Investigation of bacterial cell parameters during the spring phytoplankton bloom 2020 at Helgoland / North Sea using fluorescence in situ hybridisation and epifluorescence microscopy

Antje.Wichels [ at ]


Temperate coastal environments like the German Bight of the North Sea are characterised by annually occurring spring phytoplankton blooms. In spring, when temperature and sunlight increae, phytoplankton grow to high abundances within a matter of days. This triggers the growth of heterotrophic bacteria, which are specialised in the degradation of algal-derived polysaccharides. Even though these blooms are highly variable phenomena, recurring patterns have been identified. Typically, catalysed reporter deposition fluorescence in situ hybridisation (CARDFISH) is used to identity and quantify the bacteria involved in phytoplankton blooms. CARD-FISH is favoured for applications on environmental samples because it produces exceptionally bright signals, even in very small and slow-growing cells. To date, it is not known if CARD-FISH can also be used as a reliable source for the analyses of additional cell parameters such as cell size and ribosomal content. This concern partially originates from an unknown factor during the signal amplification step. Without the exact value of this factor, the definite correlation between signal intensity and amount of ribosomal content cannot be determined. Another cause for this concern stems from the fluorescent tyramides used in the the amplification step. Occasionally, the tyramides can be found outside the cell, suggesting the signal leaked out of the cell. This can result in an overestimation of the cell size. This study aimed to investigate the influence of variable CARD-FISH signals on the measurements of signal area and signal intensity during the spring phytoplankton bloom of 2020 at Helgoland / North Sea. This was achieved by comparing CARDFISH and Tetra-FISH experiments. In Tetra-FISH experiments, four-fold fluorescent labelled oligonucleotide probes are used. These probes are not known to have the same signal variabilities as CARD-FISH. This thesis revealed that in both methods the signal area and intensity follow similar trends in the four investigated taxa Bacteroidetes, Candidatus Prosiliicoccus, Gammaproteobacteria, and SAR11. Additionally, measurements were examined in relation to the cell size, ribosomal content, cell counts and the cell cycle at large. Consistent with the current understanding of the bacterial cell cycle, cell numbers were found to increase after cells reached a maximum in size and ribosomal content. Observations concerning the growth rate agree with previous studies, showing that Bacteroidetes are the fastest growing, followed by Gammaproteobacteria, and SAR11 as the slowest-growing. The finding that the CARD-FISH does not bias measurements of different cell parameters opens the possibility of further spring phytoplankton bloom studies regarding additional cell parameters. The fact that only one CARD-FISH experiment is needed for these analyses also allows the comparison of such parameters, opening the possibility for further cell cycle investigations. Future studies could include the investigation of the frequency of dividing cells or the influence of the exposure times during the imaging of FISH samples.

Item Type
Thesis (Master)
Primary Division
Primary Topic
Helmholtz Cross Cutting Activity (2021-2027)
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Bauer, L. (2021): Investigation of bacterial cell parameters during the spring phytoplankton bloom 2020 at Helgoland / North Sea using fluorescence in situ hybridisation and epifluorescence microscopy , Master thesis, University of Bremen.

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