Comparison of techniques for counting prokaryotes in marine planktonic and biofilm samples


  • Vanessa Ochi Agostini Laboratory of Zooplankton, Universidade Federal do Rio Grande (FURG), Institute of Oceanography (IO) - Laboratory of Biofilms and Microbial Diversity, Universidade Federal do Rio Grande do Sul (UFRGS) - Programa de Pós-Doutorado Empresarial do Conselho Nacional de Desenvolvimento Científico e Tecnológico (PDI-CNPq), Regenera Moléculas do Mar
  • Letícia Terres Rodrigues Laboratory of Cell Culture, Universidade Federal do Rio Grande do Sul (UFRGS)
  • Alexandre José Macedo Laboratory of Biofilms and Microbial Diversity, Universidade Federal do Rio Grande do Sul (UFRGS), Pharmacy Faculty and Biotechnology Centre
  • Erik Muxagata Laboratory of Zooplankton, Universidade Federal do Rio Grande (FURG), Institute of Oceanography (IO)



ecology, bacteria enumeration, flow cytometry, microbial methods, microscopy


Though a large number of techniques are available for the study of aquatic bacteria, the aim of this study was to establish a technique for analysing free-living and biofilm prokaryotic cells through laboratory assays. In particular, we wished to analyse the efficiency of ultrasound to detach and disrupt biofilm, to obtain an efficient stain treatment for quantifying free-living and biofilm prokaryotes in flow cytometry (FC), and to compare epifluorescence microscopy (EFM), scanning electron microscopy (SEM) and FC for quantifying free-living and biofilm prokaryotes#. Marine-grade plywood substrates were immersed in natural marine water that was conditioned for 12 days. At 6 and 12 days, water aliquots and substrates were removed to estimate free-living and biofilm prokaryote density. Ultrasound efficiently removed marine biofilm from substrates (up to 94%) without cell damage. FC analysis (unstained) reliably quantified marine plankton and young or mature biofilm prokaryotes compared with other staining (acridine orange, 4′,6-diamidino-2-phenylindole, propidium iodide and green fluorescent nucleic acid), EFM or SEM techniques. FC and SEM achieved similar results, while a high variability was observed in the EFM technique. FC was faster and more precise than SEM because the count is not dependent on the observer.


Download data is not yet available.


Agostini V.O., Macedo A.J., Muxagata E.M. 2016. Evaluation of antibiotics as a methodological procedure to inhibit free-living and biofilm bacteria in marine zooplankton culture. An. Acad. Bras. Cienc. 88: 733-746. PMid:27168369

Agostini V.O., Ritter M.N., Macedo A.J., et al. 2017. What determines sclerobiont colonization on marine mollusk shells? PLoS ONE 12: e0184745. PMid:28902894 PMCid:PMC5597280

Agostini V.O., Macedo A.J., Muxagata E.M. 2018a. Inhibition of biofilm bacteria and adherent fungi from marine plankton cultures using an antimicrobial combination. Inter. Aquatic. Res. 10: 165-177.

Agostini V.O., Macedo A.J., Muxagata E.M. 2018b. Effect of antimicrobials, salinity, and contamination by air on bacterial and fungal growth in cyprid cultures of Amphibalanus improvisus. Mar. Ecol. 39: e12523.

Alsharif R., Godfrey W. 2002. Bacterial Detection and Live/Dead Discrimination by Flow Cytometry. BD Biosciences, San Jose, CA, 6 pp.

Amalfitano S., Fazi S. 2008. Recovery and quantification of bacterial cells associated with streambed sediments. J. Microbiol. Meth. 75: 237-243. PMid:18602952

Ambriz-Aviña V., Contreras-Garduño J.A., Pedraza-Reyes M. 2014. Applications of flow cytometry to characterize bacterial physiological responses. BioMed Res. Int. 14: 461941. PMid:25276788 PMCid:PMC4174974

Beniac D.R., Hiebert S.L., Siemens C.G., et al. 2015. A mobile biosafety microanalysis system for infectious agents. Sci. Rep. 5: 9505. PMid:25820944 PMCid:PMC4377622

Berney M., Hammes F., Bosshard F., et al. 2007. Assessment and interpretation of bacterial viability by using the LIVE/DEAD BacLight kit in combination with flow cytometry. Appl Environ Microbiol 73: 3283-3290. PMid:17384309 PMCid:PMC1907116

Boulos L., Prévost M., Barbeau B., et al. 1999. LIVE/DEAD BacLightE: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J. Microbiol. Methods 37: 77-86.

Bouvier T., Troussellier M., Anzil A., et al. 2011. Using light scatter signal to estimate bacterial Bbiovolume by flow cytometry. Cytometry 44: 188-194.<188::AID-CYTO1111>3.0.CO;2-C

Bunse C., Pinhassi J. 2017. Marine bacterioplankton seasonal succession dynamics. Trends Microbiol 25: 494-505. PMid:28108182

Combs C.A. 2010. Fluorescence microscopy: a concise guide to current imaging methods. Curr. Protoc. Neurosci. 2: Unit2.1. PMid:20066655 PMCid:PMC3805368

Cos P., Tote K., Horemans T., et al. 2010. Biofilms: an extra hurdle for effective antimicrobial therapy. Curr Pharm Des 16: 2279-2295. PMid:20433417

Crawford G.N.C, Barer R. 1951. The Action of Formaldehyde on Living Cells as Studied by Phase-contrast Microscopy. Q. J. Microsc. Sci., 92(part 4): 403-52.

