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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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Blastocystis STs display various shapes from both viable and non-viable populations.Image gallery of Blastocystis cells showing round and irregular shapes from viable and non-viable populations. Each cell shown in the brightfield (BF) view has corresponding images which display CFSE and PI staining. The latter was used to determine viability. Irregular-shaped cells show elongated cells and amoeboid forms. Amoeboid forms with prominent pseudopodia and filamentous attachments are restricted to non-viable forms ST1-NUH9 and ST4-WR1. There are rare (0.1%) cells in viable ST7-B population that show an amoeboid-like morphology.
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pone.0143974.g003: Blastocystis STs display various shapes from both viable and non-viable populations.Image gallery of Blastocystis cells showing round and irregular shapes from viable and non-viable populations. Each cell shown in the brightfield (BF) view has corresponding images which display CFSE and PI staining. The latter was used to determine viability. Irregular-shaped cells show elongated cells and amoeboid forms. Amoeboid forms with prominent pseudopodia and filamentous attachments are restricted to non-viable forms ST1-NUH9 and ST4-WR1. There are rare (0.1%) cells in viable ST7-B population that show an amoeboid-like morphology.

Mentions: Using imaging flow cytometry, both round and irregular shapes were found in all Blastocystis isolates (Figs 2A and 3). The proportion of these shapes however differs from one subtype to another. Viable Blastocystis ST1-NUH9 is exclusively round, but only 76% round (the rest are irregular) in non-viable cells. Viable Blastocystis ST4-WR1 and ST7-B isolates are 83% and 92% round-shaped, respectively. These proportions are slightly lower (82% for ST4-WR1 and 88% for ST7-B) in non-viable cells (Fig 2B).


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

Yason JA, Tan KS - PLoS ONE (2015)

Blastocystis STs display various shapes from both viable and non-viable populations.Image gallery of Blastocystis cells showing round and irregular shapes from viable and non-viable populations. Each cell shown in the brightfield (BF) view has corresponding images which display CFSE and PI staining. The latter was used to determine viability. Irregular-shaped cells show elongated cells and amoeboid forms. Amoeboid forms with prominent pseudopodia and filamentous attachments are restricted to non-viable forms ST1-NUH9 and ST4-WR1. There are rare (0.1%) cells in viable ST7-B population that show an amoeboid-like morphology.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143974.g003: Blastocystis STs display various shapes from both viable and non-viable populations.Image gallery of Blastocystis cells showing round and irregular shapes from viable and non-viable populations. Each cell shown in the brightfield (BF) view has corresponding images which display CFSE and PI staining. The latter was used to determine viability. Irregular-shaped cells show elongated cells and amoeboid forms. Amoeboid forms with prominent pseudopodia and filamentous attachments are restricted to non-viable forms ST1-NUH9 and ST4-WR1. There are rare (0.1%) cells in viable ST7-B population that show an amoeboid-like morphology.
Mentions: Using imaging flow cytometry, both round and irregular shapes were found in all Blastocystis isolates (Figs 2A and 3). The proportion of these shapes however differs from one subtype to another. Viable Blastocystis ST1-NUH9 is exclusively round, but only 76% round (the rest are irregular) in non-viable cells. Viable Blastocystis ST4-WR1 and ST7-B isolates are 83% and 92% round-shaped, respectively. These proportions are slightly lower (82% for ST4-WR1 and 88% for ST7-B) in non-viable cells (Fig 2B).

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