The overall goal of this procedure is to observe spatial distribution of microbial strains during swarming, sliding, or biofilm formation using fluorescent microscopy. This method can help answer key questions in the social microbiology field, such as how spatial segregation influences microbial interaction. The main advantage of this technique is that the fitness and spatial distribution of microbial strains can be easily assessed using microscopic techniques and subsegment image analysis.
Demonstrating the procedure will be Theresa Holscher, a PhD researcher from my laboratory. On the day before the experiment, add three milliliters of LB medium to a culture tube and inoculate it from a freezer stock of B.subtilis. Repeat this inoculation with a separate tube for each strain of interest.
Place the tubes into a horizontal shaking incubator at 37 degrees Celsius overnight. To prepare plates for swarming and sliding experiments, pour 20 milliliters of tempered LB medium into a 90 millimeter polystyrene Petri dish. Close the dish immediately and them place it to one side of the sterile hood to cool for at least one hour.
Next, prepare plates for colony biofilm experiments by pouring 25 milliliters of agar two times SG medium into a 90 millimeter Petri dish. Close the plates immediately and set them to cool in stacks of four or less for at least one hour. To begin, place the LB agar swarming and sliding plates in a laminar flow hood and remove the lids.
Allow the plates to dry for 20 minutes. The humidity of the plates in these experiments is critical. While excess moisture allows bacteria to swim, prolonged drying prevents swarming and sliding.
Similarly, colony biofilm structure depends on the dryness of the medium. Using a spectrophotometer, determine the optical densities of overnight starter cultures at 600 nanometers. In a 1.5 milliliter microcentrifuge tube, mix density normalized quantities of the green and red fluorescent protein producing strains for a total of 200 microliters.
Vortex the tube for three seconds. Using a micro-pipette, spot two microliters of the mixed culture onto the center of a pre-dried 90 millimeter LB agar plate in the laminar flow hood. Place the plate to dry for 10 minutes.
Finally, incubate the plates at 37 degrees Celsius in an upright position to prevent condensation from collecting on the agar's surface. First, place the two times SG agar plates in a laminar flow hood for 15 minutes to dry. Next, add 100 microliters each of the green and red fluorescent protein producing biofilm strain, B.subtilis 168 starter cultures, into a 1.5 milliliter microcentrifuge tube.
Vortex the tube for three seconds. Prepare tubes with 100 microliters of LB medium and using the mixed culture, create a 10 fold dilution series of inoculates. Using a micro-pipette, spot two microliters of the non-diluted mixed culture onto the two times SG agar plate.
Repeat this with inoculates for the 10 to the one, 10 to the two, 10 to the three, and 10 to the four dilutions, spacing spots at equal distances to allow six to nine colonies to be grown on a single dish. Incubate the plates upright at 30 degrees Celsius for one to three days. To begin, place plates on the imaging platform of a fluorescence detecting microscope.
For sliding and swarming experiments, set the origin of inoculation, the middle of the Petri dish, to the corner of the visible field to allow for the measurement of radial colony expansion. Adjust the magnification to the highest resolution that allows imaging of the biggest possible region of the 90 millimeter plate. Adjust the exposure time appropriately, depending on the strength of the fluorescent signal.
For biofilm analysis, position a colony of interest in the center of the field of view. Set the magnification at the highest resolution that allows the entire colony to be viewed. Colonies are now ready for measurement and analysis.
Finally, analyze the data as described in the text protocol. This figure shows the growth of a typical two strain co-inoculated Bacillus subtilis colony. Swarming, which is a flagellant dependent collective surface movement results in a highly mixed population.
In this time-lapse video, the initial inoculant was placed in the center of the plate spread. Thin layer swarming is clearly observed and as the colony develops, the two red and green fluorescent strains appear mixed and overlapping. Conversely, images of sliding behavior show defined sectors of growth corresponding to the differently labeled fluorescent strains.
This video shows the growth of a sliding co-inoculated colony in which the strains lack functioning flagella. These colonies are still able to spread using a combination of secreted exo-polysaccharide, hydrophobin, and surfactin. Resulting colonies display distinct spatial growth assortment compared to the swarming strain.
Biofilm producing colonies starting cell densities heavily influenced the spatial assortment of the resulting colonies. Colonies initiated with high cell density of the mixed populations, showed minor or no spatial assortment. In contrast, when cell density at initiation was low clear green and red fluorescent sectors could be detected.
After watching this video you should have a good understanding of how to determine the relative abundances of fluorescently labeled strains in microbial colonies, examine spatial segregation, and study the influence of founder cell density.
