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Visualizing aquatic bacteria by light and transmission electron microscopy

Antonie van Leeuwenhoek volume 105, pages 1–14 (2014)Cite this article

Abstract

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

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References

  • Azam F (1998) Microbial control of oceanic carbon flux: the plot thickens. Science 280:694–696

    Article  CAS  Google Scholar 

  • Azam F, Fenchel T, Field JG, Gray JS, Meyer-Rell LA, Thingstad F (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 10:257–263

    Article  Google Scholar 

  • Barros NO, Farjalla VF, Soares MC, Melo RCN, Roland F (2010) Virus-bacterium coupling driven by both turbidity and hydrodynamics in an Amazonian floodplain lake. Appl Environ Microbiol 76(21):7194–7201. doi:10.1128/AEM.01161-10

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Beveridge TJ (1999) Structures of gram-negative cell walls and their derived membrane vesicles. J Bacteriol 181(16):4725–4733

    CAS  PubMed Central  PubMed  Google Scholar 

  • Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R (1999) LIVE/DEAD® BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Methods 37:77–86

    Article  CAS  PubMed  Google Scholar 

  • Chapin-Robertson K, Dahlberg SE, Edberg SC (1992) Clinical and laboratory analyses of cytospin-prepared Gram stains for recovery and diagnosis of bacteria from sterile body fluids. J Clin Microbiol 30(2):337–380

    Google Scholar 

  • Cowen JP (1992) Morphological study of marine bacterial capsules: implications for marine aggregates. Mar Biol 114:85–95

    Google Scholar 

  • Decamp O, Rajendran N (1998) Assessment of bacterioplankton viability by membrane integrity. Mar Pollut Bull 36(9):739–741

    Article  CAS  Google Scholar 

  • del Giorgio PA, Bird DF, Prairie YT, Planas D (1996) Flow cytometric determination of bacterial abundance in lanke plankton with the green nucleic acid SYTO 13. Limnol Oceanogr 41(4):783–789

    Article  Google Scholar 

  • Esteves FA, Thomas SM, Roland F (1994) Comparison of metabolism of two floodplain lakes of the Trombetas River (Pará, Brazil) based on a study of diel variation. Amazoniana XIII:33–46

    Google Scholar 

  • Ferrao-Filho A, Esteves FA (1994) Nutritive value and sedimentation rates of particulate matter in the course of two flood pulses in an Amazonian Várzea lake. Arch Hydrobiol 130:325–337

    Google Scholar 

  • Franklin RB, Campbell AH, Higgins CB, Barker MK, Brown BL (2011) Enumerating bacterial cells on bioadhesive coated slides. J Microbiol Methods 87(2):154–160. doi:10.1016/j.mimet.2011.08.013

    Article  CAS  PubMed  Google Scholar 

  • Freese HM, Karsten U, Schumann R (2006) Bacterial abundance, activity, and viability in the eutrophic River Warnow, northeast Germany. Microb Ecol 51(1):117–127. doi:10.1007/s00248-005-0091-5

    Article  CAS  PubMed  Google Scholar 

  • Gasol JM, Zweifel UL, Peters F, Fuhrman JA, Hagström Å (1999) Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria. Appl Environ Microbiol 65(10):4475–4483

    CAS  PubMed Central  PubMed  Google Scholar 

  • Grossart HP (2010) Ecological consequences of bacterioplankton lifestyles: changes in concepts are needed. Environ Microbiol Rep 2(6):706–714

    Article  PubMed  Google Scholar 

  • Hahn MW, Lunsdorf H, Janke L (2004) Exopolymer production and microcolony formation by planktonic freshwater bacteria: defence against protistan grazing. Aquat Microb Ecol 35:297–308

    Article  Google Scholar 

  • Hartmann M, Berditsch M, Hawecker J, Ardakani MF, Gerthsen D, Ulrich AS (2010) Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob Agents Chemother 54(8):3132–3142. doi:10.1128/AAC.00124-10

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Heissenberger A, Leppard GG, Herndl GJ (1996) Relationship between the intracellular integrity and the morphology of the capsular envelope in attached and free-living marine bacteria. Appl Environ Microbiol 62(12):4521–4528

    CAS  PubMed Central  PubMed  Google Scholar 

  • Higgins ML, Shockman GD, Ryter A (1971) Procaryotic cell division with respect to wall and membranes. Crit Rev Microbiol 1(1):29–72. doi:10.3109/10408417109104477

    Article  CAS  Google Scholar 

  • Higgins ML, Tsien HC, Daneo-Moore L (1976) Organization of mesosomes in fixed and unfixed cells. J Bacteriol 127(3):1519–1523

    CAS  PubMed Central  PubMed  Google Scholar 

  • Hobbie JE, Daley RJ, Jasper S (1977) Use of nucleopore filters for counting bacteria by fluorescence microscopy. Appl Environ Microbiol 33(5):1225–1228

