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Objectives
1. Be able to explain the evolutionary and ecological importance of cyanobacteria.
2. Be able to recognize and know the function of the basic components of a cyanobacterial cell.
3. Be able to recognize the diversity of cyanobacterial forms and know the function of any specialized cell types
that they use.
4. Understand the importance of cyanobacteria that occur in symbiotic relationships.
5. Be able to sight ID to genus the list of cyanobacteria included in this section.
6. Be able to explain and/or identify the terms in the Vocabulary list at the end of this laboratory.
Background
The cyanobacteria, which are also called cyanophytes or blue‐green algae, are a group of oxygen
producing photosynthetic Bacteria. Referring to them as blue‐green algae is fine so long as you remember that
most algae are eukaryotes, whereas blue‐green algae are not. The cyanobacteria are arguably one of the most
important groups of organisms to ever evolve. They have radically altered the path of evolutionary events over the
last 3.5 billion years, and they continue to be pivotal organisms in today’s ecosystems.
Cyanobacteria were the first organisms to evolve the capacity to use water as an electron donor during the light
reactions of photosynthesis, rather than electron donors such as hydrogen sulfide that other bacterial
photosynthetic predecessors used. The switch to water meant that oxygen was now the waste product of
photosynthesis. As oxygen levels accumulated in the atmosphere, the older groups of bacteria were relegated to
refugia where oxygen could not penetrate; such as in sediments – and these refugia are where you find these older
forms today. Another reason that cyanobacteria are considered so important to the evolution of other life forms is
that the buildup of oxygen meant that it was possible for ozone to form, and that meant that less destructive
ultraviolet radiation was reaching the earth’s surface.
Cyanobacteria are also very important ecologically because they are one of the few prokaryotic groups capable of
nitrogen fixation, or the conversion of atmospheric nitrogen (N2) to ammonia (NH3) by nitrogenase. The ammonia
can then be used by the cyanobacteria, other prokaryotes and by eukaryotes. No eukaryotes are capable of
nitrogen fixation!
The cyanobacteria are also important because they can produce blooms in ponds and lakes some of which are
lethal or very damaging to many organisms including vertebrates. Other cyanobacteria, like Spirulina, are sold in
health food stores, and this genus is also used by farmers to increase the protein content in their cattle feed.
Modern concepts of cyanobacteria include the prochlorophytes. These are oxygen producing photosynthetic
bacteria that have chlorophyll’s a and b, no phycobilisomes, and stacked thylakoids. In contrast, most
cyanobacteria have only chlorophyll a, phycobilisomes, and therefore unstacked thylakoids. Despite these
phenotypic differences between prochlorophytes and classic cyanobacteria, the prochlorophytes are now
considered cyanobacteria because gene data show that ‘prochlorophytes’ have evolved from blue‐green
cyanobacteria at least three times (See dark boxes on the right).
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There are no synapomorphies for all cyanobacteria but a
blue‐green cyanobacterium can be easily identified by
using a combination of homologous characters.
Prokaryotic cellular organization (thus no double
membrane bound organelles, mitosis, cytokinesis,
sex, or microtubules)
O2 evolving photosynthesis
No flagella; do have coccoid cells, colonies, and
filaments (sheath + trichome); filaments can be
uni‐, bi‐ or multiseriate.
Unstacked thylakoids
Phycobilisomes w/ chlorophyll a and accessory
phycobiliprotein pigments (phycocyanin,
allophycocyanin, phycoerythrin)
Often blue/green (cyan) in color
Cyanophycean starch
Nitrogen fixation w/ nitrogenase
Heterocyst present in derived clades
Asexual reproduction by endospores, exospores, hormogonia, or akinetes
The Basic Cyanobacterial Cell
Use the electron microscope images on the bench in order to be able to recognize and know the function of the
following cellular features:
Mucilagenous sheath: Keeps cells together for colonial taxa; UV absorption; captures micronutrients; may detain
herbivores
Cell wall (outer membrane, peptidoglycan layer, plasmalemma): Cyanobacteria are gram‐negative bacteria
because, along with other gram‐negative bacteria that are not cyanobacteria, they have an outer membrane
(OM) that prevents the gram stain from reacting with the peptidoglycan in the underlying wall. The
peptidoglycan layer in cyanobacteria is particularly thick. There is another membrane to the inside of the
peptidoglycan, called the plasmalemma or inner membrane (IM).
