This work was supported by NIH grants DP2DK116645 (to B.S.S.), Dunn Foundation pilot award and NASA grant 80NSSC22K0250 (to B.S.S.). This project was also supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the CPRIT Core Facility Support Award (CPRIT-RP180672), the NIH (S10 OD025251, CA125123, and RR024574), and the assistance of Joel M. Sederstrom, plus an instrumentation grant for the LPS NIH grant (S10 OD025251). Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1. Preparation of microbiome mixture
<ol>
	<li>Stamp or streak out bacteria from a glycerol freezer stock onto a lysogeny broth (LB) plate, or appropriate growth medium, and grow overnight at an optimal temperature based on bacterial strains of interest (typically 25 &#176;C for C. elegans natural microbes).</li>
	<li>From the LB plate, use a single colony from each bacterial isolate (e.g., 12 bacteria of the CeMbio collection) to inoculate 800 &#181;L of LB medium in separate wells of a 1 mL deep-well plate. Incubate overnight at the optimal temperature with shaking at 250 rpm. 
	NOTE: This protocol is optimized for use with defined natural microbiomes (e.g., CeMbio), but can be easily adjusted for individual microbes by growth under appropriate conditions or other culture vessels (tubes).</li>
	<li>Assess the growth of each microbe by spectrophotometry. Transfer a 20 &#181;L aliquot to 80 &#181;L of LB and add the solution to a clear flat bottom 96-well plate. Measure the OD600 on a plate reader.</li>
	<li>After overnight growth, centrifuge the deep-well culture plate at 4,000 x g for 10 min to pellet bacteria and remove the supernatant by aspiration using a 96-well aspiration manifold or multichannel pipette.</li>
	<li>Using OD600 values, normalize the concentration of each microbe to a final OD600 of 1.0 using sterile M9 medium.</li>
	<li>If combining microbes, determine the amount of bacterial culture needed for the experiment and create a microbiome master mix by combining equal volumes of each bacterial strain into a tube of sufficient size (e.g., 5 mL or 15 mL tubes).</li>
	<li>Spot 30-50 &#181;L of the microbes with a final OD600 of 1.0 onto the center of each nematode growth medium (NGM) agar-containing plate or well18. Allow the seeded microbiome to grow overnight at an optimal temperature (25 &#176;C here). 
	&#8203;NOTE: Prepare NGM agar plates in advance according to the instructions in the Wormbook or using pre-mixed NGM powder18. For example, 3 mL of NGM is added for 12-well plates, and 1.5 mL is added for 24-well plates.</li>
</ol>
2. Preparation of synchronized C. elegans for growth on the microbiome
<ol>
	<li>Grow approximately 100 worms on NGM plates (6 cm diameter) seeded with Escherichia coli OP50, until worms reach gravid adulthood. For the purposes of this protocol, the C. elegans N2 strain was used, though the protocol can easily be adapted to other nematode strains.</li>
	<li>Add 6 mL of M9 buffer (plus 0.01% triton X-100; M9-TX) to the plate, pipetting up and down to wash off approximately 100 gravid adult worms into a 15 mL conical tube.</li>
	<li>Prepare freshly made bleach solution by mixing two parts of bleach with one part of 5 M NaOH.</li>
	<li>Centrifuge the worms at 3,000 x g for 30 s to pellet the worms. Aspirate to bring the volume down to 4 mL, and then add 2 mL of bleach solution.</li>
	<li>Close the lid tightly and shake the tube. Monitor the adult worms in the solution under a stereo microscope at 10x magnification. When adult bodies start to break apart, typically after 3.5 min, stop shaking and centrifuge at 3,000 x g for 30 s.</li>
	<li>Aspirate to a minimal volume with a pipette. Resuspend, centrifuge, and aspirate four times in M9-TX to wash out the bleach.</li>
	<li>Resuspend in 4-6 mL of M9-TX and place the tube on a rotator overnight, allowing eggs to hatch and synchronize at the L1 stage.</li>
	<li>The next day, take 10 &#181;L of L1 worms in M9-TX from the tube and drop them on a clean Petri dish. Count the number of living L1 worms under a stereo microscope at 20x total magnification.</li>
	<li>Based on the L1 density, pipette the appropriate volume to drop ~50 synchronized L1 larvae on the edge of the microbiome seeded NGM plates and incubate at 20 &#176;C. Allow worms to grow until the desired age (Day 2 or Day 3 of adulthood).</li>
