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Use of sodium hypochlorite solutions to obtain axenic cultures of Nostoc strains (Cyanobacteria)

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Abstract

A method based on the treatment of akinetes using diluted solutions of sodium hypochlorite (SH solution) was developed to obtain axenic cultures of Nostoc strains (Nostoc Vaucher ex Bornet & Flahault, 1886:181). Three strains were independently grown on liquid BG-110 medium (BG-11 without a nitrogen source) until a massive differentiation of akinete cells took place. Samples of these akinete-rich cultures were homogenized and treated with diluted SH solutions (1, 2, and 3 %) for 10, 20, and 30 s. Subsequently, the treated akinetes were spread onto BG-110-agar plates and incubated under standard conditions for at least 2 weeks. Both the axenicity and the degree of contamination were monitored for each treatment by inoculating spring colonies in nutrient-broth or R2A-agar plates. Axenic cultures were obtained for the strains Nostoc sp. CCLFM I (with 1 % SH solution), Nostoc sp. CCLFM VIII (2 and 3 %) and Nostoc sp. CCLFM XXI (3 %), only applying 10 s of treatment exposure. This strategy was proven to be efficient for Nostoc cultures, as all of the tested strains became axenic. This method can be applied to virtually any strains that are capable of performing massive akinete differentiation; furthermore, it is a fast, inexpensive, and antibiotic-free approach.

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References

  • Adams DG, Duggan PS (1999) Heterocysts and akinete differentiation in cyanobacteria. New Phytol 144:1–33

    Article  Google Scholar 

  • Allen MB (1968) Simple conditions for growth of unicellular blue-green algae on plates. J Phycol 4:1–4

    Article  CAS  Google Scholar 

  • Allen MM (1973) Methods for cyanophyceae. In: Stein JK (ed) Handbook of phycological methods, culture methods and growth measurements. Cambridge University, New York, pp 127–138

    Google Scholar 

  • Aráoz R, Nghiêm H, Rippka R, Palibroda N, Tandeau de Marsac N, Herdman M (2005) Neurotoxin in axenic oscillatorian cyanobacteria: coexistence of anatoxin-a and homoanatoxin-a determined by ligand-binding assay and GC/MS. Microbiology 151:1263–1273

    Article  PubMed  Google Scholar 

  • Argueta C, Yuksek K, Patel R, Summers ML (2006) Identification of Nostoc punctiforme akinete-expressed genes using differential display. Mol Microbiol 61(3):748–757

    Article  CAS  PubMed  Google Scholar 

  • Bowyer JW, Skerman VBD (1968) Production of axenic cultures of soil-borne and endophytic blue-green algae. J Gen Microbiol 54:299–306

    Article  CAS  PubMed  Google Scholar 

  • Caire GZ, Cano MS, Mulé MCZ, Palma RM, Colombo K (1997) Exopolysaccharide of Nostoc muscorum (Cyanobacteria) in the aggregation of soil particles. J Appl Phycol 9:249–253

    Article  Google Scholar 

  • Carmichael WW, Gorham PR (1974) An improved method for obtaining axenic clones of planktonic blue-green algae. J Phycol 10:238–240

    Google Scholar 

  • Cho JY, Choi JS, Kong IS, Park SI, Kerr RG, Hong YK (2002) A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures. J Appl Phycol 14:385–390

    Article  CAS  Google Scholar 

  • Choi JS, Cho JY, Jin LG, Jin HJ, Hong YK (2002) Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis. J Appl Phycol 14:115–121

    Article  Google Scholar 

  • Choi GG, Bae MS, Ahn CY, Oh HM (2008) Induction of axenic culture of Arthrospira (Spirulina) platensis based on antibiotic sensitivity of contaminating bacteria. Biotechnol Lett 30:87–92

    Article  CAS  PubMed  Google Scholar 

  • Ferris MJ, Hirsch CF (1991) Method for isolation and purification of Cyanobacteria. Appl Environ Microbiol 57:1448–1452

