Fluorescence Time-Lapse Imaging of the Streptomyces venezuelae Life Cycle

Take a microfluidic plate with a well containing spores of Streptomyces venezuelae, a hyphae-forming bacterium.

Other designated wells contain either nutrient-rich or spent medium.

The spores are derived from a strain expressing fluorescent DivIVA and FtsZ proteins, which localize to hyphal tips and cross-walls, respectively.

Apply immersion oil to the objective lens of an inverted microscope and to the plate’s imaging window.

Mount the plate on the microscope stage and begin time-lapse imaging.

Load the spores into the plate’s culture chamber.

Then, introduce the nutrient-rich medium to initiate spore germination.

As the hyphae grow, DivIVA accumulates at the hyphal tips, while FtsZ forms scattered rings at the cross-walls.

Next, switch to the spent medium. The lack of nutrients in this medium supports sporulation.

In sprouting hyphae, DivIVA foci disappear, and FtsZ reorganizes into a regular ladder of division rings, forming sporulation septa.

Over time, the disappearance of FtsZ fluorescence in the same hyphae confirms completion of sporulation.

Pre-warm the environmental chamber to 30 degrees Celsius in advance.

Then turn on the microscope and the microscope control software. Put a high n