This study investigates the phenotypic differences between wild-type and mutant strains of Streptomyces coelicolor, focusing on their colony morphology. The mutants exhibit variations in developmental genes, impacting their growth and appearance.
Begin with cultures of wild-type and mutant strains of Streptomyces coelicolor, a filamentous soil bacterium.
The mutant strains carry mutations in developmental genes that cause phenotypic differences from the wild type.
Reculture the colonies onto fresh plates to minimize the accumulation of spontaneous mutations with age.
Streak single colonies of the strains onto separate regions of a plate in a wedge pattern to avoid cross-contamination
Incubate the plate to promote bacterial growth.
Wild-type strains form aerial filaments that develop into long chains of gray-pigmented spores, giving the colonies a fuzzy appearance.
In contrast, mutant strains either fail to develop aerial filaments, resulting in a bald appearance, or produce fewer pigmented spores, causing the aerial filaments to appear white.
Place the Petri dish on a uniform background to visualize colony morphology.
Capture images to visually compare the phenotypic differences between the wild-type and mutant strains.
Use a standard method, such as the quadrant streak method demonstrated in Saunders 2012 to streak Streptomyces strains onto MS agar plates and culture into single colonies.
Every four to six days, pick a single colony and re-streak it onto a fresh plate. As colonies age, they become prone to random mutation. After carrying out mutagenesis according to the text protocol, take note of colony morphology for each mutant as compared to the wild-type parent strain.
When characterizing new Streptomyces species, compare the new isolate to that of a well studied species noting the basic shape, surface of colony, opacity, elevation and pigmentation. Label an MS agar plate and streak wild-type and mutant strains in wedge patterns. Be careful that no strain touches another strain to avoid cross-contamination.
Note the date and time that strains are streaked onto the plate. Then incubate the plates at 30 degrees Celsius. Remember, it is extremely important to always protect your Streptomyces sample from contamination, use your best sterile technique for all steps opening plates and tubes for the least amount of time possible.
Place grown Petri dishes on colored or white paper to homogenize the background. Write on the paper the strain name, date, incubation temperature, and time from first plate streaking.
Take digital pictures of the plate with the strain information written on the paper background so that confusion is minimal.
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