Visual and Microscopic Evaluation of Streptomyces Developmental Mutants

The overall goal of the following techniques is to train beginning researchers to work with Streptomyces and other related species in the university teaching laboratory and high school classroom settings. Phenotypic observation is important for the many researchers entering the field of Streptomyces research. This video describes methods for bacterial propagation, storage and visual and microscopic examination.

The phenotpying methods described here use Streptomyces coelicolor as a model. However, the methods are applicable to all members of the large genus as well as closely related Actinomyces. The main advantage of these techniques is that they are designed to be accessible to novice researchers in a high school classroom or undergraduate teaching laboratory as well as experienced laboratory personnel.

After watching this video and reading the accompanying protocol, new researchers should be able to manipulate their Streptomyces strains, store them properly and begin visual characterization experiments. Demonstrating the procedures will be Garret Kandell and Sean Kirk, undergraduate research students from my laboratory at Otterbein University. To begin, 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. Sterilize tweezers by washing them in 70%ethanol and then passing them through a Bunsen burner flame to evaporate the ethanol. Sterilize coverslips by washing them in 70%ethanol and then allowing them to dry in a sterile Petri dish.

Next, 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.

Using sterile tweezers, prepare a coverslip lift from an MS agar plate as before where bacterial growth is evident gently touching the back of the coverslip with the tweezers to ensure sufficient transfer of bacterial spores and aerial filaments. Pick up the coverslip from the plate and place it on card stock so that the side with cell material is facing up for ease and manipulation of the coverslip. With ice cold methanol, flood the coverslip and let it sit for five minutes.

Using PBS wash the coverslip two times by gently dispensing and removing the solution. Add 15 microliters of a 10 micrograms per milliliter propidium iodide solution and or 10 micrograms per milliliter of WGA-FITC to a slide. Place the coverslip face down on a drop of fluorescent stain.

Incubate the samples in a darkened or dimly lit room for 15 minutes. Stocks of the fluorescent stains and stain samples should be protected from the light. It is recommended that researchers use conditions of low light while preparing samples and use a dark box to hold samples once they are prepared.

Observe the slides under a 100x objective lens using a phase contrast or DIC microscope equipped with epifluorescence excitation cubes for propidium iodide and FITC. Analyze the images according to the text protocol. Shown here are examples of Streptomyces mutants resulting from transposon mutagenesis.

A lighter colored aerial mycelium may indicate a lower level of gray pigment caused by a defect in sporulation. Or the lack of a fuzzy appearance is indicative of a block in aerial mycelium formation. As seen by phase contrast microscopy, wild type S.coelicolor colonies typically produce an aerial mycelium by about two days of growth.

And long chains of spores by three days of growth. White mutants may either be delayed for spore formation, show a reduction in the abundance of spores produced, produce spores with shape and or size defects or simply produce lower levels of the mature, gray spore pigment. Shown here using DIC and fluorescence microscopy with PI staining, regularly spaced staining for a chain of spores in a wild type sample is compared to the intermittent staining pattern for two transposon insertion mutants that display a chromosome segregation defect.

Using WGA-FITC to stain the cell wall wild type strain S.venezuelae is present as smooth aerial filaments among chains of spores. And exhibits the lateral like array of division septa that are typically seen during sporulation. Working with a filamentous organism is very different from working with a well known, rod shaped bacteria.

The video protocols here, should serve as an important resource for brand new researchers entering the field of Streptomyces research. After watching this video, you should have a good understanding of how to begin studying Streptomyces species and other related bacteria. Following these initial experiments, a variety of techniques can be used in a research laboratory to learn more about your Streptomyces strains.

For example, some of the advanced techniques might be protein tagging or gene expression analyses such as real time PCR and RNA seq. Initial phenotyping experiments are used to identify and characterize new strains and discern the typical role of a particular gene. These methods have already been used in university teaching laboratories to identify and characterize a wide variety of Streptomyces strains.