Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Epifluorescence Microscopy with Image Analysis as a Promising Method for Multispecies Biofilm Quantification

  • Ji Won Lee (Department of Microbiology, Pusan National University) ;
  • So-Yeon Jeong (Department of Microbiology, Pusan National University) ;
  • Tae Gwan Kim (Department of Microbiology, Pusan National University)
  • Received : 2022.09.28
  • Accepted : 2023.01.10
  • Published : 2023.03.28
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Abstract

Epifluorescence microscopy with image analysis was evaluated as a biofilm quantification method (i.e., quantification of surface area colonized by biofilms), in comparison with crystal violet (CV) staining. We performed different experiments to generate multispecies biofilms with natural and artificial bacterial assemblages. First, four species were inoculated daily in 16 different sequences to form biofilms (surface colonization, 0.1%-56.6%). Second, a 9-species assemblage was allowed to form biofilms under 10 acylase treatment episodes (33.8%-55.6%). The two methods comparably measured the quantitative variation in biofilms, exhibiting a strong positive relationship (R2 ≥ 0.7). Moreover, the two methods exhibited similar levels of variation coefficients. Finally, six synthetic and two natural consortia were allowed to form biofilms for 14 days, and their temporal dynamics were monitored. The two methods were comparable in quantifying four biofilms colonizing ≥18.7% (R2 ≥ 0.64), but not for the other biofilms colonizing ≤ 3.7% (R2 ≤ 0.25). In addition, the two methods exhibited comparable coefficients of variation in the four biofilms. Microscopy and CV staining comparably measured the quantitative variation of biofilms, exhibiting a strongly positive relationship, although microscopy cannot appropriately quantify the biofilms below the threshold colonization. Microscopy with image analysis is a promising approach for easily and rapidly estimating absolute quantity of multispecies biofilms.

Keywords

Acknowledgement

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A1A01065541).

