Using Light and Electron Microscopy to Evaluate the Taphonomy of
Modern and Fossil Microbes
GEMS research project report
Ashley Manning
Department of Geosciences
Faculty Advisor: Julie K. Bartley
June 26, 2007
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Introduction
The earliest fossil record consists entirely of microfossils. The oldest microfossils are
about 3.5 billion years old and are unicellular bacteria (Schopf, 1992). The first
eukaryotes appear in the fossil record about 2 billion years ago (Javaux, 2001). These
single-celled eukaryotes are called acritarchs, which are circular or elliptical unicellular
plankton of uncertain affinity. We are interested in acritarchs and other early microbial
fossils because of the questions that surround them. Both prokaryotes and eukaryotes
occur as fossils; however, we do not always know which groups they belong to because
of their relatively simple morphology. It is believed that acritarchs are eukaryotes
because of their relatively large size, their ornamentation (processes, spines, membranes,
and complex wall structure; Javaux, 2001), as well as the fact that they are
morphologically diverse. Another important question revolves around what causes
acritarch ultrastructure. Is the observed ultrastructure a characteristic the microbe
possessed while it was alive, or is the ultrastructure a feature produced after death? In this
project, I am examining microbial fossils in two ways: first, by observing changes that
occur to modern microbes immediately after death, during the earliest stages of
decomposition; and second, by observing the preserved morphology and ultrastructure in
ancient acritarchs.
Methods
Cultures
Cultures were grown in sterile environments. Gloeocapsa, Vaucheria sessilis,
Saprolegina, and Chlorella pyrenoidosa were maintained in a freshwater Alga-Gro
medium. Bangia, Enteromorpha intestinals, and Dunaliella salina were maintained in
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saltwater Alga-Gro medium. Culture media and algae were purchased from Carolina
Biological Company. Each medium was autoclaved before the algae were added to
ensure that there would be no bacteria in the cultures.
Cultures containing the heterotrophs were maintained in an autoclaved beef broth,
purchased from Carolina. Culture broth was mixed with water from Lake Carroll and
stored in a dark incubator at approximately 20° C in order to prevent photosynthesis.
About ten milliliters of cultured pond heterotrophs in beef broth are added to fresh
cultures of cyanobacteria (Gloeocapsa) or eukaryotic algae (Vaucheria sessilis). These
infected cultures are also kept in the incubator, in darkness, so that no new photosynthesis
occurs. Subsamples of decomposing cultures are evaluated at regular intervals to evaluate
morphological change occurring as a result of decomposition.
Algae
Healthy Cultures
Decaying Cultures
Chlorella
Vaucheria
Entermorpha
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Gloeocapsa
Bangia
Evaluation of Cells
Using the same procedure as Bartley (1996), I was able to evaluate the preservation
of these algae. Samples of the decomposing algae were evaluated weekly using both the
light microscope and the ESEM. The evaluation was based on characteristics such as cell
morphology, sheath morphology and terminal cell morphology.
Cell morphology was examined by light microscopy and scanning electron
microscopy. Because cells are transparent under the light microscope, whole-cell
morphology can be evaluated. For each species, cell wall integrity, cell shape, cell
contents, and sheath integrity (if applicable) is evaluated. At least 250 individual cells are
examined by moving the microscope stage at random intervals and scoring cell
morphology on a scale of 1 (good preservation) to 3 (poor preservation). The electron
microscope can be used to evaluate changes in sheath or cell wall ultra structure. If ultra
structure changes during composition, such a change would not be easily visible by light
microscopy. Because cells are opaque to the SEM, surface features are visible.
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Good
Poor
Fair
Results and Discussion
We focused on decomposition in order to quantify the post-mortem changes with both
light microscopy and electron microscopy. Decay did not occur as we expected. The data
should have formed a pattern that started at the top of the ternary diagram and moved
toward the left corner and then over to the right. The best examples in our data are
Gloeocapsa Cell Decay and Bangia Cell Decay. The decay data is scattered due to using
a new sample from the decaying beaker every observation. With all of our samples, the
decomposition experiments showed that the cells decay much faster than the sheaths. We
had hoped that we could observe the cell wall ultra structure of the decaying algae using
an electron microscope; this would have allowed us to relate the cell level decay to the
cell wall ultra structure. During each of our attempts, only the sheaths were able to be
observed. The sheaths showed no ultrastructure and prevented us from seeing the cell
wall, thus we have been unable to obtain useful ultrastructural data.
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Chlorella Cell Decay
Good
1 week
10 weeks
11 weeks
12 weeks
2 weeks
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
Fai r
9 weeks
Poor
Chlorella Sheath Decay
Good
1 week
2 weeks
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
9 weeks
Fai r
Poor
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Enteromorpha Cell Decay
Good
1 week
10 weeks
11 weeks
Only cell structure
was quantifiable.
12 weeks
2 weeks
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
9 weeks
Fai r
Poor
Gloeocapsa Cell Decay
Good
1 week
2 weeks
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
9 weeks
Fai r
Poor
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Gloeocapsa Sheath Decay
Good
1 week
2 weeks
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
9 weeks
Fai r
Bangia Cell Decay
Poor
Good
1 week
2 weeks
Weeks 7 and 8 could I
could not find 260 cells
3 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
Fai r
Poor
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Vaucheria Cell Decay
Good
10 weeks
4 weeks
5 weeks
6 weeks
7 weeks
8 weeks
9 weeks
Fai r
Poor
Vaucheria Sheath Decay
Good
10 weeks
4 weeks
5 weeks
7 weeks
8 weeks
9 weeks
Fai r
Poor
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Additional Work
We are going to examine the ultrastructure of acritarchs currently cataloged in our
lab. These acritarchs are from the Grand Canyon and are about 750 million years old. The
ultrastructural data that we observe from these acritarchs will then be used to select a new
set of modern algae to study.
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