Fixation of<i>Chattonella antiqua</i>and<i>C. marina</i>(Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy

Katano, Toshiya; Yoshida, Makoto; Lee, Juyun; Han, Moonsik; Hayami, Yuichi

doi:10.2216/08-102.1

Cited by 29 publications

(17 citation statements)

References 38 publications

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“…The cells are morphologically plastic and extremely delicate, and tend to disintegrate with nearly all common fixatives, although Katano et al (2009) recently reported that HEPES-buffered paraformaldehyde and glutaraldehyde deliver promising results for short-term fixation of Chattonella species. As a consequence of the lack of a rigid cell wall, raphidophytes are highly variable in cell shape, depending on physiological conditions (Imai, 2000;Band-Schmidt et al, 2004;Bowers et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

Klöpper

John

Zingone

et al. 2013

European Journal of Phycology

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We analysed the molecular and morphological features of strains of Chattonella subsalsa isolated from the western Adriatic coast (Mediterranean Sea), with the aim of confirming their classification and elucidating their phylogenetic positions within the Raphidophyceae. We sequenced parts of the ribosomal operon, including the small subunit (SSU), the internal transcribed spacer region (ITS) and the large subunit (LSU) of the rDNA. Additionally, we analysed sequences of the chloroplast-encoded subunit psaA of Photosystem I (PSI) and rbcL, encoding the large subunit of the Rubisco gene. For three phylogenetic markers (LSU, ITS, rbcL), the sequences of the strains from the Adriatic Sea were identical and for two markers (SSU, psaA) only minor differences occurred. All strains were sister to, but well separated from, sequences from isolates in culture collections and from GenBank, thus far classified as belonging to C. subsalsa. Light and electron microscopy provided evidence for morphological differences between a strain of C. subsalsa (CCMP217) from the Gulf of Mexico and the isolates from the Adriatic Sea. Differences concerned the shape and arrangement of chloroplasts and the presence of mucocysts and other surface microstructures, which were only observed in isolates from the Adriatic Sea. This is the first evidence for two different taxa classified as C. subsalsa, which are clearly separated on the basis of several genetic markers and also show morphological differences. As compared with strains assigned to C. subsalsa from the NCMA (formerly CCMP) culture collection, the Adriatic strains more closely match the original species description. This would imply that strain CCMP217 and other genetically similar strains should be described under a new species name. Nevertheless, given the high morphological plasticity of Chattonella species, the definition of the true C. subsalsa must be decided based on detailed morphological and molecular analysis of more strains from other geographical areas.

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Section: Introductionmentioning

confidence: 99%

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

Klöpper

John

Zingone

et al. 2013

European Journal of Phycology

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“…1), se hicieron recorridos en la embarcación para detectar una posible mortandad de peces. También se recolectaron muestras superficiales de fitoplancton con botellas de 1 L, las que se fijaron con una solución de lugol ácido y/o paraformaldehído al 1% (Katano et al 2009) para conteo y clasificación taxonómica de la especie dominante, basada en Margalef (1961), Fukuyo et al (1990) y Steidinger & Tangen (1996). Las muestras extraídas del florecimiento se depositaron en frascos de 2 L, sin fijar, para su identificación taxonómica y minutos antes de su clasificación las células vivas fueron inmovilizadas con paraformaldehído al 1%.…”

Section: Muestreounclassified

Condiciones ambientales durante un florecimiento de Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) en la Ensenada de La Paz, Golfo de California

López-Cortés

Band‐Schmidt²,

Bustillos-Guzmán

et al. 2014

Rev. biol. mar. oceanogr.

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“…However, there was no record about fixation of cryptomonads with glutaraldehyde for light microscopy and DNA extraction from algae preserved with glutaraldehyde up till now. Besides, the combination of glutaraldehyde and formaldehyde has been tried to preserve several algal samples, including Raphidophytes, and positive results were recorded [12,24]. Thus, it may be practical to use glutaraldehyde or the combination of glutaraldehyde and formaldehyde to preserve cryptomonad samples.…”

mentioning

confidence: 99%

“…Moreover, buffer and osmotic adjustment are two important factors that affect the preservation of cell morphology during the fixation procedure [24]. PBS (phosphate-buffered saline) is a buffer solution commonly used in biological research.…”

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Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification

Xia

Cheng²,

Zhu³

et al. 2013

J. Microbiol. Biotechnol.

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Cryptomonads are unicellular, biflagellate algae. Generally, cryptomonad cells cannot be preserved well because of their fragile nature, and an improved methodology should be developed to identify cryptomonads from natural habitats. In this study, we tried using several cytological fixatives, including glutaraldehyde, formaldehyde, and their combinations to preserve field samples collected from various waters, and the currently used fixative, Lugol's solution was tested for comparison. Results showed that among the fixatives tested, glutaraldehyde preserved the samples best, and the optimal concentration of glutaraldehyde was 2%. The cell morphology was well preserved by glutaraldehyde. Cells kept their original color, volume, and shape, and important taxonomic features such as furrow/gullet complex, ejectosomes, as well as flagella could be observed clearly, whereas these organelles frequently disappeared in Lugol's solution preserved samples. The osmotic adjustments and buffers tested could not preserve cell density significantly higher. Statistical calculation showed the cell density in the samples preserved by 2% glutaraldehyde remained stable after 43 days of the fixation procedure. In addition, DNA was extracted from glutaraldehyde preserved samples by grinding with liquid nitrogen and the 18S rDNA sequence was amplified by PCR. The sequence was virtually identical to the reference sequence, and phylogenetic analyses showed very close relationship between it and sequences from the same organism. To sum up, the present study demonstrated that 2% unbuffered glutaraldehyde, without osmotic adjustments, can preserve cryptomonads cells for identification, in terms of both light microscopy and phylogenetic analyses based on DNA sequences.

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Fixation ofChattonella antiquaandC. marina(Raphidophyceae) using Hepes-buffered paraformaldehyde and glutaraldehyde for flow cytometry and light microscopy

Cited by 29 publications

References 38 publications

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

Phylogeny and morphology of a Chattonella (Raphidophyceae) species from the Mediterranean Sea: what is C. subsalsa?

Condiciones ambientales durante un florecimiento de Cochlodinium polykrikoides (Gymnodiniales, Dinophyceae) en la Ensenada de La Paz, Golfo de California

Improved Methodology for Identification of Cryptomonads: Combining Light Microscopy and PCR Amplification

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