Biopolym. Cell. 2011; 27(3):199-205.
Molecular and Cell Biotechnologies
Up-dating the Cholodny method using PET films to sample microbial communities in soil
1Moshynets O. V., 2Koza A., 2Dello Sterpaio P., 1Kordium V. A., 2, 3Spiers A. J.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. The SIMBIOS Centre,
    University of Abertay Dundee
    Bell St., Dundee DD1 1HG, UK
  3. Division of Forensics and Bio Sciences, School of Contemporary Sciences,
    University of Abertay Dundee
    Bell St., Dundee DD1 1HG, UK

Abstract

The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitats is essential to understand microbial associations and interactions in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension measurements; molecular techniques: DNA extraction, PCR; confocal laser scanning microscopy (CLSM); micro-focus X-ray computed tomography (µCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly, we demonstrate that µCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm and community development can be investigated. The use of these films with μCT imaging in soil will enable a better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this complex environment.
Keywords: Pseudomonas, soil, buried slide, PET film

References

[1] Compant S., Duffy B., Nowak J., Clement C., Barka E. A. Use of plant growth-promoting bacteria for biocontrol of plant diseases: principles, mechanisms of action, and future prospects Appl. Environ. Microbiol 2005 71, N 9 P. 4951–4959.
[2] Compant S., Clement C., Sessitsch A. Plant growth-promoting bacteria in the rhizoand endosphere of plants: their role, colonization, mechanisms involved and prospects for utilization Soil Biol. Biochem 2010 42, N 5 P. 669–678.
[3] Dubey S. K., Tripathi A. K., Upadhyay S. N. Exploration of soil bacteria communities for their potential as bioresource Bioresour. Technol 2006 97, N 17 P. 2217–2224.
[4] Fisk A. C., Murphy S. L., Tate R. L. Microscopic observations of bacterial sorption in soil cores Biol. Fertility Soils 1999 28, N 2 P. 111–116.
[5] Li Y., Dick W. A., Tuovinen O. H. Evaluation of fluorochromes for imaging bacteria in soil Soil Biol. Biochem 2003 35, N 6 P. 737–744.
[6] Kabir M., Chotte J. L., Rahman M., Bally R., Monrozies L. J. Distribution of soil fractions and location of soil bacteria in a vertisol under cultivation and perennial grass Plant Soil 1994 163, N 2 P. 243–255.
[7] Gilmore A. E. A soil sampling tube for soil microbiology Soil Sci 1959 87, N 2 P. 95–99.
[8] Dennis P. G., Miller A. J., Clark I. M., Taylor R. G., Valsami-Jones E., Hirsch P. R. A novel method for sampling bacteria on plant root and soil surfaces at the microhabitat scale J. Microbiol. Meth 2008 75, N 1 P. 12–18.
[9] Cholodny N. Uber eine neue Methode zur Untersuchung der Bodenmikroflora Arch. Microbiol 1930 1, N 1 P. 620–652.
[10] Cholodny N. G. A soil chamber as a method for the microscopic study of the soil microflora Arch. Microbiol 1934 5, N 1 P. 148–156.
[11] MacKintosh E. E., Patel J. D., Marchant R. E., Anderson J. M. Effects of biomaterial surface chemistry on the adhesion and biofilm formation of Staphylococcus epidermidis in vitro J. Biomed. Mater. Res 2006 78, N 4 P. 836–842.
[12] Tatchou-Nyamsi-Konig J. A., Dague E., Mullet M., Duval J. F., Gaboriaud F., Block J. C. Adhesion of Campylobacter jejuni and Mycobacterium avium onto polyethylene terephtalate (PET) used for bottled waters Water Res 2008 42, N 19 P. 4751– 4760.
[13] Wang J., Huang N., Yang P., Leng Y. X., Sun H., Liu Z. Y., Chu P. K. The effects of amorphous carbon films deposited on polyethylene terephthalate on bacterial adhesion Biomaterials 2004 25, N 16 P. 3163–3170.
