View of the Underwater World: Water Analysis via Light Microscopy

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Example of water monitoring – the “River Hygiene” project

For routine water investigations, in addition to general abiotic values such as temperature, conductivity and pH values and biotic parameters such as chlorophyll values, the abundancy and diversity of living protists are also determined with the aid of easy-to-operate light microscopes. Here, the microscopes that are used are preferably portable and can be easily taken to the locations where the samples are taken.

This type of investigation takes place as part of the so-called “River Hygiene” project, a joint project conducted by the regional water resources management departments for sustainable water protection in Germany (ReWaM), which is sponsored by the Federal Ministry of Education and Research (BMBF) and as part of which water samples are taken across the whole of Germany from different watercourses (the Spree/Havel system in Berlin, the Ruhr in North Rhine-Westphalia, the Rhine and Moselle in Rhineland-Palatinate and the Isar and Ilz in Bavaria). In the urban area of Berlin (Spree/Havel system), sampling is performed together with water research institutes, the affected health and environmental authorities and the water supply and wastewater disposal companies (among others). The purpose of the “River Hygiene” joint project is to gain increased knowledge of the entry mechanisms and dynamics of hygienic burdens in flowing waters.

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Sampling, preparation and detection technology

The methodology adopted during the investigation of living protists involves taking water samples from the relevant bodies of water with a Ruttner water sampler (e.g. three replicates of 100 ml unfiltered water for analysis of the heterotrophic flagellates). These samples are then stored in a cool, dark location where they are protected from light until the microscopic investigation of the living organisms is performed. This investigation needs to take place as quickly as possible – within one hour of sampling – to prevent the risk of organisms dying and to minimize the chance of interaction between the organisms. For microscopic determination, multiple parallel samples of e.g. a 5 to 10 μl water sample are placed on a slide. Two cover glasses to the left and right and a third cover glass over the drop prevent the organisms from being crushed. The heterotrophic flagellates are investigated alive with 200 to 400-times magnification and with the aid of phase contrast (see Fig. 2). Phase contrast is essential for detection of the shape and movement of the flagella and of other special cell components (frills, loricas, ejectisomes etc.). Specific reference literature is consulted during determination of the flagellates (e.g. [5]).

Afterwards, the detected numbers of protists are extrapolated to the volume of 1 mm in order to estimate abundance and diversity. Based on the flagellate groups that are found, it is then possible to draw conclusions about the elimination of pathogens. Some organisms (e.g. kinetoplastids) consume fewer bacteria per unit of time than others (e.g. choanoflagellates with their strong swirling movements and relatively large flagellum, refer to [3] for the external differences).

By determining abundances, biovolumes and the relevant feeding types and with the aid of abiotic and biotic parameters and bacteria abundance data, light-microscopy water investigations of living single-cell organisms can supply initial approximate values for the assessment of bacterial consumption / protist grazing in the relevant waters under investigation. These consumption rates can be used as a basis for recommendations for action to improve the internal capacity of waters to eliminate harmful germs that are potentially present; these recommendations may then also potentially be transferable to other types of water bodies.

The more dissolved organic material (DOM) and bacteria that are present in a body of water and/or are transported from outside into a body of water e.g. as a result of severe precipitation events and overflowing sewage systems caused by this, the more protists will be detected in some cases. Protists feed on bacteria that, in turn, consume DOM. With the aid of simple, routinely used lens systems, these small organisms, which are often very underestimated in terms of their ecological and evolutionary significance, can be quickly determined and counted. By using easy-to-operate light microscopes, it is possible to draw valuable conclusions about the quality of bodies of water and gain new findings about potential measures for improving them..

Additional Information

Microscopy at 1000 meters below sea level

Since 2013, the German husband-and-wife team of Joachim and Kirsten Jakobsen have been diving with their 7.5 m long LULA 1000 submersible down into a fascinating world: the deep sea off the Azores. Only ten submarines in the world are capable of descending to a depth of one kilometer and withstanding the resulting pressure of 100 bar. Spotlights and good cameras open up this fascinating world – and recently also a routine Zeiss Primovert microscope.

Also “on board” is the author of this article, Prof. Hartmut Arndt. In cooperation with the Jakobsens, he is hoping to research the diversity of this previously largely ignored nanofauna together with his department at the Zoological Institute of the University of Cologne. The focus is on deep sea protists with a size of a few micrometers and their importance for the carbon balance of deep seas.

The Jakobsens offered the researchers from Cologne the ideal opportunity to observe and investigate these organisms in their natural environment. When the marine organisms swim past the Perspex window, the Jakobsens photograph and film them in as much detail as possible. Many species are yet to be documented and are still un-named. Single-cell organisms that are not visible to the naked eye can now be examined by the Jakobsens under a microscope. They use a type of water tap to capture the seawater directly inside the submarine. The samples are then investigated immediately on-site to check for single-cell organisms.

Recommended microscope equipment

The typical microscope contrast methods that are used for aquatic organisms are brightfield, phase contrast and darkfield. These can be set up easily with Zeiss Axio Lab.A1 and an achromatic-aplanatic universal condenser. The size of the protists ranges from 1 to 150 μm and can therefore be successfully detected with 10x, 20x and 40x lenses. Good optical correction (e.g. N-Achroplan lenses) is important for detailed detection of protist features. Phase contrast is essential for characterization, as it is used to visualize the cell components.

Differential interference contrast (DIC) can be used for even deeper insight into the cell interior, as this can contrast even the finer cell components. This method is particularly suited to the investigation of larger ciliates and amoebas.

References:

[1] Arndt, H., Dietrich, D., Auer, B., Cleven, E., Gräfenhan, T., Weitere, M., Mylnikov, A. (2000) Functional Diversity of Het-erotrophic Flagellates in Aquatic Ecosystems. In LeadbeaterBSC, Green JC (eds) The Flagellates. Taylor & Francis, London, 240 – 268.

[2] Azam, F., Fenchel, T., Field, J. G., Gray, J. S.,Meyer-Reil, L. A., Thingstad, F. (1983) The ecological role of water-column microbes in the sea. Marine Ecology Progress Series 10: 257 – 263.

[3] Boenigk, J., Arndt, H. (2000) Comparative studies on the feeding behavior of two heterotrophic nanoflagellates: the filter-feeding choanoflagellate Monosiga ovata and the raptorial-feeding kinetoplastid Rhynchomonas nasuta. Aquatic Microbial Ecology 22: 243 – 249.

[4] Deng, L., Krauss, S., Feichtmayer, .J, Hofmann, R., Arndt, H., Griebler, C. (2014) Grazing of heterotrophic flagellates on viruses driven by feeding behaviour. Environmental Microbiology Series 6: 325 – 330.

[5] Jeuck, A., Arndt, H. (2013) A short guide to common heterotrophic flagellates of freshwater habitats based on the morphology of living organisms. Protist 164: 842 – 860.

[6] Richter, D. J., King, N. (2013) The genomic and cellular foundations of animal origins. Annual Review of Genetics 47: 509 – 537.

* Dr. A. Jeuck, Prof. Dr. H. Arndt: Allgemeine Ökologie, Institut für Zoologie, Universität zu Köln 50674 Köln

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