    CAS  PubMed Central  PubMed  Google Scholar 

  • Hoppert M, Mayer F (1999) Principles of macromolecular organization and cell function in bacteria and archaea. Cell Biochem Biophys 31:247–285

    Article  CAS  PubMed  Google Scholar 

  • Joux F, Lebaron P (2000) Use of fluorescent probes to assess physiological functions of bacteria at single-cell level. Microbes Infect 2:1523–1535

    Article  CAS  PubMed  Google Scholar 

  • Kepner RL Jr, Pratt JR (1994) Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol Rev 58(4):603–615

    CAS  PubMed Central  PubMed  Google Scholar 

  • Kerfeld CA, Heinhorst S, Cannon GC (2010) Bacterial microcompartments. Annu Rev Microbiol 64:391–408

    Article  CAS  PubMed  Google Scholar 

  • Kirchman DL (1993) Statistical analysis of direct counts of microbial abundance. In: Kemp PF, Sherr BF, Sherr EB, Cole JJ (eds) Handbook of methods in aquatic microbial ecology, vol 1. Lewis Publishers, Florida, pp 117–120

    Google Scholar 

  • Kulp A, Kuehn MJ (2010) Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol 64:163–184. doi:10.1146/annurev.micro.091208.073413

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Li Z, Clarke J, Beveridge TJ (1998) Gram-negative bacteria produce membrane vesicles which are capable of killing other bacteria. J Bacteriol 180(20):5478–5483

    CAS  PubMed Central  PubMed  Google Scholar 

  • Li X, Feng HQ, Pang XY, Li HY (2008) Mesosome formation is accompanied by hydrogen peroxide accumulation in bacteria during the rifampicin effect. Mol Cell Biochem 311(1–2):241–247. doi:10.1007/s11010-007-9690-4

    Article  CAS  PubMed  Google Scholar 

  • Liberton M, Howard Berg R, Heuser J, Roth R, Pakrasi HB (2006) Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. Protoplasma 227(2-4):129–138. doi:10.1007/s00709-006-0145-7

    Article  PubMed  Google Scholar 

  • McBroom AJ, Kuehn MJ (2007) Release of outer membrane vesicles by Gram-negative bacteria is a novel envelope stress response. Mol Microbiol 63(2):545–558. doi:10.1111/j.1365-2958.2006.05522.x

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Melo RCN, Spencer LA, Perez SA, Ghiran I, Dvorak AM, Weller PF (2005) Human eosinophils secrete preformed, granule-stored interleukin-4 through distinct vesicular compartments. Traffic 6(11):1047–1057. doi:10.1111/j.1600-0854.2005.00344.x

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Melo RCN, Rosa PG, Noyma NP, Pereira WF, Tavares LER, Parreira GG, Chiarini-Garcia H, Roland F (2007) Histological approaches for high-quality imaging of zooplanktonic organisms. Micron 38(7):714–721

    Article  CAS  PubMed  Google Scholar 

  • Melo RCN, Spencer LA, Perez SA, Neves JS, Bafford SP, Morgan ES, Dvorak AM, Weller PF (2009) Vesicle-mediated secretion of human eosinophil granule-derived major basic protein. Lab Investig J Tech Methods Pathol 89(7):769–781. doi:10.1038/labinvest.2009.40

    Article  CAS  Google Scholar 

  • Miller KR (1979) Structure of a bacterial photosynthetic membrane. Proc Natl Acad Sci USA 76(12):6415–6419

    Article  CAS  PubMed  Google Scholar 

  • Moriarty DJW, Hayward AC (1982) Ultrastructure of bacteria and the proportion of gram-negative bacteria in marine sediments. Microb Ecol 8:1–14

    Article  CAS  PubMed  Google Scholar 

  • Nevot M, Deroncele V, Lopez-Iglesias C, Bozal N, Guinea J, Mercade E (2006) Ultrastructural analysis of the extracellular matter secreted by the psychrotolerant bacterium Pseudoalteromonas antarctica NF3. Microb Ecol 51(4):501–507. doi:10.1007/s00248-006-9065-5

    Article  PubMed  Google Scholar 

  • Paul JH (1982) Use of Hoestcht Dyes 33258 and 33342 for enumeration of attached and planktonic bacteria. Appl Environ Microbiol 43(4):939–944

    CAS  PubMed Central  PubMed  Google Scholar 

  • Pomeroy LR (1974) The ocean food web a changing paradigm. Bioscience 24:499–504

    Article  Google Scholar 

  • Pomeroy LR, Williams PJL, Azam F, Hobbie JE (2007) The microbial loop. Oceanography 20(2):28–33

    Article  Google Scholar 

  • Porter KS, Feig YS (1980) The use of DAPI for identifying and counting aquatic microflora. Limnol Oceanogr 25(5):943–948

    Article  Google Scholar 

  • Roberts IS (1996) The biochemistry and genetics of capsular polysaccharide production in bacteria. Annu Rev Microbiol 50:285–315

