, Volume 51, Issue 1, pp 131–138

How endo- is endo-? Surface sterilization of delicate samples: a Bryopsis (Bryopsidales, Chlorophyta) case study

  • Joke Hollants
  • Frederik Leliaert
  • Olivier De Clerck
  • Anne Willems


In the search for endosymbiotic bacteria, elimination of ectosymbionts is a key point of attention. Commonly, the surface of the host itself or the symbiotic structures are sterilized with aggressive substances such as chlorine or mercury derivatives. Although these disinfectants are adequate to treat many species, they are not suitable for surface sterilization of delicate samples. In order to study the bacterial endosymbionts in the marine green alga Bryopsis, the host plant’s cell wall was mechanically, chemically and enzymatically cleaned. Merely a chemical and enzymatic approach proved to be highly effective. Bryopsis thalli treated with cetyltrimethylammonium bromide (CTAB) lysis buffer, proteinase K and bactericidal cleanser Umonium Master showed no bacterial growth on agar plates or bacterial fluorescence when stained with a DNA fluorochrome. Moreover, the algal cells were intact after sterilization, suggesting endophytic DNA is still present within these algae. This new surface sterilization procedure opens the way to explore endosymbiotic microbial communities of other, even difficult to handle, samples.


Bryopsis Endosymbionts Endosymbiotic bacteria Marine green algae Surface sterilization 