</ol>
3. Collecting worm population for gut microbiome analyses
<ol>
	<li>Wash the worms off the bacterial lawn with 1-2 mL of M9-TX to a sterilized 2 mL 96-well deep plate. 
	NOTE: For microbial strains that create significant biofilms, shake the plate with liquid for 5 min to break up microbial debris.</li>
	<li>Centrifuge the deep-well plate at 300 x g for 1 min to pellet the worms, and then remove the liquid using an aspirating manifold. 
	NOTE: Users can use a multichannel pipette to manually remove the liquid if an aspirating manifold is not available. Make sure the pipette tips do not touch the bottom of the wells to avoid the loss of worm samples.</li>
	<li>Add 1.8 mL of M9-TX to each well of the deep-well plate using a 1.2 mL multichannel pipette. Mix by pipetting up and down a couple of times, and centrifuge at 300 x g for 1 min. Repeat this step four times to remove bacteria in the liquid.</li>
	<li>After the final wash, bring the volume in each well to 100 &#181;L using the aspirating manifold. 
	NOTE: To separate adults from the larvae using gravity, leave the plate on the bench for 30-60 s, allowing adult worms to sink to the bottom of the well. Then, aspirate the plate to remove larvae from the suspension.</li>
	<li>Add 100 &#181;L of 10 mM levamisole in M9-TX to each well and allow the worms to paralyze for 5 min. After the levamisole treatment, add 4% of the bleach solution described in step 2.3 to each well for 2 min to reduce bacterial clumps, if needed. Wash with M9-TX twice following the bleach treatment and aspirate to a final volume of 100 &#181;L after the second wash.</li>
	<li>Transfer worms to a flat bottom 96-well plate containing 150 &#181;L of 10 mM levamisole in M9-TX. Keep the worm density at 50-100 per well and split the worms into multiple wells if the population is too crowded.</li>
</ol>
4. Setting up the large particle sorter and autosampler
<ol>
	<li>Turn on the air compressor, computer, and LPS instrument. Check and empty the waste tank. Then, add 500 mL of bleach to the empty waste tank. Check and refill sheath and water tanks if the volume is low. Ensure that the 250 &#181;m fluidic and optical core assembly (FOCA) is in place.</li>
	<li>Run the control particles for quality control. Open the LPS instrument software. On the software window, check Enable Lasers to turn on the 488 nm and 561 nm lasers. 
	NOTE: It is possible to skip steps 4.2-4.5 if the control particles have been run within the previous 2 weeks and FOCA has not been changed in that time.</li>
	<li>Select Set Up &#62; Control Particles. Select No in the prompt to go to the default settings.&#8203;&#8203; Vortex the control particles (10 mL dH2O and 10 mL control particles in a 50 mL conical tube) before each use and load them onto the sample port.</li>
	<li>When ready, click on Acquire to record 500 events. Ensure the event rate reads between 15&#8211;30 events/s. 
	NOTE:&#160;If the event rate is not 15&#8211;30 events/s, an engineer should be contacted to adjust machine settings.</li>
	<li>Examine coefficient of variation (CV) for all parameters. If CV &#60; 15%, the quality control is passed and the LPS software can be closed. If CV &#62; 15%, run again and adjust micrometers with freshly made control particles to align the laser with the flow cell.</li>
	<li>Connect and turn on the autosampler. Open the autosampler instrument software. This opens the autosampler and the LPS software. On the LPS software window, go to File &#62; New Experiment then File &#62; New Sample. Check Enable Lasers to turn on 488 nm and 561 nm lasers if it has not been done already.</li>
	<li>Create templates for acquisition dot plots using time of flight (TOF), extinction (EXT), and fluorescent channels in the LPS software window. A good starting maximum number to capture all the worms is 8,000 for TOF and EXT. Fluorescence scales will vary based on each microbe. Include control worm samples grown on non-fluorescent microbe (e.g., OP50) and each fluorescent microbe alone (if mixing microbes) as controls with every experiment.
	<ol>
		<li>Right-click within the body of a graph to modify the scaling. Use control animals such as Day 1 adults or synchronized L1s (or population of interest) to assist with TOF and EXT gating selection.</li>