    Article  CAS  PubMed  Google Scholar 

  • Romanova ND, Sazhin AF (2011) Methodological aspects of the determination of the bacterioplankton number, biomass, and production. Oceanology 51(3):518–527

    Article  Google Scholar 

  • Ryter A (1968) Association of the nucleus and the membrane of bacteria: a morphological study. Bacteriol Rev 32(1):39–54

    CAS  PubMed Central  PubMed  Google Scholar 

  • Santhana Raj L, Hing HL, Omar B, Hamidah T, Aida Suhana R, Nor Asiha CP, Paramsarvaram S, Sumarni G, Hanjeet K (2007) Mesosomes are a definite event in antibiotic-treated Staphylococcus aureus ATCC 25923. Trop Biomed 24(1):105–109

    CAS  PubMed  Google Scholar 

  • Sawstrom C, Pearce I, Davidson AT, Rosen P, Laybourn-Parry J (2008) Influence of environmental conditions, bacterial activity and viability on the viral component in 10 Antarctic lakes. FEMS Microbiol Ecol 63(1):12–22

    Article  PubMed  Google Scholar 

  • Schooling SR, Beveridge TJ (2006) Membrane vesicles: an overlooked component of the matrices of biofilms. J Bacteriol 188(16):5945–5957. doi:10.1128/JB.00257-06

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Seo EY, Ahn TS, Zo YG (2010) Agreement, precision, and accuracy of epifluorescence microscopy methods for enumeration of total bacterial numbers. Appl Environ Microbiol 76(6):1981–1991. doi:10.1128/AEM.01724-09

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Smith EM, Del Giorgio PA (2003) Low fractions of active bacteria in natural aquatic communities? Aquat Microb Ecol 31:203–208

    Article  Google Scholar 

  • Soares MC, Lobão LM, Vidal LO, Noyma NP, Barros NO, Cardoso SJ, Roland F (2011) Light microscopy in aquatic ecology: methods for plankton communities studies. Methods Mol Biol 689:215–227

    Article  PubMed  Google Scholar 

  • Stoderegger KE, Herndl GJ (2001) Visualization of the exopolysaccharide bacterial capsule and its distribution in oceanic environments. Aquat Microb Ecol 26:195–199

    Article  Google Scholar 

  • Stokes BO (2004) Principles of cytocentrifugation. Lab Med 35(7):434–437. doi:10.1309/ftt59gwkdwh69fb0

    Article  Google Scholar 

  • Thompson SS, Naidu YM, Pestka JJ (1985) Ultrastructural localization of an extracellular protease in pseudomonas fragi by using the peroxidase–antiperoxidase reaction. Appl Environ Microbiol 50(4):1038–1042

    CAS  PubMed Central  PubMed  Google Scholar 

  • Wang Y, Hammes F, De Roy K, Verstraete W, Boon N (2010) Past, present and future applications of flow cytometry in aquatic microbiology. Trends Biotechnol 28(8):416–424. doi:10.1016/j.tibtech.2010.04.006

    Article  CAS  PubMed  Google Scholar 

  • Yurkov VV, Beatty JT (1998) Aerobic anoxygenic phototrophic bacteria. Microb Mol Biol 62(3):695–724

    CAS  Google Scholar 

  • Zweifel UL, Hagstrom A (1995) Total counts of marine bacteria include a large fraction of non-nucleoid-containing bacteria (ghosts). Appl Environ Microbiol 61(6):2180–2185

    CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The work of the authors is supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil).

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Authors and Affiliations

  1. Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, 36036-900, Brazil

    Thiago P. Silva, Thabata L. A. Duque, Juliana P. Gamalier & Rossana C. N. Melo

  2. Laboratory of Aquatic Ecology, Department of Biology, Federal University of Juiz de Fora (UFJF), Juiz de Fora, MG, 36036-900, Brazil

    Natália P. Noyma, Luciana O. Vidal, Lúcia M. Lobão & Fábio Roland

  3. Laboratory of Structural Biology and Reproduction, Department of Morphology, Federal University of Minas Gerais (UFMG), Belo Horizonte, MG, 31.270-901, Brazil

    Hélio Chiarini-Garcia

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  1. Thiago P. Silva
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  2. Natália P. Noyma
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  3. Thabata L. A. Duque
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  4. Juliana P. Gamalier
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  5. Luciana O. Vidal
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  8. Fábio Roland
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  9. Rossana C. N. Melo
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Correspondence to Rossana C. N. Melo.

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Silva, T.P., Noyma, N.P., Duque, T.L.A. et al. Visualizing aquatic bacteria by light and transmission electron microscopy. Antonie van Leeuwenhoek 105, 1–14 (2014). https://doi.org/10.1007/s10482-013-0047-6

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  • DOI: https://doi.org/10.1007/s10482-013-0047-6

Keywords

  • Aquatic bacteria
  • Ultrastructure
  • Fluorescence microscopy
  • Aquatic ecosystems
  • Cell viability
  • Electron microscopy