Abstract
We developed an alternative method of staining cell nuclei and chloroplast nucleoids of algal cells using SYBR Green I (the fluorescent dye used commonly for detecting dsDNA in agarose and polyacrylamide gels as an alternative to highly mutagenic ethidium bromide and for DNA staining of viruses and bacteria followed by flow cytometry, digital image analysis or real-time PCR), which enabled routine stainingin vivo. Cells do not need to be fixed or treated chemically or physically before staining, thus the shape, size and position of DNA-containing structures are not affected. The fluorescence signal is sharp and reproducible. Examples of application of the method are shown in color microphotographs for representatives of eukaryotic algae from the taxaChlorophyta, Rhodophyta and the prokaryoticCyanophyta. The method is also useful for studying progress of the cell cycle in algal cells dividing by multiple fission, as shown by observation of changes in nuclear number during the cell cycle of the green algaChlamydomonas reinhardtii andScenedesmus quadricauda. Staining with SYBR Green I can be recommended as a fast, safe and efficient method for the detection of DNA-containing structuresin vivo.
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Aldea C., Alvarez C.P., Folgueira L., Delgado R., Otero J.R.: Rapid detection of herpes simplex virus DNA in genital ulcers by real-time PCR using SYBR Green I dye as the detection signal.J.Clin.Microbiol.40, 1060–1062 (2002).
Bišová K., Hendrychová J., Cepák V., Zachleder V.: Cell growth and division processes are differentially sensitive to cadmium inScenedesmus quadricauda.Folia Microbiol.48, 805–816 (2003).
Broadaway S.C., Barton S.A., Pyle B.H.: Rapid staining and enumeration of small numbers of total bacteria in water by solid-phase laser cytometry.Appl.Environ.Microbiol.69, 4272–4273 (2003).
Cepák V.: Variability of nucleoid morphology of some cyanophytes growing under various growth conditions.Algol.Stud.81, 39–52 (1996).
Cepák V., Zobačová M., Zachleder V.: The effect of cadmium ions on the cell cycle of the green flagellateChlamydomonas noctigama.Arch.Hydrobiol.Algol.Stud.106 (Suppl. 144), 117–129 (2002).
Cepák V., Přibyl P., Vítová M.: The effect of light of differing spectral qualities on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal algaScenedesmus obliquus. Folia Microbiol.51, in press (2006).
Chen B., Rodbard D., Chrambach A.: Polyacrylamide gel electrophoresis with optical scanning, using multiphasic buffer systems.Anal.Biochem.89, 596–608 (1978).
Coleman A.W.: Visualization of chloroplast DNA with two fluorochromes.Exp.Cell Res.114, 95–100 (1978).
Coleman A.W.: The use of fluorochrome 4′,6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.J.Cell Biol.82, 299–305 (1979).
Coleman A.W., Maguire M.J.: A microspectrophotometric analysis of nuclear and chloroplast DNA inVolvox.Dev.Biol.94, 441–450 (1982).
Coleman A.W., Maguire M.J., Coleman J.R.: Mithramycin DNA and 4′,6-diamidino-2-phenylindole DNA staining for fluorescence micro-spectrophotometric measurement of DNA in nuclei plastids and virus particles.J.Histochem.Cytochem.29, 959–968 (1981).
Dallman T., Ares M., Howell S.H.: Analysis of transcription during the cell cycle in toluenizedChlamydomonas reinhardtii cells.Mol.Cell.Biol.3, 1537–1539 (1983).
Hashimoto H., Murakami S.: Chloroplast replication and loss of chloroplast DNA induced by nalidixic acid inEuglena gracilis.Cell Struct.Funct.7, 111–120 (1982).
Hull H.M., Hoshaw R.W., Wang J.C.: Cytofluorometric determination of nuclear DNA in living and preserved algae.Stain Technol.57, 273–282 (1982).
Kuroiwa T.: Mitochondrial nuclei.Internat.Rev.Cytol.75, 1–59 (1982).
