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.


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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. Sci. mar. [Internet]. 2021Sep.2 [cited 2024Feb.22];85(3):211-20. Available from:




Funding data

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