This video demonstrates a procedure for studying the effects of sheer stress on bacterial adhesion. During a severe infection with a bacterial pathogen such as nice sirium mens, the pathogen reaches the bloodstream there, it attaches to and colonizes host cells while under mechanical stress from flowing blood following proliferation, some bacteria detach and colonize new sites and the cycle begins again to study factors that affect bacterial adhesion to host cells. Endothelial cells are introduced to disposable flow chambers and culture to allow the cells to reach confluence.
Fluorescent bacteria are added and sheer stress is experimentally controlled. Using a syringe pump adhesion of the bacteria to host cells and proliferation is followed microscopically. The resulting videos are analyzed to determine the effect of shear stress on the process of endothelium colonization.
This procedure can be very useful to study the pathogenesis of nice meningitis, but it can also be used for other bacteria that are submitted to shear stress throughout the pathogenesis process. This technique is, can be a little bit tricky to do in particular when it comes to introducing bubbles into the system, which can be a problem. So today demonstrating the procedure will be mega, a PhD student working my laboratory.
The experiments described in this video are performed using cells cultured in an IDI micro slide six 0.4 consisting of a disposable sterile plastic slide containing six 17 millimeter long and 3.8 millimeter wide channels that are 0.4 millimeters deep. Each channel has two access ports with lure adapters, one on each end to introduce uve cells, pipette 30 microliters of a one times 10 to the six cell suspension into each of the channels. On the slide, allow the cells to adhere for three hours at 37 degrees and a humidified incubator with an atmosphere of 5%CO2.
Following the incubation, add an additional 120 microliters of endo SFM. To fill the wells cells should form a sub confluent monolayer after about 20 hours. In the meantime, streak GFP expressing nice sirium meningitis onto APLs, the bacteria is a human pathogen.
Therefore, any manipulation of this microorganism should be performed in a level two security facility with trained personnel wearing a gown and gloves, vaccination of personnel against the sero group used is recommended. Here's strain 8 0 1 3. Expressing GFP is cultured under the control of an IPDG inducible promoter streak.
The bacteria on GC BPLs containing Kellogg supplements and five micrograms per milliliter of chloramphenicol in a 37 degree Celsius 5%CO2 incubator for 16 hours. Once the mammalian cell cultures are established, bacteria are added to the host cells in the channel. In preparation for the flow assay, using an inoculating loop, collect the bacteria from the Petri dish, transfer them to a 50 milliliter conical tube containing prewarm, supplemented endothelial cell culture.
Medium Endo SFM. Adjust the optical density to an OD 600 of 0.05 by diluting the sample in the same medium to a final volume of five milliliters. To induce GFP expression, add IPTG to a final concentration of one millimolar and incubate the culture for 120 minutes.
In a humidified 37 degrees Celsius 5%CO2 incubator with gentle shaking. Place the micro slide with a cultivated endothelial cells prepared earlier on the stage of an inverted microscope equipped with a heated platform to maintain the sample temperature at 37 degrees Celsius. Pour prewarm endo SFM supplemented with 2%FBS into a sterile glass speaker and fill the 50 milliliter syringe by pulling on the plunger.
The entry tubing is connected to a three-way stop cock. Next, attach the entry tubing to the syringe and to press the plunger to fill the tubing with medium. Carefully connect the other end of the entry tubing to the micro slide using the provided right angle connectors.
Avoid introducing air into the chamber. Place the syringe on the pump, then place a cut one milliliter syringe on the available slot on the stop cock as a reservoir. Then affix the exit tube to the other end of the channel and carefully fill the channel with medium by pushing on the syringe plunger to approximately one centimeter distance from the chamber exit.
Measure the bacterial OD 600 and adjust to 0.15 and endo SFM containing 2%FBS as before vigorously vortex the sample to disrupt bacterial aggregates. Next, using a disposable plastic pasture pipette transfer 100 microliters of endos fm, supplemented with 2%FBS medium into the reservoir. Then aspirate 100 microliters of the bacterial solution from the top of the vortex solution and add it to the reservoir.
Carefully turn the stopcock, inject the bacteria into the chamber of the micro slide to allow adhesion to proceed in the presence of controlled share stress level. Introduce endo SFM containing 2%FBS into the chamber using the syringe pump at 3.5 milliliters per hour for 15 minutes. This video is accelerated 60 times.