    CAS  PubMed Central  PubMed  Google Scholar 

  • Flores E, Herrero A (2010) Compartmentalized function through cell differentiation in filamentous cyanobacteria. Nat Rev 8:39–49

    CAS  Google Scholar 

  • Fogg GE (1942) Studies on nitrogen fixation by blue-green algae. I. Nitrogen fixation by Anabaena cylindrica Lemm. J Exp Biol 19:78–87

    CAS  Google Scholar 

  • Frontier S (1985) Diversity and structure in aquatic ecosystems. Oceanogr Mar Biol Annu Rev 23:253–312

    Google Scholar 

  • Hoiczyk E (1998) Structural and biochemical analysis of the sheath of Phormidium uncitatum. J Bacteriol 180:3923–3932

    CAS  PubMed Central  PubMed  Google Scholar 

  • Hoiczyk E, Baumeister W (1995) Envelope structure of four gliding filamentous cyanobacteria. J Bacteriol 177:2387–2395

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kim J-S, Park Y-H, Yoon B-D, Oh H-M (1999) Establishment of axenic cultures of Anabaena flos-aquae and Aphanothece nidulans (Cyanobacteria) by lysozyme treatment. J Phycol 35:865–869

    Article  Google Scholar 

  • McDanile HR, Middlebrook JB, Bowman RO (1962) Isolation of pure cultures of algae from contaminated cultures. Appl Microbiol 10:223

    Google Scholar 

  • Meeks JC, Campbell EL, Summers ML, Wong FC (2002) Cellular differentiation in the cyanobacterium Nostoc punctiforme. Arch Microbiol 178:395–403

    Article  CAS  PubMed  Google Scholar 

  • Melo RS, Neves MHCB (1849) Baptista OR (2011) Cultivo axênico das cianobactérias marinhas Aphanothece halophytica Frémy, 1933 e Chroococcus minutus (Kützing) Nägeli. Acta Bot Bras 25(1):234–240

    Article  Google Scholar 

  • Parker DL (1982) Improved procedures for the cloning and purification of Microcystis cultures (cyanophyta). J Phycol 18:471–477

    Article  Google Scholar 

  • Rejmankova E, Harbin-Ireland A, Lege M (2000) Bacterial abundance in larval habitats of four species of Anopheles (Diptera: Culicidae) in Belize, Central America. J Vec Ecol 25:229–239

    CAS  Google Scholar 

  • Rippka R (1988) Isolation and purification of cyanobacteria. Method Enzymol 167:3–27

    Article  CAS  Google Scholar 

  • Rippka R, Waterbury JB, Stanier RY (1981) Isolation and purification of cyanobacteria: some general principles. In: Starr MP, Stolp H, Trüper HG, Balows A, Schlegel HC (eds) The prokaryotes. A handbook on habitats, isolation and identification of bacteria. Spring-Verlag, New York, pp 212–220

    Google Scholar 

  • Sena L, Rojas D, Montiel E, González H, Moret J, Naranjo L (2011) A strategy to obtain axenic culture of Arthrospira spp. cyanobacteria. World J Microbiol Biotechnol 27:1045–1053

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Shih PM, Wu D, Latifi A, Axen SD, Fewer DP, Talla E, Calteau A, Cai F, Tandeau de Marsac N, Rippka R, Herdman M, Sivonen K, Coursin T, Laurent T, Goodwin L, Nolan M, Davenport KW, Han CS, Rubin EM, Eisen JA, Woyke T, Gugger M, Kerfeld CA (2013) Improving the coverage of the Cyanobacterial phylum using diversity-driven genome sequencing. Proc Natl Acad Sci 110:1053–1058

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sili C, Ena A, Materassi R, Vincenzini M (1994) Germination of desiccated aged akinetes of alkaliphilic cyanobacteria. Arch Microbiol 162:20–25

    Article  CAS  Google Scholar 

  • Smith TW, Walker ED, Kaufman MG (1998) Bacterial density and survey of cultivable heterotrophs in the surface water of a freshwater marsh habitat of Anopheles quadrimaculatus larvae (Diptera:Culicidae). J Am Mosq Control Assoc 14:70–72