Binary fission: The type of cell division done by prokaryotic cells; the new cell wall (septum, cross wall) forms from
the outside towards the inside of the cell. The parent cell divides into two daughter cells of equal size.
Unstacked thylakoid membranes: These membranes, in and upon which the light reactions of photosynthesis
occur, are unstacked because the surface phycobilisomes prevent stacking. The thylakoids are parallel to each
other and are free in the cytoplasm – i.e. they are not surrounded by a membrane system.
Phycobilisomes: Form a half sphere of accessory pigments on the surface of the thylakoids; they are made of 3
pigments: phycoerythrin, phycocyanin, allophycocyanin; they absorb blue‐green to orange wavelengths and
pass that energy to chlorophyll a.
Carboxysome (Polyhedral body): Made of Ribulose bis‐phosphate Carboxylase/Oxygenase (rubisco) and is the site
of carbon fixation in the cyanobacterial cell.
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Nucleoid: The cluster of prokaryotic DNA (i.e. circular chromosomes) in a prokaryotic cell. The DNA is not directly
visible in the micrographs; the nucleoid is evident as a clear or open space surrounded by other cytoplasmic
constituents, like 70s ribosomes.
70s ribosomes: The site of protein synthesis (i.e. translation); prokaryotic ribosomes are slightly smaller than
eukaryotic (80s) ribosomes.
Gas vesicles: Are found in some planktonic species and, when present, confer positive buoyancy to the cell; they are
made of protein and not membranes (and so should not be called vacuoles); they are formed during low light
in order to get cells higher in the water column where there is more light; greater photosynthesis produces
more cyanophycean starch which in turn results in more osmotic pressure within the cells and so the cylindrical
vesicles collapse and the cells sink again.
Setting up Kohler Illumination on the Compound Microscope
1. Get a prepared slide and get it in focus with the 10x objective.
2. Rotate the black ring at the bottom of the microscope until the resulting black polygon is as small as it can be in
your field of view.
3. Use the knob for the substage condenser to raise or lower the condenser to the point where the edges of the
black polygon form a sharp black edge with a slight blue tinge. The light is now focused on the same plane as the
specimen on your slide.
4. Use the black ring at the bottom again to open the polygon so that it is close to the outer edge of the field of
view.
5. Now center the polygon in your field of view by gently turning the two silver knurled knobs under the stage. The
light is now centered in your field of view. This completes Kohler illumination.
6. Don’t forget to make further light adjustments as necessary. Light quantity is controlled by the dial on the
microscope arm whereas the amount of contrast is determined by the iris diaphragm, which is a single lever
located just underneath the stage.
Diversity
Traditionally, cyanobacteria have been classified on the basis on cell shape, how they reproduce, whether
they are unicellular or colonial, the presence or absence of a sheath and the presence of specialized cells. Gene
based analyses of phylogenetic relationships among cyanobacteria have shown that some of traditional groups
actually are monophyletic, whereas other are polyphyletic. Not surprisingly, taxa with simple forms (e.g. unicells)
appear to have evolved multiple times, whereas taxa with specialized cells (e.g. heterocysts, akinetes) form natural,
monophyletic groups. However, taxonomic keys must rely on phenotypic characters for pragmatic reasons and
so what follows is an array of cyanobacteria organized by the forms they take.
Unicellular and colonial forms lacking specialized cells or reproduction
Be able to recognize the plate‐like colony of coccoid cells that is typical for Merismopedia; be able to recognize
this taxon as an example of a colonial rather than a multicellular taxon; be able to find examples of binary fission.
