Marine Microbial Ecology

How are microbes studied?

Various techniques have to be used for studying microbes, for no one technique can measure everything. All are selective and have particular advantages and disadvantages.

Microscopy

Light microscopy

Both conventional compound microscopes and inverted microscopes are used to examine samples. These are used in transmitted light, Normaski differential interference contrast, phase contrast and epifluorescent modes to distinguish cellular organelles, appendages, and stained or autofluorescent cells (red autofluorescence typically denotes cellular chlorophyll).

Images are obtained using fine grained photographic film in microscope-mounted cameras or broadcast-quality video.

Living organisms are routinely examined at sea using microscopes that are isolated from the ship's vibrations. Live material can also be examined using microscopes in a cold (0ºC) room.

Light micrographs (LM) of various organisms are shown below.

LM of the Antarctic diatom Corethron criophilum
Image: Harvey Marchant

LM of the diatom Asteromphalus sp.
Image: Fiona Scott

The faint rings around the Thaumatomastix cells are surface body scales, easily seen using an electron microscope (see TEM image)
Image: Fiona Scott

Two euglenoid cells with ingested diatoms
Image: Fiona Scott

Click on image below to view QuickTime movie

Cells of the diatom Corethron criophilum. Image: Harvey Marchant

Light microscopy allows direct identification of microplankton but does not have sufficient resolution to identify most nanoplankton (especially those smaller than 10 µm). It allows cell behaviour (swimming, feeding etc.) to be studied as well as direct counts of phytoplankton. However, plankton counting is extremely time consuming, and "a task which cannot be completed without ruin of mind and body" (Haeckel, 1890).

Fluorescent stains are used to distinguish live and dead bacteria (BacLite) and highlight DNA and RNA of protists, bacteria and viruses (SYBR Green 1).

Electron microscopy

Electron microscopy is vital for the identification of small cells as well as examining details of larger cells. Much of the taxonomy of protists is based on fine structural details such as scales, flagella, surface patterning etc.

Scanning electron microscopy (SEM) shows the surface detail of cells.

The diatom Thalassiosira gravida has intricate surface ornamentation
Image: Fiona Scott

Hooks at one end of the common Antarctic diatom, Corethron criophilum
Image: Fiona Scott

SEM of the flagellated prasinophyte Pyramimonas gelidicola
Image: Sandy Melloy

SEM of the dinoflagellate Protoperidinium defectum
Image: Fiona Scott

Kakoeca antarctica, an endemic antarctic choanoflagellate
Image: Fiona Scott

Emiliania huxleyi a Southern Ocean coccoithophorid
Image: Fiona Scott

Transmission electron microscopy (TEM) is used to examine either thin slices of material to show internal details of cells, or shadow-cast material to reveal fine surface structures of scales, flagella and other external cell components.

Thaumatomastix sp. is covered by intricate body scales
Image: Fiona Scott

Organic body scales of Chrysochromulina vexillifera
Image: Fiona Scott

Details of choanoflagellate lorica as seen with TEM
Image: Fiona Scott

Scales from the flagellum and body of Pyramimonas sp.
Image: Rick van den Enden

Flow cytometry

A Flow Cytometer analyses particles by passing them in single file through a laser beam. It can count up to 1000 cells per second, measuring for each one the light scattering properties (indicating size and complexity), as well as yellow-green, orange, and red fluorescence. Autofluorescence (from chlorophylls and other pigments) as well as the use of fluorescent stains help distinguish cell types.

The capacity to rapidly count large numbers of particles greatly increases the reliability and precision of cell concentration estimates, however the cells are not directly identified and must be examined by microscopy.

Click on image below to view a larger version

Flow cytometer output of BacLight® stained marine bacteria. BacLight® is a dual nucleic acid stain that causes live bacteria to fluoresce green and dead bacteria to fluoresce red when excited with the 488 nm argon laser of the flow cytometer. The upper coloured plot shows the dead bacterial population in red and the live in green. These populations are also shown as a contour plot (upper right). The lower plots show these populations as counts of green (FL1) and red (FL3) fluorescent particles.

Pigment analysis

Phytoplankton, like all photosynthetic plants, use chlorophylls and carotenoids to absorb light for photosynthesis. Chlorophyll a, or a derivative, is present in all types of phytoplankton and is commonly used as an indicator of the phytoplankton biomass. It enables living phytoplankton to be distinguished from zooplankton, detritus and dead phytoplankton. It is highly fluorescent and can be measured in unconcentrated seawater samples. Other chlorophylls and carotenoids are present in phytoplankton, many of which have restricted taxonomic distribution. These pigments can be used as quantitative markers for particular taxonomic groups.

