Enirchment of Photosynthetic Nitrogen-fixing Cyanobacteria: Methods

Prepared a liter each of cyanobacteria media and trace metal media both for liquid media and solid plates
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Collected samples from still pools of water in direct sunlight or from a fish tank in light
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Placed samples in liquid media to allow the concentration of organisms to increase
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Allowed one week for growth at room temperature under lights (2,000 to 3,000 lux)
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50ul taken from each liquid sample and plated onto cyanobacteria and trace metal media
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Allowed growth on plates for one week at room temperature under light (2,000 to 3,000 lux), with plates wrapped in parafilm to prevent excess drying
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Appearance of green growth on cyanobacteria plates indicated successful enrichment of culture
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Streak for isolation, grow on plates for one week under same growth conditions
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Repeat until target organism is isolated

Conducted Gram stain to confirm that organism was Gram negative
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Conducted extensive microscopy to identify which type of nitrogen-fixing, photosynthetic, filamentous cyanobacteria was isolated
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Procedure for Identification (see below)**
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Searched for cell-differentiation in isolated cyanobacteria

**Procedure: Identifying Genera of the Order Nostocales (filamentous, nitrogen-fixing, photosynthetic cyanobacteria)
If reproduction is by trichome breakage (hormogonia not produced – no developmental stage) or by germination of akinetes (spore like cells), go to 2

1.If reproduction is by hormogonia that are distinguishable morphologically from the trichomes (adult filamentous chains of cells), with reproduction by trichome breakage and akinete germination also possible, go to 4
2.Terminal heterocysts (differentiated, nitrogen-fixing cells), possibly at both ends of trichomes. Akinetes (if present) are next to heterocysts, genus Cylindrospermum
Heterocysts interspersed or terminal, akinete (if present) location varies, go to 3
Spherical, oval, or cylindrical vegetative cells, genus Anabaena
3.Disk-shaped, wider than they are short vegetative cells, genus Nodularia
4.Terminal heterocysts; tapering in mature trichomes from heterocyst to tip, genus Calothrix
Untapered trichomes, go to 5
Heavily sheathed trichomes, false branch may occur at intercalary heterocyst, heterocysts at one end of hormogonia filaments only, genus Scytonema
5. Unsheathed trichomes, no false branching, heterocysts at both ends of hormogonia filaments, genus Nostoc