Open AccessComparison of techniques for counting prokaryotes in marine planktonic and biofilm samples
Author(s) -
Vanessa Ochi Agostini,
Letícia Rodrigues,
Alexandre José Macêdo,
Erik Muxagata
Publication year - 2021
Publication title -
scientia marina
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.51
H-Index - 68
eISSN - 1886-8134
pISSN - 0214-8358
DOI - 10.3989/scimar.05117.019
Subject(s) - biofilm , acridine orange , propidium iodide , staining , biology , stain , marine bacteriophage , bacteria , flow cytometry , fluorescence microscope , microbiology and biotechnology , microorganism , nucleic acid , fluorescence , biochemistry , apoptosis , genetics , physics , quantum mechanics , programmed cell death
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.