References

  1. Battin TJ, Besemer K, Bengtsson MM, Romani AM, Packmann AI. 2016. The ecology and biogeochemistry of stream biofilms. Nat. Rev. Microbiol. 14: 251-263. https://doi.org/10.1038/nrmicro.2016.15
  2. Donlan RM. 2002. Biofilms: microbial life on surfaces. Emerg. Infect. Dis. 8: 881-890. https://doi.org/10.3201/eid0809.020063
  3. Flemming H-C and Wingender J. 2010. The biofilm matrix. Nat. Rev. Microbiol. 8: 623-633. https://doi.org/10.1038/nrmicro2415
  4. Flemming H-C and Wuertz S. 2019. Bacteria and archaea on Earth and their abundance in biofilms. Nat. Rev. Microbiol. 17: 247-260. https://doi.org/10.1038/s41579-019-0158-9
  5. Ramage G, Mowat E, Jones B, Williams C, Lopez-Ribot J. 2009. Our current understanding of fungal biofilms. Crit. Rev. Microbiol. 35: 340-355. https://doi.org/10.3109/10408410903241436
  6. Hall-Stoodley L, Costerton JW, and Stoodley P. 2004. Bacterial biofilms: from the natural environment to infectious diseases. Nat. Rev. Microbiol. 2: 95-108. https://doi.org/10.1038/nrmicro821
  7. Elias S and Banin E. 2012. Multi-species biofilms: living with friendly neighbors. FEMS Microbiol. Rev. 36: 990-1004. https://doi.org/10.1111/j.1574-6976.2012.00325.x
  8. Tolker-Nielsen T and Molin S. 2000. Spatial organization of microbial biofilm communities. Microb. Ecol. 40: 75-84. https://doi.org/10.1007/s002480000057
  9. Allison DG. 2003. The biofilm matrix. Biofouling 19: 139-150. https://doi.org/10.1080/0892701031000072190
  10. Stewart PS and Franklin MJ. 2008. Physiological heterogeneity in biofilms. Nat. Rev. Microbiol. 6: 199-210. https://doi.org/10.1038/nrmicro1838
  11. Azeredo J, Azevedo NF, Briandet R, Cerca N, Coenye T, Costa AR, et al. 2017. Critical review on biofilm methods. Crit. Rev. Microbiol. 43: 313-351. https://doi.org/10.1080/1040841X.2016.1208146
  12. Wilson C, Lukowicz R, Merchant S, Valquier-Flynn H, Caballero J, Sandoval J, et al. 2017. Quantitative and qualitative assessment methods for biofilm growth: A mini-review. Res. Rev. J. Eng. Technol. 6. PMID: 30214915.
  13. Stepanovic S, Vukovic D, Dakic I, Savic B, and Svabic-Vlahovic M. 2000. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J. Microbiol. Methods 40: 175-179. https://doi.org/10.1016/S0167-7012(00)00122-6
  14. Li X, Yan Z, Xu J. 2003. Quantitative variation of biofilms among strains in natural populations of Candida albicans. Microbiology 149: 353-362. https://doi.org/10.1099/mic.0.25932-0
  15. Ren D, Madsen JS, Sorensen SJ, Burmolle M. 2015. High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation. ISME J. 9: 81-89. https://doi.org/10.1038/ismej.2014.96
  16. Jeong S-Y and Kim TG. 2022. Effects of dispersal on species distribution, abundance, diversity and interaction in a bacterial biofilm metacommunity. J. Appl. Microbiol. 132: 459-469. https://doi.org/10.1111/jam.15194
  17. Stewart PS, Murga R, Srinivasan R, de Beer D. 1995. Biofilm structural heterogeneity visualized by three microscopic methods. Water Res. 29: 2006-2009. https://doi.org/10.1016/0043-1354(94)00339-9
  18. Reichhardt C and Parsek MR. 2019. Confocal laser scanning microscopy for analysis of Pseudomonas aeruginosa biofilm architecture and matrix localization. Front. Microbiol. 10: 677.
  19. Kim TG and Knudsen GR. 2011. Comparison of real-time PCR and microscopy to evaluate sclerotial colonisation by a biocontrol fungus. Fungal Biol. 115: 317-325. https://doi.org/10.1016/j.funbio.2010.12.008
  20. Djordjevic D, Wiedmann M, McLandsborough L. 2002. Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation. Appl. Environ. Microbiol. 68: 2950-2958. https://doi.org/10.1128/AEM.68.6.2950-2958.2002
  21. Staudt C, Horn H, Hempel D, Neu T. 2004. Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms. Biotechnol. Bioeng. 88: 585-592. https://doi.org/10.1002/bit.20241
  22. Kim TG, Yi T, Lee E-H, Ryu HW, Cho K-S. 2012. Characterization of a methane-oxidizing biofilm using microarray, and confocal microscopy with image and geostatic analyses. Appl. Microbiol. Biotechnol. 95: 1051-1059. https://doi.org/10.1007/s00253-011-3728-y
  23. Abramoff MD, Magalhaes PJ, Ram SJ. 2004. Image processing with ImageJ. Biophotonics Int. 11: 36-42.
  24. Noh YJ, Jeong S-Y, and Kim TG. 2021. Effects of different heterotrophic bacteria on phototrophic activity of Chlorella sp. MF1907. Microbiol. Biotechnol. Lett. 49: 101-110. https://doi.org/10.48022/mbl.2009.09001
  25. Jeong S-Y, Cho K-S, Kim TG. 2014. Density-dependent enhancement of methane oxidation activity and growth of methylocystis sp. by a non-methanotrophic bacterium Sphingopyxis sp. Biotechnol. Rep. 4: 128-133. https://doi.org/10.1016/j.btre.2014.09.007
  26. Jeong S-Y, Cho K-S, Kim TG. 2018. Adverse effect of the methanotroph Methylocystis sp. M6 on the non-methylotroph Microbacterium sp. NM2. J. Microbiol. Biotechnol. 28: 1706-1715. https://doi.org/10.4014/jmb.1804.04015
  27. O'Toole GA. 2011. Microtiter dish biofilm formation assay. J. Vis. Exp. 30: 2437.
  28. Shechtman O. 2013. The coefficient of variation as an index of measurement reliability, pp. 39-49. Methods of clinical epidemiology, Ed. Springer.
  29. Ludecke C, Jandt KD, Siegismund D, Kujau MJ, Zang E, Rettenmayr M, et al. 2014. Reproducible biofilm cultivation of chemostat-grown Escherichia coli and investigation of bacterial adhesion on biomaterials using a non-constant-depth film fermenter. PLoS One 9: e84837.
  30. Rumbaugh KP and Sauer K. 2020. Biofilm dispersion. Nat. Rev. Microbiol. 18: 571-586. https://doi.org/10.1038/s41579-020-0385-0
  31. Blackman IC and Frank JF. 1996. Growth of Listeria monocytogenes as a biofilm on various food-processing surfaces. J. Food Prot. 59: 827-831. https://doi.org/10.4315/0362-028X-59.8.827
  32. Wirtanen G and Mattila-Sandholm T. 1993. Epifluorescence image analysis and cultivation of foodborne biofilm bacteria grown on stainless steel surfaces. J. Food Prot. 56: 678-683. https://doi.org/10.4315/0362-028X-56.8.678
  33. Wang Z-W, Lee S-H, Elkins JG, and Morrell-Falvey JL. 2011. Spatial and temporal dynamics of cellulose degradation and biofilm formation by Caldicellulosiruptor obsidiansis and Clostridium thermocellum. AMB Express 1: 30.