[14] O'Donnell A. G., Young I. M., Rushton S. P., Shirley M. D., Crawford J. W. Visualization, modelling and prediction in soil microbiology Nat. Rev. Microbiol 2007 5, N 9 P. 689– 699.
[15] Baveye P. C., Laba M., Otten W., Bouckaert, L., Dello Sterpaio P., Goswami R. R., Grinev D., Houston A., Hu Y., Liu J., Mooney S., Pajor R., Sleutel S., Tarquis A., Wang W., Wei Q., Sezgin M. Observer-dependent variability of the thresholding step in the quantitative analysis of soil images and X-ray microtomography data Geoderma 2010 157, N 1–2 P. 51–63.
[16] Rainey P. B., Bailey M. J. Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome Mol. Microbiol 1996 19, N 3 P. 521–533.
[17] De Brujin I., de Kock M. J., Yang M., de Waard P., van Beek T. A., Raaijmakers J. M. Genome-based discovery, structure prediction and functional analysis of cyclic lipopeptide antibiotics in Pseudomonas species Mol. Microbiol 2007 63, N 2 P. 417–428.
[18] Koza A., Hallett P. D., Moon C. D., Spiers A. J. Characterization of a novel air-liquid interface biofilm of Pseudomonas fluorescens SBW25 Microbiology 2009 155, Pt 5 P. 1397–1406.
[19] Nelson K. E., Weinel C., Paulsen I. T., Dodson R. J., Hilbert H., Martins dos Santos V. A., Fouts D. E., Gill S. R., Pop M., Holmes M., Brinkac L., Beanan M., DeBoy R. T., Daugherty S., Kolonay J., Madupu R., Nelson W., White O., Peterson J., Khouri H., Hance I., Chris Lee P., Holtzapple E., Scanlan D., Tran K., Moazzez A., Utterback T., Rizzo M., Lee K., Kosack D., Moestl D., Wedler H., Lauber J., Stjepandic D., Hoheisel J., Straetz M., Heim S., Kiewitz C., Eisen J. A., Timmis K. N., Dusterhoft A., Tummler B., Fraser C. M. Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440 Environ. Microbiol 2002 4, N 12 P. 799– 808.
[20] King E. O., Ward M. K., Raney D. E. Two simple media for the demonstration of pyocyanin and fluorescin J. Lab. Clin. Med 1954 44, N 2 P. 301–307.
[21] Spiers A. J., Kahn S. G., Bohannon J., Travisano M., Rainey P. B. Adaptive divergence in experimental populations of Pseudomonas fluorescens. I. Genetic and phenotypic bases of wrinkly spreader fitness Genetics 2002 161, N 1 P. 33–46.
[22] Widmer F., Seidler R. J., Gillevet P. M., Watrud L. S., Di Giovanni G. D. A highly selective PCR protocol for detecting 16S rRNA genes of the genus Pseudomonas (sensu stricto) in environmental samples Appl. Environ. Microbiol 1998 64, N 7 P. 2545–2553.
[23] Hallett P. D., Young I. M. Changes to water repellence of soil aggregates caused by substrate-induced microbial activity Eur. J. Soil Sci 1999 50, N 1 P. 35–40.
[24] Johnson G. D., Nogueira Araujo G. M. A simple method of reducing the fading of immunofluorescence during microscopy J. Immunol. Meth 1981 43, N 3 P. 349–350.
[25] Espinosa-Urgel M., Salido A., Ramos J. L. Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds J. Bacteriol 2000 182, N 9 P. 2363–2369.
[26] Bos R., van der Mei H. C., Busscher H. J. Physico-chemistry of initial microbial adhesive interactions – its mechanisms and methods for study FEMS Microbiol. Rev 1999 23, N 2 P. 179–230.
[27] Bantinaki E., Kassen R., Knight C. G., Robinson Z., Spiers A. J., Rainey P. B. Adaptive divergence in experimental populations of Pseudomonas fluorescens. III. Mutational origins of wrinkly spreader diversity Genetics 2007 176, N 1 P. 441–453.
[28] Gottenbos B., Van der Mei H. C., Busscher H. J. Initial adhesion and surface growth of Staphylococcus epidermidis and Pseudomonas aeruginosa on biomedical polymers J. Biomed. Mater. Res 2000 50, N 2 P. 208–214.
[29] Moshynets O. V., Shpylova S. P., Spiers A. J., Kosakivska I. V. The phytosphere of Brassica napus L. as a niche for Pseudomonas fluorescens SBW25 Rep. Natl Acad. Sci. Ukraine 2010 N 12 P. 150–153.