  1. Amann RI, Ludwig W, Schleifer K (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev 59:143–169PubMedGoogle Scholar
  2. Andersen RA (2005) Algal culturing techniques. Academic, New YorkGoogle Scholar
  3. Ashen JB, Goff LJ (2000) Molecular and ecological evidence for species specificity and coevolution in a group of marine algal-bacterial symbioses. Appl Environ Microbiol 66:3024–3030CrossRefPubMedGoogle Scholar
  4. Berger S, Kaever MJ (1992) Dasycladales: an illustrated monograph of a fascinating algal order. Thieme, StuttgartGoogle Scholar
  5. Burke C, Kjelleberg S, Thomas T (2009) Selective extraction of bacterial DNA from the surfaces of macroalgae. Appl Environ Microbiol 75:252–256CrossRefPubMedGoogle Scholar
  6. Burr FA, West JA (1970) Light and electron microscope observations on the vegetative and reproductive structures of Bryopsis hypnoides. Phycologia 9:17–37Google Scholar
  7. Chisholm JRM, Dauga C, Ageron E, Grimont PAD, Jaubert JM (1996) ‘Roots’ in mixotrophic algae. Nature 381:382CrossRefGoogle Scholar
  8. Connell TD (1981) A new technique for surface sterilization of insect eggs. J Kansas Entomol Soc 54:124–128Google Scholar
  9. Croft MT, Lawrence AD, Raux-Deery E, Warren MJ, Smith AG (2005) Algae acquire vitamin B12 through a symbiotic relationship with bacteria. Nature 483:90–93CrossRefGoogle Scholar
  10. Dawes CJ, Lohr CA (1978) Cytoplasmic organization and endosymbiotic bacteria in the growing points of Caulerpa prolifera. Rev Algol 13:309–314Google Scholar
  11. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
  12. Droop MR (1967) A procedure for routine purification of algal cultures with antibiotics. Br Phycol Bull 3:295–297CrossRefGoogle Scholar
  13. Fisher MM, Wilcox LW, Graham LE (1998) Molecular characterization of epiphytic bacterial communities on Charophycean green algae. Appl Environ Microbiol 64:4384–4389PubMedGoogle Scholar
  14. Fries L, Iwasaki H (1976) p-hydroxyphenylacetic acid and other phenolic compounds as growth stimulators of the red alga Porphyra tenera. Plant Sci Lett 6:299–307CrossRefGoogle Scholar
  15. Head WD, Carpenter EJ (1975) Nitrogen fixation associated with the marine macroalga Codium fragile. Limnol Oceanogr 20:815–823CrossRefGoogle Scholar
  16. Kan Y, Fujita T, Sakamoto B, Hokama Y, Nagai H, Kahalalide K (1999) A new cyclic depsipeptide from the Hawaiian green alga Bryopsis species. J Nat Prod 62:1169–1172CrossRefPubMedGoogle Scholar
  17. Kim GH, Klotchkova TA, Kang Y (2001) Life without a cell membrane: regeneration of protoplasts from disintegrated cells of the marine green alga Bryopsis plumosa. J Cell Sci 114:2009–2014PubMedGoogle Scholar
  18. Kooistra W, Boelebos SA, Stam WT (1991) A method for obtaining axenic algal cultures using the antibiotic cefotaxime with emphasis on Cladophoropsis membranacea (Chlorophyta). J Phycol 27:656–658CrossRefGoogle Scholar
  19. Lodewyckx C, Vangronsveld J, Porteous F, Moore ERB, Taghavi S, Mezgeay M, van der Lelie D (2002) Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606CrossRefGoogle Scholar
  20. Marshall K, Joint I, Callow ME, Callow JA (2006) Effect of marine bacterial isolates on the growth and morphology of axenic plantlets of the green alga Ulva linza. Microb Ecol 52:302–310CrossRefPubMedGoogle Scholar
  21. Meyer JM, Hoy MA (2008) Removal of fungal contaminants and their DNA from the surface of Diaphorina citri (Hemiptera: Psyllidae) prior to a molecular survey of endosymbionts. Fla Entomol 91:702–705CrossRefGoogle Scholar
  22. Mine I, Menzel D, Okuda K (2008) Morphogenesis in giant-celled algae. Int Rev Cell Mol Biol 266:37–83CrossRefPubMedGoogle Scholar
  23. Provasoli L, Pintner IJ (1980) Bacteria induced polymorphism in an axenic laboratory strain of Ulva lactuca (Chlorophyceae). J Phycol 16:196–201CrossRefGoogle Scholar
  24. Staufenberger T, Thiel V, Wiese J, Imhoff JF (2008) Phylogenetic analysis of bacteria associated with Laminaria saccharina. FEMS Microbiol Ecol 64:65–77CrossRefPubMedGoogle Scholar
  25. Tatewaki M, Provasoli L, Pintner IJ (1983) Morphogenesis of Monostroma oxyspermum (Kütz.) Doty (Chlorophyceae) in axenic culture, especially in bialgal culture. J Phycol 19:409–416CrossRefGoogle Scholar
  26. Temmerman R, Scheirlinck I, Huys G, Swings J (2003) Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis. Appl Environ Microbiol 69:220–226CrossRefPubMedGoogle Scholar
  27. Turner JB, Friedmann EI (1974) Fine structure of capitular filaments in the coenocytic green alga Penicillus. J Phycol 10:125–134Google Scholar
  28. van den Hoek C, Mann DG, Jahns HM (1995) Algae. An introduction to phycology. Cambridge University Press, Cambridge, pp 419–428Google Scholar
  29. Weinberger F, Beltran J, Correa JA, Lion U, Pohnert G, Kumar N, Steinberg P, Kloareg B, Potin P (2007) Spore release in Acrochaetium sp. (Rhodophyta) is bacterially controlled. J Phycol 43:235–241CrossRefGoogle Scholar
  30. West JA, McBride DL (1999) Long-term and diurnal carpospore discharge patterns in the Ceramiaceae, Rhodomelaceae and Delesseriaceae (Rhodophyta). Hydrobiologia 398(399):101–113CrossRefGoogle Scholar
  31. Yu Z, Morrison M (2004) Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 70:4800–4806CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Joke Hollants
    • 1
    • 2
  • Frederik Leliaert
    • 2
  • Olivier De Clerck
    • 2
  • Anne Willems
    • 1
  1. 1.Laboratory of Microbiology, Biochemistry and Microbiology Department (WE10)Ghent UniversityGhentBelgium
  2. 2.Phycology Research Group and Center for Molecular Phylogenetics and Evolution, Biology DepartmentGhent UniversityGhentBelgium

Personalised recommendations