	</ol>
	</li>
	<li>On the autosampler window, select Prime. Go to File &#62; Open Script to select the correct built-in script, and then click on OK.
	<ol>
		<li>For analysis only, select the script with Acquire. For sorting, select the script with Dispense. For acquisition from 96-well plates, select the script with 96 well; for acquisition from 384-well plates, select the script with 384 well. For sample volume (40 or 100 &#181;L), select the script with the corresponding volume.</li>
	</ol>
	</li>
	<li>Go to Plate Template on the autosampler software menu to select the desired wells for analysis. Load the control samples. After the plate is loaded onto the autosampler stage and secured, press Run Plate on the autosampler window. Save the file when prompted. 
	NOTE:&#160;Control samples and settings can be done with a 50 mL conical tube on the LPS sample port before attaching the LP sampler.</li>
	<li>When the acquisition is finished, click on the Store Data button on the top ribbon in the LPS software window and save the data again.</li>
</ol>
5. Analysis of C. elegans features and gut microbiome levels per animal
<ol>
	<li>Raw data is stored in three file types: .lmd, .bxr3, .txt. Load the text file labeled .txt file into the R program for analysis. Complete the steps below in R19.</li>
	<li>Use the package tidyverse20 as a framework for the analysis.</li>
	<li>Using the ggplot function (included in tidyverse) plot the LPS data using the TOF values as x-axis and EXT values as y-axis. A normal plot will show two clouds of dots. Ensure one is close to the origin and x-axis, indicating smaller particles such as larvae and small debris and the second group of particles is toward the upper right of the plot and represents adult nematodes.</li>
	<li>Using this grouping as a guide, choose adequate TOF and EXT cut-off values. Gating may vary by C. elegans strains on different microbes based on changes in physiology (e.g., dark vs. clear animals or changes in egg carriage may alter EXT coefficients).</li>
	<li>Use the function subset to separate adults and larvae by TOF and EXT gating. In the example here, use TOF &#62; 1,200 and EXT &#62; 1,000 settings to gate for adult animals.</li>
	<li>Analyze the resulting datasets for statistical differences; use the t-test function in R for this experiment.
	<ol>
		<li>To determine the impact of microbiome on the size and optical density of C. elegans, compare the TOF and EXT values, respectively, between conditions. TOF is a proxy for worm size and can indicate changes in the growth rates of the animals at specific times. EXT changes are greatest between the end of development and adulthood due to the light scattered by eggs and can be used to assess changes in the timing of that transition and fecundity. Assess the proportion and distributions of the progeny if they were retained during the analyses.</li>
		<li>To determine changes in gut microbiome colonization levels, first normalize fluorescence values (e.g., red or green columns) by dividing with individual TOF values for each animal. As worms grow in size and become adults, the volume of their gut gets larger as well. Normalization helps to reduce artifacts from differences in sample animal size distributions. Use normalized animals to assess changes in gut microbiome colonization across conditions. 
		&#8203;NOTE: A raw data example and corresponding R scripts can be found in the Supplementary File 1 JOVE_worm_microbiome.txt and Supplementary File 2 JOVE_worm_microbiome.r. See Figure 2 and Figure 3 for a representative example of these analyses of the host and the microbiome using two fluorescent bacteria.</li>
	</ol>
	</li>
</ol>
6. Sorting of C. elegans animals by gut microbiome features
<ol>
	<li>Collect a C. elegans population grown on fluorescent-tagged microbes (e.g., RFP) as described in Step 3. Worms should be placed into 50 mL conical tubes with a minimum of 5 mL of sample. Aim for approximately 2,000 worms/mL including all adults and offspring. 
	NOTE:&#160;This can also be done in a flat bottom 96-well plate using the LP sampler. Keep the worm density at 50&#8211;100 per well.
	<ol>
		<li>If LP sampler has been used then turn off and disconnect the LP sampler. Reconnect the sample and purge lines to the LPS before sorting.</li>
