The overall goal of this method is to induce cellular differentiation of the bacterium Vibrio parahaemolyticus and the formation of a population of differentiated swarmer cells easily available for single cell analysis by fluorescence microscopy. This method will help scientists understand how bacteria regulate the intracellular organization during cellular differentiation and morphogenesis. Since imaging cells within bacterial communities can be challenging, the main advantage of this technique is that it produces a population of differentiated swarmer cells that exist in a single cell layer readily available for single cell microscopy analysis.
The implications of this technique extend toward a better understanding of how bacteria regulate changes in the cell cycle, cell morphology, and intracellular organization during cellular differentiation. Demonstration of this method is critical as reliable induction of cellular differentiation and the formation of cells available for single cell analysis are difficult to achieve. To prepare the agar solution for making swarming plates, suspend four grams of heart infusion, or H.I.agar powder, and 100 milliliters of double distilled water.
Carefully boil the H.I.agar solution in a microwave oven, shaking the bottle from time to time until the powder is dissolved. Autoclave the agar solution at 121 degrees Celsius for 20 minutes, cool down the agar solution to 60 t0 65 degrees Celsius with constant stirring. The next step involves handling 2, 2'Bipyridyl, which is highly toxic, so gloves and safety goggles must be worn.
Add 2, 2'Bipyridyl to a final concentration of 50 micromolar. Add calcium chloride to a final concentration of four millamolar. Inoculate a small amount of V.parahaemolyticus cells from the edge of a single colony into 5 millilters of LB broth.
Incubate the culture at 37 degrees Celsius in a shaker incubator until it reaches an optical density at 600 nanometers of 0.8. This usually takes around two to three hours. While the cell culture is growing, pipette 30 to 35 milliliters of the final H.I.agar solution into a round 150 millimeter petri dish, and let the agar solidify.
Right before spotting the cell culture, dry the agar plate upright with an open lid at 37 degrees Celsius for at least 10 minutes, or until any liquid residues have disappeared, but no longer. Spot one microliter of cell culture at the center of the H.I.agar swarming plate and let the spot dry. Seal the plate with clear plastic tape, taking care to avoid the formation of air bubbles and folds to the tape.
Incubate the plate at 24 degrees Celsius overnight. This will result in the formation of a V.Parahaemolyticus swarm colony with swarm flares extending from the center of the colony, as shown in this time lapse video clip. The first step in this procedure is to prepare agarose slides for microscopy.
Add one gram of agarose to 100 milliliters of a 20%PBS and a 10%LB broth solution. Carefully boil the solution in the microwave to dissolve the agarose, and then let it cool down to 65 to 70 degrees Celsius while mixing with a magnetic stir bar. Place a clean microscope slide on to a perfectly level section of a working bench.
Fix the slide to the bench by covering both ends of the slide with two layers of general purpose laboratory labeling tape. The distance between the two pieces of tape should be around three centimeters. Pipette about 250 microliters of the previously prepared agarose solution onto the non-covered glass surface.
Place a second microscope slide in the same orientation as the bottom slide on top and slightly press down so that the residual agarose can flow out to the sides. Let the agarose solidify for one to two minutes. Use a scalpel to cut off any residual agarose that is attached to the sides of the microscope slide.
When the agarose has solidified, slowly pull the top glass slide off of the bottom one in a horizontal movement. Slowly remove the tape from the ends of the microscope slide. The agarose slide should be used as soon as possible, as it will slowly begin to dry out.
Immediately before the transfer of swarmer cells to the microscope slide, Use a scalpel to cut the plastic tape off the previously prepared swarmer plate. During the excision of the swarming agar, it is important to direct the cut from the outside to the inside of the swarming colony to avoid contaminating the swarming flares with cells that originate from the middle of the colony. Use a fresh scalpel to cut out a three millimeter by 10 millimeter piece of swarming agar from the most outer edge of a swarming colony, making sure to direct the cuts from the outside of the swarming colony edge to the inside of the colony.
Transfer the piece of swarming agar to the microscope agarose slide with the cells facing the agarose pad to imprint the swarming cells onto the agarose pad. After waiting for about 30 seconds, remove the agar piece carefully from the agarose pad. Place a glass coverslip on the agarose pad where the cells were imprinted.
The cells are now ready for fluorescence microscopy. Stereo-microscopy imaging of a V.parahaemolyticus swarmer colony in increasing magnification shows swarm flares extending from the periphery of the swarm colony. Cells in the flares are grouped in mono or bilayers only, while cells in the middle of the swarm colony are stacked in multiple layers.
DIC microscopy imaging revealed the flares only consist of a fully differentiated population of swarmer cells, while cells from the middle of the swarm colony are significantly shorter, and likely represent a mixture of swimmer and swarmer cells. As proof of principle, DIC and fluorescence microscopy imaging was performed on wild-type V.Parahaemolyticus swimmer and swarmer cells, ectopically expressing an essential chemotaxis protein, YFP-CheW. In swimmer cells, YFP-CheW localizes in a unipolar and bipolar manner, but in swarmer cells, YFP-CheW is always bipolarly localized and also forms clusters positioned randomly along the cell length.
Changes in the localization pattern of YFP-CheW during swimmer to swarmer cell differentiation, were also shown by displays of foci distribution and demographic analyses of fluorescence intensity profiles. After its development, this technique paved the way for researchers in the field of microbial cell biology to explore how the bacteria Vibrio parahaemolyticus regulates its internal organization during its distinct differentiated states of swimmer and swarmer cells. After watching this video, you should have a good understanding of how to induce cellular differentiation of the bacteria Vibrio parahaemolyticus and how to prepare a population of differentiated swarmer cells for single cell analysis by fluorescence microscopy.
Don't forget that Vibrio parahaemolyticus is a human pathogen and proper procedures and regulations on how to handle BSL 2 pathogens in your region should be applied.
