This study outlines a method for synchronizing the cell cycle of cyanobacteria using a nutrient-rich agar medium and alternating light-dark conditions. The process involves monitoring DNA compaction through fluorescence microscopy after specific treatments.
Begin with a nutrient-rich agar plate supplemented with thiosulfate.
Streak the cyanobacteria and incubate under alternating light and dark periods to maintain the circadian cycle.
During the light period, nutrient uptake and thiosulfate support bacterial growth and the condensation of DNA.
In the dark period, the cells conserve energy and divide.
The alternating light-dark cycle synchronizes cells to the same stage of growth and division.
After the light period, add a sucrose solution and pipette repeatedly to release cyanobacteria, causing a color change. Transfer the cells into a tube.
Add a DNA-specific fluorescent dye and incubate in the dark. The dye binds to the cyanobacterial DNA.
Centrifuge to separate the cyanobacteria and remove the supernatant.
Resuspend the cyanobacteria in sucrose solution, transfer the suspension to a slide, and cover it.
Under a fluorescence microscope, the blue condensed DNA in cells confirms the synchronized, circadian-regulated progression of the cyanobacterial cell cycle.
Begin with growing cyanobacteria on nine-centimeter, sterilized BG-11 plates containing 1.5% agar and 0.3% sodium thiosulfate. Incubate the plates at 23 degrees Celsius under a 12-12 light cycle with 50 micro-E of light per square meter per second. To maintain the cells, transfer them onto fresh BG-11 agar plates weekly.
Within one week, cultures appear as green bands. Transfer the green clumps of cells using a flame-sterilized loop, and streak them onto the new plate. To observe DNA compaction, collect the cells cultured for six days at the end of the light period.
To release the cells from the plate, add one milliliter of 0.2-molar sucrose. Then, collect the cell suspension, and repeat the sucrose addition and removal until the solution turns green. The color change indicates that most cells have been released.
Now, transfer 500 microliters of suspended-cell solution to a Microfuge tube, and add Hoechst stain for a final concentration of one microgram per milliliter. Then, let the cells incubate in the dark for 10 minutes. Next, spin down the cells, discard the supernatant, and resuspend them in 10 microliters of 0.2-molar sucrose.
Next, transfer one microliter of the suspension to a glass slide, put on a cover slip, and observe the cells under a fluorescence microscope equipped with a UV filter. Using a 100 times oil immersion objective, look for evidence of DNA compaction, which should be observable in most of the cells.
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