Dang H., Lovell C.R. 2002. Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization. Appl. Environ. Microbiol. 68: 496-504. PMid:11823183 PMCid:PMC126732

Davey H.M., Kell D.B. 1996. Flow cytometry and cell sorting of heterogeneous microbial populations: the importance of single cell analyses. Microbiol. Rev. 60: 641-696. PMid:8987359 PMCid:PMC239459

Davey H.M., Kell D.B. 1997. Fluorescent brighteners: novel stains for the flow cytometric analysis of microorganisms. Cytometry 28: 311-315.<311::AID-CYTO6>3.0.CO;2-E

Falcioni T., Papa S., Gasol J.M. 2008. Evaluating the flow-cytometric nucleic acid double-staining protocol in realistic situations of planktonic bacterial death. Appl. Environ. Microbiol. 74: 1767-1779. PMid:18223113 PMCid:PMC2268295

Felip M., Andreatta S., Sommaruga R., et al. 2007. Suitability of flow cytometry for estimating bacterial biovolume in natural plankton samples: comparison with microscopy data. Eng. Fail. Anal. 73: 4508-4514. PMid:17513595 PMCid:PMC1932810

Fischer E.R., Hansen B.T., Nair V., et al. 2012. Scanning electron microscopy. Curr. Protoc. Microbiol. 2B: 2. PMid:22549162 PMCid:PMC3352184

Flemming H-C., Wingender J. 2010. The biofilm matrix. Nat. Rev. 8: 623-633. PMid:20676145

Franklin R.B., Campbell A.H., Higgins C.B., et al. 2011. Enumerating bacterial cells on bioadhesive coated slides. J. Microbiol. Methods 87: 154-160. PMid:21893107

Freitas V. da R., Sand S.T., Simonetti A.B. 2010. Formação in vitro de biofilme por Pseudomonas aeruginosa e Staphylococcus aureus na superfície de canetas odontológicas de alta rotação. Revista de Odontologia da UNESP 39: 193-200.

Gant V.A., Warnes G., Phillips I., et al. 1993. The application of flow cytometry to the study of bacterial responses to antibiotics. J. Med. Microbiol. 39: 147-154. PMid:8345510

Garren M., Azam F. 2010. New method for counting bacteria associated with coral mucus. Appl. Environ. Microbiol. 76: 6128-6133. PMid:20656857 PMCid:PMC2937480

Gasol J.M., Giorgio P.A. del. 2000. Using flow cytometry for counting natural planktonic bacteria and understanding the structure of planktonic bacterial communities. Sci. Mar. 64: 197-224.

Golladay S.W., Sinsabaugh R.L. 1991. Biofilm development on leaf and wood surfaces in a boreal river. Freshwater Biol. 25: 437-450.

Harrison J.J., Ceri H., Yerly J., et al. 2006. The use of microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms cultivated in the Calgary Biofilm Device. Biol. Proced. 8: 194-215. PMid:17242736 PMCid:PMC1779619

Hobbie J.E., Daley R., Jasper S. 1977. Use of Nuclepore filters for counting bacteria by fluorescence microscopy. Appl. Environ. Microbiol. 33: 1225-1228. PMid:327932 PMCid:PMC170856

Jin Y., Zhang T., Samaranayake Y.H., et al. 2005. The use of new probes and stains for improved assessment of cell viability and extracellular polymeric substances in Candida albicans biofilms. Mycopathologia 159: 353-360. PMid:15883718

Jochem F.J. 2001. Morphology and DNA content of bacterioplankton in the western Gulf of Mexico: Analysis by epifluorescence microscopy and flow cytometry. Aquat. Microb. Ecol. 25: 179-194.

Kepner R.L.Jr., Pratt J.R. 1994. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol. Mol. Biol. Rev. 58: 603-615. PMid:7854248 PMCid:PMC372983

Kerstens M., Boulet G., Van Kerckhoven M., et al. 2015. A flow cytometric approach to quantify biofilms. Folia Microbiol. 60: 335-342. PMid:25948317

Kim S.K., Lee J.H. 2016. Biofilm dispersion in Pseudomonas aeruginosa. J. Microbiol. 54: 71-85. PMid:26832663

Lopes L.F.P., Agostini V.O., Guimarães S.S, et al. 2018. Evaluation of the effect of antimicrobials in marine cultures, using the copepod Acartia tonsa as a bioindicator. Chem. Ecol. 34: 747-761.

McKenzie A.T. 2019. Glutaraldehyde: A review of its fixative effects on nucleic acids, proteins, lipids, and carbohydrates.