Kuroiwa T., Suzuki T.: An improved method for the demonstration of thein situ chloroplast nuclei in higher plants.Cell Struct. Funct.5, 195–197 (1980).
Kuroiwa T., Nishibayashi S., Kawano S., Suzuki T.: Visualization of DNA in various phages (T4, χ, T7, ϕ29) by ethidium bromide epifluorescent microscopy.Experimentia37, 969–970 (1981a).
Kuroiwa T., Suzuki T., Ogawa K., Kawano S.: The chloroplast nucleus: distribution, number, size and shape, and a model for the multiplication of the chloroplast genome during chloroplast development.Plant Cell Physiol.22, 381–396 (1981b).
Lüttke A., Bonotto S.: Chloroplast DNA ofAcetabularia mediterranea: cell cycle related changes in distribution.Planta153, 536–542 (1981).
Meinhardt L.W., Bellato C.M., Tsai S.M.: SYBR Green I used to evaluate the nuclei number of fungal mycelia.BioTechnology31, 42–46 (2001).
Sulek J.: Nuclear division inScenedesmus quadricauda (Turp.)Breb.Arch.Hydrobiol.Algol.Stud.12, 224–258 (1975).
Suzuki T., Kawano S., Kuroiwa T.: Structure of three-dimensionally rod-shaped mitochondrial nucleoids isolated from the slime mouldPhysarum polycephalum.J.Cell Sci.58, 241–261 (1982).
Suzuki T., Fujikura K., Higashiyama T., Kuniaki Takata K.: DNA staining for fluorescence and laser confocal microscopy.J.Histochem.Cytochem.45, 49–53 (1997).
Tschermak-Woess E., Scholler A.: Verteilung und Aufteilung der DNS bei cinigen Cyanophyceen, festgestellt durch ihrc DAPI-Fluorescenz.Plant Syst.Evol.140, 207–223 (1982).
Williamson Q.H., Fennel O.J.: The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA.Meth.Cell Biol.12, 335–351 (1975).
Zachleder V., Cepák V.: Visualization of DNA containing structures by fluorochrome DAPI in those algal cells which are not freely permeable to the dye.Arch.Hydrobiol. Algol.Stud.47, 157–168 (1987).
Zachleder V., Šetlík I.: Effect of irradiance on the course of RNA synthesis in the cell cycle ofScenedesmus quadricauda.Biol.Plant.24, 341–353 (1982).
Zachleder V., Kawano S., Kuroiwa T.: Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the algaScenedesmus quadricauda.Protoplasma192, 228–234 (1996).
Zachleder V., Kawano S., Cepák V., Kuroiwa T.: The effect of nalidixic acid on growth and reproductive events in nucleocytosolic and chloroplast compartments in the algaScenedesmus quadricauda.Folia Microbiol.49, 441–451 (2004).
Žižka Z., Hostounský Z., Kalalová S.: RCH-microscopy used in microbiological studies.Folia Microbiol.44, 328–332 (1999).
Žižka Z., Hostounský Z., Kálalová S.: Morphological details of microorganisms revealed by RHC-microscopy at high magnification — a ready-to-use adaptation of a light microscope.Folia Microbiol.46, 495–503 (2001).
Žižka Z., Hostounský Z., Gabriel J.: ARC-microscopy — a novel microscopic technique used in microbiological studies.Folia Microbiol.48, 829–838 (2003).
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This work was supported by theGrant Agency of the Czech Republic (grants 204/02/1438 and 204/03/1113), by theGrant Agency of Academy of Sciences of the Czech Republic (grant no. B5020305), and byInstitutional Research Concept (no. AV 0Z 502 0903).
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Vítová, M., Hendrychová, J., Cepák, V. et al. Visualization of DNA-containing structures in various species ofChlorophyta, Rhodophyta andCyanophyta using SYBR green I dye. Folia Microbiol 50, 333–340 (2005). https://doi.org/10.1007/BF02931414
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DOI: https://doi.org/10.1007/BF02931414