the lowest DNA content. Bar graphs show the average Hoechst-staining of the three groups of Blastocystis cells based on circularity (C). Differences between groups based on circularity in each ST were found to be significant (p < 0.05) using ANOVA.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143974.g004: Round Blastocystis have higher DNA content.Blastocystis cells were plotted according to circularity using the software’s shape wizard. The population of cells was then divided into three groups: low, mid and high circularity (A). A dot-plot was generated to analyze the relationship between circularity and DNA content indicated by Hoechst staining of Blastocystis cells (B). The dot-plot shows that some highly circular cells have the highest DNA content while the least circular cells have the lowest DNA content. Bar graphs show the average Hoechst-staining of the three groups of Blastocystis cells based on circularity (C). Differences between groups based on circularity in each ST were found to be significant (p < 0.05) using ANOVA.
Mentions: Analysis of single Hoechst-staining was also done to further correlate Blastocystis shape and reproductive status. Hoechst-staining is used for cell-cycle analysis. Actively dividing cells register higher fluorescence compared to inert cells. In all the isolates studied, round-shaped cells had higher average Hoechst-staining (Fig 4). This finding links well with the observation using PI-staining that most irregularly-shaped may not have biological or reproductive roles at all.

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