The gut microbiome plays important roles in shaping host's physiology. This protocol, in particular, allows for high throughput assessment of fluorescent gut microbe colonization levels in single, cellular animals on a large scale. One major benefit of this method is the ability to monitor the gut microbiome of live animals in real time, which allows for the identification and correlation of any changes with the host's physiology.
Demonstrating the procedure will be Dana Blackburn, a research associate from my laboratory. Begin by washing the worms off the bacterial lawn with one to two milliliters of M9-TX. Transfer the worms to a sterilized two milliliter 96 well deep plate.
Pellet them at 300 G for one minute and remove the liquid using an aspirating manifold. Using a 1.2 milliliter multi-channel pipette, add 1.8 milliliters of M9-TX to each well of the deep well plate mixed by pipetting a couple of times and centrifuge at 300 G for one minute. After the final wash, bring the volume in each well to 100 microliters, using the aspirating manifold.
Add 100 microliters of 10 millimolar levamisole and M9-T to each well, and allow the worms to paralyze for five minutes. Then add 4%bleach solution to each well for two minutes to reduce bacterial clumps. After the bleach treatment, add M9-TX into each well of the plate to wash the worms.
Centrifuge the plate and aspirate to a final volume of 100 microliters after the second wash. Transfer the worms to a flat bottom 96 well plate, containing 150 microliters of 10 millimole of levamisole and M9-TX. Keep the worm density at 50 to 100 worms per well and split the worms into multiple wells if the population is too crowded.
Turn on the air compressor, computer and LPS instrument. Check and empty the waste tank. Check and refill sheath and water tanks if the volume is low.
Ensure the 250 micrometers fluidic and optical core assembly is in place. Connect and turn on the autosampler. Open the autosampler instrument software to open the autosampler and the LPS software.
On the LPS software window, go to file and click new experiment, then select file again and click new sample check. Enable lasers to turn on 488 and 561 nanometer lasers if it has not been done already. Now create templates for acquisition dot plots, using time of flight, extinction and fluorescent channels in the LPS software window.
Right click within the body of a graph to modify the scaling. Next, load the control samples. On the autosampler window, select prime, go to file, click open script to select the correct built-in script and click okay.
Go to plate template on the sampler software menu to select the desired wells for analysis. After loading and securing the plate onto the autosampler stage, press run plate on the autosampler window and save the file when prompted, then click the store current data button on the top ribbon in the LPS software window and save the data again. Open the LPS software.
Navigate to file and select new experiment, then go back to file and click new sample. Create a dot plot with the time of flight on the X axis and extinction on the Y axis. Create another dot plot with the time of flight on the X axis and red on the Y axis.
Ensure to check enable lasers to activate the 488 and the 561 nanometer lasers. Position the control sample tube on the sample port, then click acquire within the acquire and dispense dialogue box. When prompted, ensure to save the file.
Once enough worms have been measured to differentiate populations, select abort in the dot plot showing the time of flight versus extinction. Draw a gate around the area representing the adult population. Then navigate to view, click gating hierarchy and check that the fluorescent gates are correctly listed under the adult population gate.
In the time of flight versus red dot plot, create gates around the high and low red value areas to highlight the regions of interest showing microbiome colonization in adult worms. Once settings are optimized, proceed to file and click save experiment, then click file and select save sample. To load the collection apparatus, select load plate A to allow the collection stage to move into a position primed for loading either a collection tube or a 96 well plate.
For dispensing to a 96 well plate, go to the setup section and select plates, then click on calibrated plates and choose 96 well plate. Next, select the wells to which the worms will be sorted and input the number of objects to be sorted into each well. Be sure to specify the gated regions of interest as well.
If there are multiple gated regions being sorted into the same plate, ensure to check the box marked gate each well. Click okay to save the changes. After sorting numbers and assigning the locations, load the sample onto the sample port and click on the fill plate buttons from the acquire and dispense dialogue box to initiate dispensing into a 96 well plate.
Higher extension and time of flight values are observed in adults compared to larvae. Progenies from two day old adults were dominated by L1 and L2 stages, while most progeny from three day old adults reached L3 and L4 stages. When grown on E.coli, the time of flight and extension values increased on day three compared to day two.
When grown on ochrobactrum, BH three and mixed cultures, cenor abdidas allegands showed differences in time of flight and extension values. Increased ochrobactrum BH3 colonization was observed in the mixed condition than in ochrobactrum BH3 alone. In contrast, green, fluorescent values indicated lower OP50 colonization in the mixed condition than in OP50 alone.
The worms are heavily skewed toward the Y axis, suggesting OP50 colonization is low in most worms, while levels of ochrobactrum, BH3 colonization are evenly distributed in the population. The relationship between ochrobactrum, BH3 colonization levels and host's development such as body density and the differences in reproduction patterns was also observed. The three-day old adults grown on the two member mix, microbiome exhibited a wide range of RFP intensity, indicating individual variations in ochrobactrum colonization within the group.
RFP images of the wells containing 15 sorted and individual worms from high and low RFP gates are shown. The most important thing is to remember to use appropriate controls for all of the steps to ensure that the protocol and the machine are working properly. In real time, animals with specific features can be used to isolate strains from host or microbial mutant pools to identify genes that regulate post microbe interactions.
Isolated animals can also be used for single animal or enriched phenotypic pool based downstream analyses, like RNA-seq or the like. Really, the opportunities are truly endless.