Walls from different cells are not physically connected, which is why this taxon is not considered multicellular.
However, Merismopedia is considered colonial because a common mucilaginous sheath holds the coccoid cells
together.
FOLLOW STEPS 1 – 9 BELOW IN ORDER TO MAKE YOUR PREPARATION AND DOCUMENT YOUR WORK WITH
Merismopedia.
1. Get a compound microscope from the cabinet in back of the room.
2. Use freshwater to make a wet mount from the Merismopedia culture. Be sure not to add too much liquid.
Your coverslip should not be sliding around on the surface of the slide.
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3. View the cells in the microscope in order to familiarize yourself with how the microscope works and to
ensure that you have some colonies present. Don’t do any drawing or photography yet.
4. Take your Merismopedia slide off of the stage and put a very small drop of India ink on one edge of the
cover slip. Use a small piece of paper towel to pull the ink underneath the coverslip.
After making a slide, you may find that a stain or some kind of background color is necessary to improve
viewing of the organism. In this case, the India ink is not staining anything. It can only occur in the space
not occupied by the cyanobacterium and its mucilaginous sheath. India ink is frequently used to detect a
sheath that might otherwise be difficult to detect by only adjusting the iris diaphragm.
5. Now view your colonies at 400X magnification. Draw some of the colonies and cells in your lab notebook,
which you may want to splice into your lab manual. Use at least a half page for your drawing. Colored
pencils can help a lot. The quality of your drawing should be good enough for you to identify features and
to help you remember information during your future studying.
Your drawing should contain a date and labels.
6. Add a scale bar to your drawing. There should be a micrometer in one of your eyepieces. Calculate the
diameter of a typical cell in your colony by using the below table. You need to know your total
magnification (eyepiece * objective lens) as well as the number of micrometer units it takes to span the
diameter of your representative cell. Then you can use the conversion factor in the below table to add a
scale bar to your drawing that describes the cell diameter in mm or um; the latter are more often used.
magnification mm/unit µm/unit
40X 0.028 28
100X 0.011 11
400X 0.0028 2.8
1000X 0.0011 1.1
7. View Merismopedia using the oil immersion lens. Before doing this, make sure that your coverslip is not
sliding around on too much liquid. After focusing on some cells with the high/dry objective, place these
cells in the center of your field and then swing out the objective. Place a SMALL DROP of immersion oil on
the cover slip spot that you were viewing. Carefully slide the immersion objective into place and find the
cells again. You will find that when you move the stage, if you have put too much water underneath the
coverslip then you will just be pushing the coverslip around the slide and you will have little control over
what you are seeing. If your slide has been on the stage a long time, the light may start to dry off the slide
and damage the alga.
If you had not been told that Merismopedia was a cyanobacterium, how could you tell from looking at it
that it is a prokaryote?
8. CLEAN THE OIL OBJECTIVE LENS WITH LENS TISSUE WHEN YOU ARE DONE. ADD FRESH WATER TO THE
LENS TISSUE AND GENTLY WIPE THE OBJECTIVES, FOLLOWED BY THE STAGE AREA. ONCE OIL GETS
BEHIND THE LENS OF ANY OBJECTIVE THAT OBJECTIVE HAS TO BE THOWN OUT – VERY EXPENSIVE!!
Make a wet mount of Microcystis with India ink as you did for Merismopedia. Note that the organization of cells
in this colony is much more amorphous than Merismopedia. This is probably the taxon that has bloomed on
occasion in our local lagoons and rivers and resulted in the death of dogs. Draw and photographically document
Microcystis as you did for Merismopedia. The toxin produced is called microcystin and is a dangerous hepatotoxin
for animals including humans.
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Filamentous cyanobacteria lacking spores, heterocysts, or akinetes
Make a wet mount of Oscillatoria and note the oscillating motion of the filaments (sheath + trichome =
f i l a m e n t ). Add India ink to your wet mount and note the lack of a mucilaginous sheath around the trichome.