The real duration is 10 minutes. After this step, the adhesion and detachment of bacteria to host cells under various conditions can be assessed as well be described in the upcoming sections of the video. To quantify the initial adhesion of individual bacteria to host cells, begin acquiring images after 15 minutes of flow while liquid is still circulating, acquire images of 10 random fields to determine the mean number of adherent bacteria per field imaged.
Begin by opening image day software and open the image to be analyzed. Start by clicking on the image menu and selecting. Adjust brightness contrast.
Optimize the visualization of individual adhering fluorescent bacteria over the background by adjusting the intensity levels by moving the cursor. Then in the plugin, analyze cell counter menu. Open the cell counter tool, initialize the image, choose object, type and click on each individual bacteria.
The total number of bacteria appears in the window. Finally, export the data for each image to excel and average to quantify the mean number of adherent bacteria per field to measure the resistance of individual adherent bacteria to flow. After the initial 15 minutes of flow program, the syringe pump setup to generate shear stress ranging from three to 100 dimes per square centimeter for five minutes.
At the end of the five minutes, stop the flow and acquire and analyze 10 random fields as before to determine the mean number of remaining bacteria per field. Following the initial 15 minutes of flow, adjust the syringe pump to the selected shear stress. Allow it to flow for several hours, recording images at a rate of one frame every five minutes.
Ideally, several positions can be followed if the microscope is equipped with a motorized XY platform. Following video microscopy, stop the sheer stress by turning off the syringe pump. Acquire two to three additional images immediately and then stop the image acquisition sequence on the software.
An example of bacterial proliferation can be seen here. Finally, to analyze images, use image J'S image adjust threshold menu to convert the images to binary. Then in the analyze menu under set measurement, select the area measurement.
Then use the analyze particle tool to automatically determine the size of the colonies. Large micro colonies form on the cellular surface after five to seven hours, whether or not flow is maintained at this point. To measure micro colony resistance to flow first, acquire two to three images of both cells by phase contrast and bacteria by GFP fluorescence.
Apply high shear stress for five minutes and take fluorescence images at one frame every five seconds. Stop the shear stress by turning off the syringe pump. Acquire two to three additional images and then stop the image acquisition sequence on the software next to collect the medium for a plating assay.
First, remove the exit tubing taking care to avoid emptying the channel. Add prewarm fresh medium to fill the wells. Then remove the entry tubing and collect the remaining medium with a micro pipet and transfer to a micro centrifuge tube To remove any remaining medium from the channel, rinse the micro slide twice by adding 120 microliters to PBS to the channel.
Tilting the slide twice and discarding the medium. To collect the infected cells, add 50 microliters of tripsin EDTA to the micro slide and incubate for five minutes at 37 degrees to detach them from the channel and mix this detached sample with a suspension collected in the previous step. After performing serial dilution in PBS plate 10 microliter fractions onto GCB agri plates and triplicate incubated 37 degrees Celsius 5%CO2 overnight.
The next day determine the number of colony forming units to assess bacterial detachment from micro colonies. Infect the cells in the flow chamber as before at low flow 3.5 milliliters per hour after 30 minutes, remove unbound bacteria by increasing flow to five milliliters per minute for a period of two minutes, and then return to low flow 3.5 milliliters per hour and allow infection to continue for two hours. Then every hour, collect a drop of medium coming out of the flow chamber in a micro centrifuge tube for four hours.
Once all samples have been collected, determine the number of colony forming units as before. This is a graphic representation of initial adhesion of bacteria on the cellular surface. Each line on the graph represents the cumulative number of bacteria bound in a given field as a function of time.
Values for three fields are indicated to give an idea of the field to field variation that is expected after proliferation on the cellular surface. The mechanical resistance of micro colonies was tested by increasing the shear stress level to 10 dines per square centimeter, but while type micro colonies are resistant, micro colonies formed by the pill v mutant are disrupted by flow increase. This mutant fails to induce the remodeling of the plasma membrane under micro colonies and is thus more highly sensitive to increase shear stress After its development.
This technique paved the way for other researchers in the infectious disease field to explore the impact of shear stress on the interaction of various pathogens with different cell types. Don't forget that working with human pathogens can be extremely hazardous and per precautions, such as level two or three measures should always be taken while performing this procedure.