	</ol>
	</li>
	<li>Open the LPS software, go to File &#62; New Experiment then File &#62; New Sample. Create a dot plot with TOF on the x-axis and Extinction on the y-axis. Create another dot plot with TOF on the x-axis and Red on the y-axis. Use the same axis scale and laser settings as used previously. Check Enable Lasers to turn on 488 nm and 561 nm lasers 
	NOTE:&#160;If these plots have been created previously go to File &#62; Open Experiment and File &#62; Open Sample to load graphs.
	<ol>
		<li>Place the control sample tube onto the sample port. In the Acquire/Dispense dialog box click Acquire. Save the file when prompted. After enough worms have been measured to distinguish populations click Abort. 
		NOTE: Flow rates should be approximately 15&#8211;30 events/s. If not, adjust worm concentrations to ensure that individual animals are analyzed or sorted per droplet.&#160;In LPS, worms pass through the path of the laser in different positions (straight versus curled or bent). They can take a different amount of time to pass the laser and detector depending on their positions.</li>
		<li>In the TOF vs Extinction dot plot draw a gate around the adult population. In the TOF vs Red dot plot draw gates around the areas with high and low red values as regions of interest for adult worms with different levels of microbiome colonization. Go to View &#62; Gating Hierarchy and ensure that the fluorescent gates are listed under the adult population gate. After settings are optimized go to File &#62; Save Experiment and File &#62; Save Sample. 
		NOTE:&#160;In the example here, select animals based on TOF/EXT gates for adults and sort into pools that exhibit either high or low colonization with red fluorescent protein-expressing bacteria. However, any combination of parameters measured by the LPS can be used to identify the population(s) of interest.</li>
	</ol>
	</li>
	<li>Prior to sorting, ensure that the proper collection apparatus has been loaded. To load the 96-well plate onto the collection apparatus, select Load Plate A &#62; Move under the Move Stage section in the Acquire/Dispense dialog box to allow the collection stage to move into a position primed for loading a 96-well plate.</li>
	<li>For bulk sorting, select the appropriate tube size and click Move to load either a 15 mL or 50 mL conical tube. In the Sorting section of the Acquire/Dispense dialog box choose the sorting gate from the drop-down list of the regions created. Input the number of objects from the defined region to dispense to a collection tube directly below the flow cell nozzle. Click on the Bulk Sort button under the Acquire/Dispense dialog box.</li>
	<li>For dispensing to a 96-well plate, go to Setup &#62; Plates &#62; Calibrated Plates &#62; 96-well Plate. Then go to View &#62; Plate Template, select the wells to which the worms will be sorted, input the number of objects to be sorted into each well, and select the gated regions of interest. If more than one gated region will be sorted into the same plate check the box Gate Each Well. Click OK to save changes.</li>
	<li>After sorting numbers and locations have been assigned, click on the Fill Plate buttons from the Acquire/Dispense dialog box to initiate dispensing to a 96-well plate.
	<ol>
		<li>Before filling the entire 96-well plate perform test sorting of a subset of animals or controls into a 96-well plate and analyze using a stereomicroscope to ensure appropriate numbers and desired animal populations have been sorted. Once accurate sorting has been confirmed repeat steps 6.3-6.6 for experimental samples. Click on the Store Data button on the top ribbon in the LPS software window to save data again. 
		NOTE:&#160;All the measurements (EXT, TOF, RFP, GFP, etc.) can also be collected for sorted animals and compared to those that were not sorted. This can allow for analysis and sorting modes to be run at the same time if desired.</li>
	</ol>
	</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIP-MAP:+High-Throughput+mapping+of+Caenorhabditis+elegans+temperature-sensitive+mutants+via+molecular+inversion+probes.">MIP-MAP: High-Throughput mapping of Caenorhabditis elegans temperature-sensitive mutants via molecular inversion probes.</a> Genetics. 207 (2), 447-463 (2017).</li></ol>

