Phase-Contrast Microscopy for Evaluating Developmental Mutants in Streptomyces

JoVE Encyclopedia of Experiments

Microbiology

0 weergaven • 2:44 min • September 26th, 2025

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Begin with agar plates containing colonies of wild-type and mutant strains of Streptomyces coelicolor, a filamentous soil bacterium.

Place a sterile coverslip over a region of dense colony growth where aerial filaments, which are filaments extending above the colony surface, are clearly visible.

Gently press the coverslip to transfer the aerial filaments and associated spores onto the coverslip surface.

Next, take a microscope slide with mounting media and mount the coverslip cell-side-down to preserve the bacterial structures.

Place the slide on a microscope stage, add a drop of immersion oil on the coverslip, and align the objective.

Using phase-contrast microscopy, acquire images of the aerial filaments from both wild-type and mutant strains.

Wild-type filaments exhibit uniformly sized, regularly spaced spores.

In contrast, mutant strains display defects, such as bulging filaments, irregular spores, or lysed spores, revealing developmental phenotypic differences between the wild type and mutants.

To prepare a coverslip lift of bacterial growth, use sterile tweezers to pick up a sterile coverslip and place the coverslip on an MS agar plate where bacterial growth is dense. Using tweezers, gently press on the back of the coverslip to ensure sufficient transfer of bacterial spores and aerial mycelium. Pick up the coverslip from the plate and place it at a 45 degree angle with the cell material facing the surface of a microscope slide containing a 15 microliter drop of 50% glycerol.

Allow the coverslip to fall on to the glycerol which will reduce air bubbles. To perform phase contrast microscopy at various time intervals, place the prepared slide on the microscope stage and add a drop of immersion oil to the center of the coverslip. Rotate the 100x phase objective in place and set the condenser turret to the proper matching phase setting.

Once the objective lens is in contact with the oil, use only the fine adjustment knob to focus the image. Examine several fields of view to discern noticeable and consistent differences between mutant strains and the wild type. Such as the ability to form spores, spore size and shape and the overall number of spores.

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Laatst bijgewerkt: 22 augustus 2026