Mohammed M.M.A., Nerland A.H., Al-Haroni M., et al. 2013. Characterization of extracellular polymeric matrix, and treatment of Fusobacterium nucleatum and Porphyromonas gingivalis biofilms with DNase I and proteinase K. J. Oral Microbiol. 5: 20015. PMid:23372876 PMCid:PMC3559756

Monfort P., Baleux B. 1992. Comparison of flow cytometry and epifluorescence microscopy for counting bacteria in aquatic ecosystems. Cytometry 13: 188-192. PMid:1547667

Muthukrishnan T., Govender A., Dobretsov S., et al. 2017. Evaluating the reliability of counting bacteria using epifluorescence microscopy. J. Mar. Sci. Eng. 5: 4.

Nebe-von-Caron G., Stephens P.J., Badley R.A. 1999. Bacterial detection ad differentiation by cytometry and fluorescent probes. Proc. Royal Soc. Lond. 34: 321-327.

Nebe-von-Caron G., Stephens P.J., Hewitt C.J., et al. 2000. Analysis of bacterial function by multi-colour fluorescence flow cytometry and single cell sorting. J. Microbiol. Methods 42: 97-114.

Oliveira S.S., Wasielesky Jr W.F.B., Ballester E.L.C., et al. 2006. Caracterização da assembléia de bactérias nitrificantes pelo método "Fluorescent in situ Hybridization" (FISH) no biofilme e água de larvicultura do Camarão-rosa Farfantepenaeus paulensis. Atlântica 28(1): 33-45.

Ophus M. 2014. Bacterial community dynamics in a biofilter exposed to a micropollutant. Norwegian University of Science and Technology. 123 pp.

Oulahal N., Martial-Gros A., Bonneau M., et al. 2004. Combined effect of chelating agents and ultrasound on biofilm removal from stainless steel surfaces. Application to "Escherichia coli milk" and "Staphylococcus aureus milk" biofilms. Biofilms 1: 65-73.

Parthasarathy R. 2018. Monitoring microbial communities using light sheet fluorescence microscopy. Curr. Opin. Microbiol. 43: 31-37. PMid:29175679 PMCid:PMC5963963

Porter K.G., Feig Y.S. 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25: 943-948.

Posch T., Loferer-Krößbacher M., Gao G., et al. 2001. Precision of bacterioplankton biomass determination: a comparison of two fluorescent dyes, and of allometric and linear volume-to-carbon conversion factors. Aquat. Microb. Ecol. 25: 55-63.

R Core Team. R. 2017. A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL . 2017.

Sailer M.F., van Nieuwenhuijzen E.J., Knol W. 2010. Forming of a functional biofilm on wood surfaces. Ecol Eng 36: 163-167.

Sgier L., Freimann R., Zupanic A., et al. 2016. Flow cytometry combined with viSNE for the analysis of microbial biofilms and detection of microplastics. Nat. Commun. 7: 11587. PMid:27188265 PMCid:PMC4873979

Shapiro H.M., Nebe-Von-Caron G. 2004. Multiparameter flow cytometry of bacteria. Methods Mol. Biol. 263: 33-44.

Shi L., Günther S., Hübschmann T., et al. 2007. Limits of propidium iodide as a cell viability indicator for environmental bacteria. Cytometry Part A 71A: 592-598. PMid:17421025

Suzuki M.T. 1993. DAPI direct counting underestimates bacterial abundances and average cell size compared to AO direct counting. Limnol. Oceanogr. 38: 1566-1570.

Troussellier M., Courties C., Lebaron P., et al. 1999. Flow cytometric discrimination of bacterial populations in seawater based on SYTO 13 staining of nucleic acids. FEMS Microbiol. Ecol. 29: 319-330.

Walberg M., Gaustad P., Steen H.B. 1996. Rapid flow cytometric assessment of mecillinam and ampicillin bacterial susceptibility. J. Antimicrob. Chemother 37: 1063-1075. PMid:8836810

Xu J., Bigelow T.A., Halverson L.J., et al. 2012. Mechanical destruction of Pseudomonas aeruginosa biofilms by ultrasound exposure. AIP Conf. Proc. 1481: 463-468.

Yu M., Wu L., Huang T., et al. 2015. Rapid detection and enumeration of total bacteria in drinking water and tea beverages using a laboratory-built high-sensitivity flow cytometer. Anal Methods 7: 3072-3079.

Zhang R., Neu T.R., Zhang Y., et al. 2015. Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus. Appl. Microbiol. Biotechnol. 99: 7343-7356. PMid:26169631

Zimmerman R., Meyer-Reil L-A. 1974. A new method for fluorescence staining of bacterial populations on membrane filters. Kiel Meeresforsch. 30: 24-27.



How to Cite

Ochi Agostini V, Terres Rodrigues L, Macedo AJ, Muxagata E. Comparison of techniques for counting prokaryotes in marine planktonic and biofilm samples. scimar [Internet]. 2021Sep.2 [cited 2021Sep.27];85(3):211-20. Available from:




Funding data

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Grant numbers 11/2014 (6/2551-000244-4)