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

Vanessa Ochi Agostini,Alexandre José Macedo,Letícia Terres Rodrigues,Erik Muxagata

doi:10.3989/scimar.05117.019

Abstract

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.

Highlights

Prokaryotes may be freely available in the water column as part of the plankton, interacting directly with chemical processes and playing a variety of roles in the food chain, or they may even be attached to living or non-living surfaces embedded in extracellular polymeric substances (EPS), forming microbial aggregates or biofilms, usually associated with matter and energy transport and biofouling (Kerstens et al 2015, Bunse and Pinhassi 2017, Agostini et al 2017)
The combined application of fluorescence staining and confocal laser scanning microscopy is useful for counting microbes in a biofilm sample, avoiding the loss of focus that is observed in traditional epifluorescence microscopy (EFM) for thick biofilm samples (Dang and Lovell 2002)
The ultrasound procedure removed 94% and 93% of biofilm bacterial density the from plywood substrates at 6 (p

Summary

Introduction

Prokaryotes may be freely available in the water column as part of the plankton, interacting directly with chemical processes and playing a variety of roles in the food chain (e.g. the microbial loop), or they may even be attached to living (organisms) or non-living (debris) surfaces embedded in extracellular polymeric substances (EPS), forming microbial aggregates or biofilms, usually associated with matter and energy transport and biofouling (Kerstens et al 2015, Bunse and Pinhassi 2017, Agostini et al 2017). Free-living and biofilm prokaryotes have ecological and economic importance, so accurate determination of their abundance and biomass are important in most microbiology applications (Alsharif and Godfrey 2002). Several methods have been proposed as alternatives for enumerating planktonic (free-living) and biofilm bacteria in natural aquatic environments and in laboratory assays (Boulos et al 1999). The ability to accurately estimate bacterial abundance and standing stock biomass in fresh and marine waters by inspecting bacterioplankton cells stained with a fluorochrome has contributed to the field of aquatic microbial ecology (Suzuki 1993). The combined application of fluorescence staining and confocal laser scanning microscopy is useful for counting microbes in a biofilm sample, avoiding the loss of focus that is observed in traditional EFM for thick biofilm samples (Dang and Lovell 2002). Microscopy techniques are time-consuming, requiring intrinsic preparation methods that can limit their use in routine analyses (Combs 2010, Beniac et al 2015)

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