Fluorometry

The concentration of chlorophyll a in surface waters is continuously monitored on cruises of the Aurora Australis by measuring the fluorescence of water pumped from an intake at 7m depth. The temperature and salinity of this water is measured simultaneously.

Vertical profiles of chlorophyll a are measured with a fluorometer attached to the CTD. This shows the vertical distribution of phytoplankton much better than discrete samples from the rosette sampler (typically collected at 15m intervals).

Note however that fluorescence is reduced by sunlight and the response per unit chlorophyll varies during the day.

Ratio of chlorophyll content measured by HPLC (correct) divided by the Fluorescence (estimate) changes dramatically with the light intensity (PAR = photosynthetically active radiation, µE.m-2.sec-1).

HPLC

HPLC (high performance liquid chromatography) is used to separate, identify and quantify the various chlorophylls and carotenoids in phytoplankton. Many of these are markers for particular taxa and can be used to estimate their contributions to the phytoplankton community.

HPLC set up in fume cupboard

HPLC is an excellent technique for mapping populations, since it is feasible to analyse more than 1000 samples per cruise. However it does not identify taxa directly and must be combined with microscopy to determine the key species present.

Special software (CHEMTAX) was developed collaboratively with CSIRO Marine Research to calculate the relative contributions of different groups of phytoplankton from the pigment content of field samples.

Pigment analysis in oceanography

Feeding experiments

Studies of feeding by protists that are heterotrophic (dependent on external food) or mixotrophic (can use photosynthesis or external food) use two approaches:

First approach

Uptake of fluorescently labelled particles (dextrans, bacteria, latex microspheres, algae) is detected using epifluorescent microscopy or flow cytometry. Results show the size spectrum and uptake rate of food available to protists. Cultures of the choanoflagellate Acanthocorbis unguiculata have been incubated with fluorescent microspheres as a surrogate food source. Smaller microspheres (0.25 µm diameter) are ingested faster, and by a larger percentage of cells, than larger FM (0.5 and 1.0 µm diameter).

SEM of the choanoflagellate Acanthocorbis unguiculata
Image: John van den Hoff

Left: Eight cells of Acanthocorbis unguiculata under transmitted light illumination
Right: The same eight cells under blue light fluorescence illumination. Each cell has attracted fluorescent microspheres (the minute yellow-green dots) which have become attached to the lorica and ingested into the cell body
Images: Fiona Scott

Time course graph of the percentage of cells of Acanthocorbis unguiculata ingesting Fluorescent Microspheres (FM) of various diameters

Click on image below to view QuickTime movie

Ciliate after consuming fluorescent microbeads - transmittance then fluorescence microscopy. Image: Harvey Marchant

Second approach

The growth rate of phytoplankton and the grazing rate of protozoa and zooplankton upon them can be measured simultaneously by incubating a series of water samples with various dilutions with filtered seawater. In brief, the phytoplankton grow at the same rate irrespective of dilution, but the grazing rate declines at higher dilutions because the food particles are further apart and harder to find. Extrapolation to infinite dilution estimates the phytoplankton growth rate in the absence of grazing. Knowing the growth rate, the grazing rate can be determined.

Response to UV irradiation

Field and laboratory studies have been undertaken to determine the tolerance of microbial species and communities to UV exposure. Ozone depletion over Antarctica enhances the UV-B (280 - 320 nm wavelength) irradiation, which penetrates near-surface waters to 50 m depth. UV radiation has been shown to reduce growth, production and survival in the top 10 -15 m of the water column.

UV light can impact all levels of the microbial community. Microbes differ greatly in their susceptibility to UV-induced damage and significant changes to community structure and function have been observed in natural assemblages exposed to antarctic sunlight.

Cultures

Controlled laboratory studies of the physiology of key protist species are performed using cultures maintained at the Australian Antarctic Division. Cultures are isolated by selecting single cells from a field sample and maintained in various nutrient-enriched culture media based on seawater supplemented with various nutrients.

Karen isolating cells in a coldroom. Image: Rick van den Enden

Visiting scientist, Dr Peter Henriksen, culturing phytoplankton in a laminar flow cabinet. Image: Simon Wright

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