The authors have no conflicts of interest to declare.

Flow vermimetry has been used to characterize C. elegans genes and pathways against pathogen colonization and toxicity in several studies21,22. Here, a high throughput amenable approach is presented that uses C. elegans to investigate how intestinal microbiomes modulate their host physiology. Compared to existing methods using colony forming units (CFU) or 16S rRNA amplicon sequencing9,16</sup...

Animal evolution is under constant microbial influence1. From diverse microbes in the environment, animal hosts acquire specific partners2 that extend the capabilities of the host and drive its physiology and susceptibility to disease3. For example, metagenomic analyses of the gut microbiome uncovered enriched metabolic classes of microbial genes that may confer greater energy harvest and storage in obese mice4, many of which are also found in the human gut microbiome5. There is still a great need to establish causal relationships and pinpoint the molecular determinants of the microbiome impact, though progress has been hampered by the microbiome complexities and tractability of host systems to large-scale screening.
The model organism C. elegans provides a platform to advance molecular understanding of links between microbiome and host physiology. C. elegans possesses 20 intestinal cells with a mucosal layer and villi structures. These cells are equipped with abundant chemoreceptor genes that sense microbial products and produce antimicrobial molecules that potentially regulate their gut colonizers6,7. This conserved biology of C. elegans has led to a tremendous number of discoveries in host signaling that regulate gut microbes, including insulin signaling, TGF-beta, and MAP Kinase8,9,10.
C. elegans utilize microbes as both their diet for growth during development and microbiome as adults. With old age, some microbes may over-accumulate in the gut lumen and the host-microbe relationship shifts from symbiosis to pathogenesis11. In their natural habitats, C. elegans encounters a wide array of bacterial species12,13. Sequencing 16S rDNA from representative samples collected in natural habitats (rotten fruits, plant stem, and animal vectors) revealed that the natural microbiome of C. elegans is dominated by four bacterial phyla: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. Within these divisions lies great variation in the diversity and richness of bacteria based on the habitat12,13,14,15. Several defined communities have been established, including the 63-member (BIGbiome)16 and 12-member (CeMbio) collections representing the top microbiome genera created for the C. elegans research community17. Both microbiomes and component strains can have a diverse impact on the physiology of C. elegans such as body size, growth rates, and stress responses9,16,17. These studies provide resources and examples to establish C. elegans as a model for microbiome research.
Here a large particle sorter (LPS) based workflow (Figure 1) is presented that utilizes the C. elegans system to simultaneously measure microbiome composition and basic measures of host physiology at the population scale. From the microbial side, the workflow is adaptable to assemble a defined microbiome or single microbes to test the robustness and plasticity of the community with increasing microbial interactions. From the host side, the workflow enables high throughput assays to measure colonization levels of fluorescent microbes in the microbiome and host physiological readout in terms of development, body size, and reproduction. Taken together, the C. elegans microbiome model enables high throughput screens to pinpoint the metabolic and genetic determinants modulating host physiology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical bottom centrifuge tubes</td>
			<td>VWR</td>
			<td>10026-076</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep-well plates (1 mL)</td>
			<td>Axygen</td>
			<td>P-DW-11-C</td>
			<td></td>
		</tr>
		<tr>
			<td>96 deep-well plates (2 mL)</td>
			<td>Axygen</td>
			<td>P-DW-20-C</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well Costar plate</td>
			<td>Corning</td>
			<td>3694</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Millipore Sigma</td>
			<td></td>
			<td>Standard bacteriology agar is also sufficient.</td>
		</tr>
		<tr>
			<td>Aspirating manifold</td>
			<td>V&#38;P scientific</td>
			<td>VP1171A</td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bleach solution&#160;</td>
			<td></td>
			<td></td>
			<td>Mix Bleach with 5M Sodium hypochlorite 2:1 (v/v)</td>
		</tr>
		<tr>
			<td>Cell Imaging Multimode Reader</td>
			<td>Biotek</td>
			<td>Cytation 5</td>
			<td>Bacterial OD measurement</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo scientific&#160;</td>
			<td>Sorvall Legend XTR</td>
			<td>For 96 well plate and conical tubes</td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Nikon</td>
			<td>TiE</td>
			<td></td>
		</tr>
		<tr>
			<td>ggplot: Various R Programming Tools for Plotting Data.</td>
			<td>R package</td>
			<td>Version 3.3.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Particle Autosampler</td>
			<td>Union Biometrica</td>
			<td>LP Sampler</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Particle Sorter</td>
			<td>Union Biometrica</td>
			<td>COPAS Biosorter</td>
			<td></td>
		</tr>
		<tr>
			<td>Levamisole</td>
			<td>Fisher</td>
			<td>AC187870100</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny Broth (LB)</td>
			<td>RPI</td>
			<td>L24066</td>
			<td>Standard LB home-made recipes using Bacto-tryptone, yeast extract, and NaCl are also sufficient.</td>
		</tr>
		<tr>
			<td>M9 solution&#160;</td>
			<td></td>
			<td></td>
			<td>22 mM KH2PO4 monobasic, 42.3 mM Na2HPO4, 85.6 mM NaCl, 1 mM MgSO4</td>
		</tr>
		<tr>
			<td>Nematode Growth Medium</td>
			<td>RPI</td>
			<td>N81800-1000.0</td>
			<td>1 mM CaCl2, 25 mM KPO4 pH 6.0, 1 mM MgSO4 added after autoclaving.</td>
		</tr>
		<tr>
			<td>RStudio</td>
			<td>GNU</td>
			<td>Version 1.3.1093</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hypochlorite</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>5M NaOH</td>
		</tr>
		<tr>
			<td>Stereo Microscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 10 cm diameter petri dishes</td>
			<td>Corning</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 12-well plates</td>
			<td>VWR</td>
			<td>10062-894</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 24-well plates</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 6 cm diameter petri dishes</td>
			<td>Corning</td>
			<td>351007</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
	</tbody>
</table>

