Cultivation of Thermophilic Cyanobacteria using Agar Plates 2
Environmental samples are cultivated on nutrient agar under warm, light-controlled conditions to enrich thermophilic cyanobacteria. Through gradual dilution and repeated re-culturing, slow-growing photosynthetic microbes are selectively favored while background organisms are reduced. This approach results in visible cyanobacterial growth and provides enriched cultures for further physiological and molecular studies.
| Item | Quantity | Supplier | Notes |
|---|---|---|---|
| Environmental Samples | — | — | Water, mat, or sediment sample |
| BG 11 and BG110 medium | — | — | BG 11 for for general cyanobacterial growth and BG 110 for Nitrogen-free; selects potential N₂-fixers |
| Agar powder | — | — | For solid medium preparation |
| Sterile spreader | — | — | For even sample spreading |
| Petri dishes | — | — | Agar plate preparation |
| Parafilm | — | — | Prevents plate drying and contamination |
1. Prepare agar plates using BG-11 or nitrogen-free BG-110 medium and allow them to solidify.
2. Gently mix the environmental sample to ensure uniform distribution.
3. Perform serial dilutions of the sample using sterile water or buffer.
4. Plate diluted samples onto agar plates using either spread-plate or spot-plate methods.
5. Allow the inoculum to absorb into the agar surface under sterile conditions.
6. Seal plates to prevent contamination and moisture loss.
7. Incubate plates under controlled light conditions at elevated temperatures (45–55 °C).
8. Monitor plates periodically for the appearance of pigmented colonies or surface growth.
9. Transfer visible growth to fresh agar plates for re-streaking and further enrichment.
10. Repeat re-streaking as needed until cleaner, more uniform growth is obtained.
This protocol results in the gradual enrichment of thermophilic, photosynthetic microorganisms on agar plates. Over time, pigmented colonies or patches become visible, and repeated re-streaking reduces background organisms. The enriched cultures obtained are suitable for microscopic observation and downstream physiological or molecular analyses.
1. No visible growth
Cause: Low cell density or unsuitable incubation conditions
Solution: Extend incubation time or adjust temperature and light intensity
2. Overgrowth by non-target organisms
Cause: Fast-growing contaminants
Solution: Use higher incubation temperatures or nitrogen-free medium
3. Agar surface drying
Cause: Prolonged incubation at high temperature
Solution: Seal plates properly and maintain humidity in the incubator
4. Uneven colony distribution
Cause: Inadequate spreading
Solution: Ensure even spreading of the inoculum across the agar surface.
Mastigocladus laminosus
A thermophilic cyanobacterium capable of nitrogen fixation that can also adapt to growth at temperatures below its optimal range. It is motile via gliding and forms branching filaments. The organism develops gelatinous, cartilaginous, or spongy mats that are dull blue-green to olive-green in color and exhibits a growth rate of approximately 1.5 doublings per day. It shows low tolerance to hydrogen sulfide and produces spores that are resistant to freezing and desiccation.
Synechococcus sp.
These organisms may be thermophilic or mesophilic and are typically obligate photoautotrophs that perform aerobic photosynthesis. Their small cells are cylindrical, ovoid, or rod-shaped and occur singly, in pairs, or in short chains, lacking a surrounding sheath. Cells often form irregular aggregates held together by a slime layer and reproduce through repeated binary fission in a single plane. Most species are nonmotile, while motile forms move by gliding and display positive phototaxis. Currently, 28 species of Synechococcus have been described.
1. Wear appropriate personal protective equipment, including lab coat, gloves, and eye protection.
2. Handle agar plates and biological samples using aseptic techniques.
3. Use caution when working at elevated incubation temperatures.
4. Dispose of biological waste and used plates according to laboratory biosafety guidelines.
5. Clean work surfaces with disinfectant before and after handling samples.
Microscopic Analysis.pdf
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