The ink technique will be a useful technique when, during the process of keying, you need to decide how much
of a mucilage layer is present.
Examine a prepared slide of Lyngbya. Locate the mucilaginous sheath around the trichome and the
hormogonia. A hormogonium is a segment of a larger filament that becomes detached (by programmed cell death)
and then glides (i.e. oscillates) out of the parent sheath to form a sheath of its own. It is a simple ‘asexual’ way of
reproducing for filamentous species.
Heterocyst and akinete producing cyanobacteria
Use the electron micrograph and light microscope images on the bench of heterocysts and akinetes in order to
learn how to recognize them and explain how they function. In comparison to other cyanobacterial cell types,
these are very specialized cells.
Heterocyst: This cell type is specialized for nitrogen fixation by the enzyme nitrogenase. This enzyme is inhibited by
oxygen, which explains the evolution of the heterocyst as a derived character within cyanobacteria. The
heterocyst has an extra thick wall to prevent the entry of oxygen, and it has fewer photosystem II light
harvesting complexes (also note the lower density of thylakoids in the micrograph) and so less oxygen will be
produced by photosynthesis inside the heterocyst. In the light microscope image, the reduction in the density
of light harvesting complexes is evident by the lighter pigmentation of the heterocyst. It is important to know
that nitrogenase occurs in species that do not have heterocysts, but in these cases the nitrogenase can only
function when the cells happen to be in a microanaerobic environment.
Akinete: This is a resting cell that is able to persist through adverse conditions. The akinete is filled with stored
nitrogen (as cyanophycin granules) and carbon (cyanophycean starch) and it has an extra thick cell wall. When
conditions improve the cytoplasm inside the akinete divides and pushes through the akinete wall.
Make a wet mount of Anabaena and locate the intercalary heterocysts. In addition to its thick walls and paler
yellow‐green color, polar nodules in the heterocyst may help to distinguish it from other vegetative cells. Akinetes
can occur in Anabaena but are often not present because the cultures have been growing in non‐stressful
conditions.
Examine the prepared slides of Cylindrospermum and locate akinetes. Akinetes are often 2‐3 times as wide as
normal vegetative cells and often 3‐4 times longer. In some taxa they have a spiny appearance.
Be able to find akinetes in the fresh material of Cylindrospermum.
Study the light microscope image of Scytonema on the bench in order to understand what is meant by “false
branching.”
Make a wet mount of Scytonema and note the heterocysts and false branching.
True‐branching cyanobacteria
Make a wet mount of Fischerella (similar to Stigonema) and note the true branching, heterocysts, and akinetes
if present. Fischerella is uniseriate whereas Stigonema is multiseriate.
Some mutualistic symbioses involving cyanobacteria
Examine the fresh material of the lichen Peltigera. Make a thin cross‐section through the thallus and be able to
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locate the layer of nitrogen fixing Nostoc.
Examine the fresh material of the water fern Azolla and locate the pockets of Nostoc. Do this by sectioning and
then gently squashing the mesophyll of the fern leaf.
Cyanobacterial Sight ID List
Anabaena
Calothrix
Cylindrospermum
Gloeocapsa
Lyngbya
Merismopedia
Microcystis
Nostoc
Oscillatoria
Scytonema
Spirulina
Stigonema
Tolypothrix
Vocabulary for cyanobacteria
70s ribosomes
Akinete
Allophycocyanin
Binary fission
Carboxysome
Chlorophyll a
Coccoid
Colonial
Cyanophycean starch
Cyanophycin granules
False branching
Filament
Gas vesicles
Heterocyst
Hormogonium (pl. hormogonia)
Inner membrane (plasmalemma)
Mucilage sheath
Nitrogenase
Nucleoid
Outer membrane
Peptidoglycan
Phycobilisome
Phycocyanin
Phycoerythrin
RubisC/O
Thylakoid
Trichome
True branching
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