application of flow vermimetry for quantification and analysis of the caenorhabditis elegans gut microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

Defining adult and larvae population gates 
Here, synchronized C. elegans L1s were grown on an NGM plate seeded with E. coli OP50 (Eco), a standard laboratory diet. C. elegans populations were collected for LPS analysis after 96 h or 120 h of growth at 20 &#176;C (Figure 2A). A dot plot of extinction (EXT, a proxy of body density) versus time-of-flight (TOF, a proxy of body length) creates two visually separated clouds of animals. Each dot rep...

Watch this Scientific Journal Video about Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome at JoVE.com

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

Caenorhabditis elegans is a powerful model to examine the molecular determinants driving host-microbiome interactions. We present a high throughput pipeline profiling the single animal levels of gut microbiome colonization together with key aspects of the C. elegans physiology.

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

application-flow-vermimetry-for-quantification-analysis

Research

JoVE Journal

Biology

650 Views.  Baylor College of Medicine. Caenorhabditis elegans is a powerful model to examine the molecular determinants driving host-microbiome interactions. We present a high throughput pipeline profiling the single animal levels of gut microbiome colonization together with key aspects of the C. elegans physiology.

Video: Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 &alpha;-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in Caenorhabditis elegans

In this paper, we describe an optimized procedure for extracting and analyzing prostaglandins and other eicosanoids from C. elegans using LC-MS/MS.

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, ...

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved in humans and are associated with metabolic syndrome or other diseases. Examination of lipid accumulation in this organism can be carried out by fixative dyes or label-free methods. Fixative stains like Nile red and oil red O are inexpensive, reliable ways to quantitatively measure lipid levels and to qualitatively observe lipid distribution across tissues, respectively. Moreover, these stains allow for high-throughput screening of various lipid metabolism genes and pathways. Additionally, their hydrophobic nature facilitates lipid solubility, reduces interaction with surrounding tissues, and prevents dissociation into the solvent. Though these methods are effective at examining general lipid content, they do not provide detailed information about the chemical composition and diversity of lipid deposits. For these purposes, label-free methods such as GC-MS and CARS microscopy are better suited, their costs notwithstanding.

Quantification of Lipid Abundance and Evaluation of Lipid Distribution in Caenorhabditis elegans by Nile Red and Oil Red O Staining

Nile red staining of fixed Caenorhabditis elegans is a method for quantitative measurement of neutral lipid deposits, while oil red O staining facilitates qualitative assessment of lipid distribution among tissues.

Caenorhabditis elegans is an exceptional model organism in which to study lipid metabolism and energy homeostasis. Many of its lipid genes are conserved ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, sequester or eliminate damaged proteins to maintain a healthy proteome, thus ensuring cellular proteostasis and preventing further protein damage. Because of redundant functions within the proteostasis network, screening for detectable phenotypes using knockdown or mutations in chaperone-encoding genes in the multicellular organism Caenorhabditis elegans results in the detection of minor or no phenotypes in most cases. We have developed a targeted screening strategy to identify chaperones required for a specific function and thus bridge the gap between phenotype and function. Specifically, we monitor novel chaperone interactions using RNAi synthetic interaction screens, knocking-down chaperone expression, one chaperone at a time, in animals carrying a mutation in a chaperone-encoding gene or over-expressing a chaperone of interest. By disrupting two chaperones that individually present no gross phenotype, we can identify chaperones that aggravate or expose a specific phenotype when both perturbed. We demonstrate that this approach can identify specific sets of chaperones that function together to modulate the folding of a protein or protein complexes associated with a given phenotype.

Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions

To study chaperone-chaperone and chaperone-substrate interactions, we perform synthetic interaction screens in Caenorhabditis elegans using RNA interference in combination with mild mutations or over-expression of chaperones and monitor tissue-specific protein dysfunction at the organismal level.

Correct folding and assembly of proteins and protein complexes are essential for cellular function. Cells employ quality control pathways that correct, ...

Automating Aggregate Quantification in Caenorhabditis elegans

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans

The following protocol describes the development and optimization of a high-throughput workflow for worm culturing, fluorescence imaging, and automated image processing to quantify polyglutamine aggregates as an assessment of changes in proteostasis.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures the interaction of homologous chromosomes is the assembly of the synaptonemal complex (SC) in meiotic prophase I. Even though there is little sequence homology between protein components within the SC among different species, the general structure of the SC has been highly conserved during evolution. In electron micrographs, the SC appears as a tripartite, ladder-like structure composed of lateral elements or axes, transverse filaments, and a central element. 
However, precisely identifying the localization of individual components within the complex by electron microscopy to determine the molecular structure of the SC remains challenging. By contrast, fluorescence microscopy allows for the identification of individual protein components within the complex. However, since the SC is only ~100 nm wide, its substructure cannot be resolved by diffraction-limited conventional fluorescence microscopy. Thus, determining the molecular architecture of the SC requires super-resolution light microscopy techniques such as structured illumination microscopy (SIM), stimulated-emission depletion (STED) microscopy, or single-molecule localization microscopy (SMLM). 
To maintain the structure and interactions of individual components within the SC, it is important to observe the complex in an environment that is close to its native environment in the germ cells. Therefore, we demonstrate an immunohistochemistry and imaging protocol that enables the study of the substructure of the SC in intact, extruded Caenorhabditis elegans germline tissue with SMLM and STED microscopy. Directly fixing the tissue to the coverslip reduces the movement of the samples during imaging and minimizes aberrations in the sample to achieve the high resolution necessary to visualize the substructure of the SC in its biological context.

Super-Resolution Microscopy of the Synaptonemal Complex Within the Caenorhabditis elegans Germline

Super-resolution microscopy can provide a detailed insight into the organization of components within the synaptonemal complex in meiosis. Here, we demonstrate a protocol to resolve individual proteins of the Caenorhabditis elegans synaptonemal complex.

During meiosis, homologous chromosomes must recognize and adhere to one another to allow for their correct segregation. One of the key events that secures ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are crucial in constituting structural components of cell membranes, storing energy, and as signaling molecules. Regulation of lipid metabolism and signaling is essential to activate distinct longevity pathways. The roundworm Caenorhabditis elegans is an excellent and powerful organism to dissect the contribution of lipid metabolism and signaling in longevity regulation. Multiple research studies have described how diet supplementation of specific lipid molecules can extend C. elegans lifespan; however, minor differences in the supplementation conditions can cause reproducibility issues among scientists in different labs. Here, two detailed supplementation methods for C. elegans are reported employing lipid supplementation either with bacteria seeded on plates or bacterial suspension in liquid culture. Also provided herein are the details to perform lifespan assays with lifelong lipid supplementation and qRT-PCR analysis using a whole worm lysate or dissected tissues derived from a few worms. Using a combination of longitudinal studies and transcriptional investigations upon lipid supplementation, the feeding assays provide dependable approaches to dissect how lipids influence longevity and healthy aging. This methodology can also be adapted for various nutritional screening approaches to assess changes in a subset of transcripts using either a small number of dissected tissues or a few animals.

Lipid Supplementation for Longevity and Gene Transcriptional Analysis in Caenorhabditis elegans

The present protocol describes lipid supplementation methods in liquid and on-plate cultures for Caenorhabditis elegans, coupled with longitudinal studies and gene transcriptional analysis from bulk or a few worms and worm tissues.

Aging is a complex process characterized by progressive physiological changes resulting from both environmental and genetic contributions. Lipids are ...