    Google Scholar 

  • Stanier RY, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Chroococcales). Bacteriol Rev 35:171–205

    CAS  PubMed Central  PubMed  Google Scholar 

  • Sutherland JM, Herdman M, Stewart WDP (1979) Akinetes of the cyanobacterium Nostoc PCC7524: macromolecular composition, structure and control of differentiation. J Gen Microbiol 115:273–287

    Article  Google Scholar 

  • Tassigny M, Laporte G, Pourriot R (1969) Recherches sur les techniques de purification des cultures algales: élaboration des méthodes applicables aux Desmidiacées et Cyanophycées filamenteuses, Diatomées. Ann Inst Past 117:64–75

    CAS  Google Scholar 

  • Thiel T, Wolk CP (1983) Metabolic activities of isolated akinetes of the cyanobacterium Nostoc spongiaeforme. J Bacteriol 156:369–374

    CAS  PubMed Central  PubMed  Google Scholar 

  • Vaara T, Vaara M, Niemela S (1979) Two improved methods for obtaining axenic cultures of Cyanobacteria. Appl Environ Microbiol 38:1011–1014

    CAS  PubMed Central  PubMed  Google Scholar 

  • Vásquez-Martínez G, Rodriguez MH, Hernández-Hernández F, Ibarra JE (2004) Strategy to obtain axenic cultures from field-collected samples of the cyanobacterium Phormidium animalis. J Microbiol Methods 57:115–121

    Article  Google Scholar 

  • Watanabe MM, Nakagawa M, Katagiri M, Aizawa K, Hiroki M, Nozaki H (1998) Purification of freshwater picoplanktonic cyanobacteria by pour-plating in ‘ultra-low-gelling-temperature agarose’. Phycol Res 46:71–75

    Article  Google Scholar 

  • Wieringa KT (1968) A new method for obtaining bacteria-free cultures of blue-green algae. Antonie Van Leeuwenhoek 34:54–56

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto Y (1975) Effect of desiccation on the germination of akinetes of Anabaena cylindrica. Plant Cell Physiol 16:749–752

    Google Scholar 

Download references

Acknowledgments

This study was funded by grants from The Brazilian National Research Council (CNPq) and M.G.M.V.Vaz was supported by CNPq scholarship (135154/2008-1) and by State of São Paulo Research Foundation (FAPESP) graduate scholarship (2010/18732-0). The authors would like to thank Dr. Rosane Aguiar (in memorian) for her great contribution on cyanobacterial studies at Federal University of Viçosa. M.G.M.V.Vaz would like to thank Msc. Diego Bonaldo Genuário for his constructive and helpful comments on the manuscript.

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Authors and Affiliations

  1. Laboratory of Molecular Ecology of Cyanobacteria, Center for Nuclear Energy in Agriculture, University of São Paulo, Piracicaba, São Paulo, 13400-970, Brazil

    Marcelo Gomes Marçal Vieira Vaz

  2. Laboratory of Microorganism Physiology, Microbiology Department, Federal University of Viçosa, Viçosa, Minas Gerais, 36570-000, Brazil

    Marcelo Gomes Marçal Vieira Vaz, Rafael Wesley Bastos, Guilherme Paier Milanez, Mariana Neves Moura, Éder Galinari Ferreira, Celia Perin, Marísia Cyreti Forte Pontes & Antônio Galvão do Nascimento

  3. Laboratory of Ecology and Physiology of Microorganism, Microbiology Department, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, 31270-901, Brazil

    Rafael Wesley Bastos

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  1. Marcelo Gomes Marçal Vieira Vaz
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  2. Rafael Wesley Bastos
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Correspondence to Marcelo Gomes Marçal Vieira Vaz.

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Vaz, M.G.M.V., Bastos, R.W., Milanez, G.P. et al. Use of sodium hypochlorite solutions to obtain axenic cultures of Nostoc strains (Cyanobacteria). Braz. J. Bot 37, 115–120 (2014). https://doi.org/10.1007/s40415-014-0055-4

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  • DOI: https://doi.org/10.1007/s40415-014-0055-4

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