Archives of Microbiology

, Volume 201, Issue 2, pp 259–266 | Cite as

High-resolution novel method for tracking bacteria in a multi-species biofilm

  • David PolakEmail author
  • Sharon Shany-Kdoshim
  • Liana Zaydel
  • Osnat Feuerstein
  • Yael Houri-Haddad
Short Communication


The aim of this study is to establish a novel high resolution tracking ability of a specific bacterium in multispecies biofilm. A periodontal multispecies biofilm was constructed with Streptococcus sanguis, Actinomyces naeslundii, Porphyromonas gingivalis and Fusobacterium nucleatum. A single species was stained with fluorescein isothiocyanate (FITC). The mature biofilm was stained for viability (propidium iodide) and analysis was performed with flow cytometry. The sensitivity of the assay was compared with colony forming units (CFU) counts. A single cell suspension of P. gingivalis was grown in broth and biofilm to identify the location of these events on side scatter and forward scatter. The sensitivity of the assay was comparable to that of the CFU counts. The assay allows quantification of the ratio of a single bacterium within the biofilm, and its viable proportion. The described method is reproducible and of high resolution, and allows the examination of microbes’ composition and viability within a biofilm structure.


Multispecies biofilm Flow cytometry Bacterial viability Periodontitis 



The study was self-supported.

Compliance with ethical standards

Conflict of interest

We hereby declare that this is an original manuscript, has not been sent elsewhere, do not have commercial or other associations that might pose a conflict of interest. All authors have contributed to, seen, and approved the final submitted version of the manuscript. The study was self-supported.


  1. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A (2017) Flow cytometry: basic principles and applications. Crit Rev Biotechnol 37(2):163–176. CrossRefGoogle Scholar
  2. Al-Ahmad A, Wunder A, Auschill TM, Follo M, Braun G, Hellwig E, Arweiler NB (2007) The in vivo dynamics of Streptococcus spp., Actinomyces naeslundii, Fusobacterium nucleatum and Veillonella spp. in dental plaque biofilm as analysed by five-colour multiplex fluorescence in situ hybridization. J Med Microbiol 56(Pt 5):681–687. CrossRefGoogle Scholar
  3. Cruz Olivo EA, Santos D, Elena de Lima M, Dos Santos VL, Sinisterra RD, Cortes ME (2016) Antibacterial effect of synthetic peptide LyeTxI and LyeTxI/betaCD association compound against planktonic and multispecies biofilms of periodontal pathogens. J Periodontol. Google Scholar
  4. Feuerstein O, Ginsburg I, Dayan E, Veler D, Weiss EI (2005) Mechanism of visible light phototoxicity on Porphyromonas gingivalis and Fusobacterium nucleatum. Photochem Photobiol 81(5):1186–1189. CrossRefGoogle Scholar
  5. Fontana CR, Song X, Polymeri A, Goodson JM, Wang X, Soukos NS (2015) The effect of blue light on periodontal biofilm growth in vitro. Lasers Med Sci. Google Scholar
  6. Guggenheim B, Giertsen E, Schupbach P, Shapiro S (2001) Validation of an in vitro biofilm model of supragingival plaque. J Dental Res 80(1):363–370CrossRefGoogle Scholar
  7. Guggenheim B, Gmur R, Galicia JC, Stathopoulou PG, Benakanakere MR, Meier A, Thurnheer T, Kinane DF (2009) In vitro modeling of host-parasite interactions: the ‘subgingival’ biofilm challenge of primary human epithelial cells. BMC Microbiol 9:280. CrossRefGoogle Scholar
  8. Hansen LB, Ren D, Burmolle M, Sorensen SJ (2017) Distinct gene expression profile of Xanthomonas retroflexus engaged in synergistic multispecies biofilm formation. ISME J 11(1):300–303. CrossRefGoogle Scholar
  9. Hope CK, Wilson M (2006) Biofilm structure and cell vitality in a laboratory model of subgingival plaque. J Microbiol Methods 66(3):390–398. CrossRefGoogle Scholar
  10. Maciorowski Z, Chattopadhyay PK, Jain P (2017) Basic multicolor flow cytometry. Curr Protocols Immunol 117:541–5438. CrossRefGoogle Scholar
  11. Rickard AH, Campagna SR, Kolenbrander PE (2008) Autoinducer-2 is produced in saliva-fed flow conditions relevant to natural oral biofilms. J Appl Microbiol 105(6):2096–2103. CrossRefGoogle Scholar
  12. Rudi K, Moen B, Dromtorp SM, Holck AL (2005) Use of ethidium monoazide and PCR in combination for quantification of viable and dead cells in complex samples. Appl Environ Microbiol 71(2):1018–1024. CrossRefGoogle Scholar
  13. Sanchez MC, Marin MJ, Figuero E, Llama-Palacios A, Leon R, Blanc V, Herrera D, Sanz M (2014) Quantitative real-time PCR combined with propidium monoazide for the selective quantification of viable periodontal pathogens in an in vitro subgingival biofilm model. J Periodontal Res 49(1):20–28. CrossRefGoogle Scholar
  14. Seinige D, Krischek C, Klein G, Kehrenberg C (2014) Comparative analysis and limitations of ethidium monoazide and propidium monoazide treatments for the differentiation of viable and nonviable campylobacter cells. Appl Environ Microbiol 80(7):2186–2192. CrossRefGoogle Scholar
  15. Shaddox LM, Alfant B, Tobler J, Walker C (2010) Perpetuation of subgingival biofilms in an in vitro model. Mol Oral Microbiol 25(1):81–87. CrossRefGoogle Scholar
  16. Thurnheer T, Gmur R, Guggenheim B (2004) Multiplex FISH analysis of a six-species bacterial biofilm. J Microbiol Methods 56(1):37–47CrossRefGoogle Scholar
  17. Valm AM, Mark Welch JL, Rieken CW, Hasegawa Y, Sogin ML, Oldenbourg R, Dewhirst FE, Borisy GG (2011) Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging. Proc Natl Acad Sci USA 108(10):4152–4157. CrossRefGoogle Scholar
  18. Walker C, Sedlacek MJ (2007) An in vitro biofilm model of subgingival plaque. Oral Microbiol Immunol 22(3):152–161. CrossRefGoogle Scholar
  19. Waters JC (2009) Accuracy and precision in quantitative fluorescence microscopy. J Cell Biol 185(7):1135–1148. CrossRefGoogle Scholar
  20. Zijnge V, van Leeuwen MB, Degener JE, Abbas F, Thurnheer T, Gmur R, Harmsen HJ (2010) Oral biofilm architecture on natural teeth. PloS One 5(2):e9321. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Periodontology, Hadassah Medical Center, Hadassah Faculty of Dental MedicineHebrew UniversityJerusalemIsrael
  2. 2.Department of Prosthodontics, Hadassah Faculty of Dental MedicineHebrew UniversityJerusalemIsrael

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