The authors would like to acknowledge H. Schulenberg and C. LaRock for their generous sharing of bacterial strains used in these experiments. This work was supported by funding from Emory University and NSF (PHY2014173).

Worms used in these experiments were obtained from the Caenorhabditis Genetic Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Bristol N2 is the wild-type. DAF-2/IGF mutants daf-16(mu86) I (CGC CF1038) and daf-2(e1370) III (CGC CB1370)&#160;are used to illustrate differences in intestinal bacterial load.
HT115(DE3) E. coli carrying the pos-1 RNAi vector is from the Ahringer library21. ...

<ol>
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The authors have no conflicts of interest.

Here data are presented on the advantages of single-worm quantification of bacterial load in C. elegans, along with a 96-well disruption protocol to allow the rapid and consistent acquisition of large data sets of this type. As compared with existing methods33, these protocols allow higher-throughput measurement of intestinal microbial communities in the worm.
This approach has plating as a rate-limiting step and is not truly &#34;high-throughput&#34;. Large-ob...

Heterogeneity in host-microbe associations is observed ubiquitously, and variation between individuals is increasingly recognized as a contributing factor in population-level processes from competition and coexistence1 to disease transmission2,3,4. In C. elegans, &#34;hidden heterogeneity&#34; within isogenic populations has been observed repeatedly, with sub-populations of individuals showing distinct phenotypes in heat shock response5,6, ageing, and lifespan7,8,9,10,11, and many other aspects of physiology and development12. Most analyses that attempt to identify sub-population structure provide evidence for two sub-populations in experimental populations of isogenic, synchronized worms5,7,8, though other data suggest the possibility of within-population distributions of traits rather than distinct groups7,12,13. Of relevance here, substantial heterogeneity in intestinal populations is observed even within isogenic populations of worms colonized from a shared source of microbes13,14,15,16, and this heterogeneity can be concealed by the batch digest measurements that are widely used17,18,19,20 for bacterial quantification in the worm.
This work presents data suggesting a need for greater reliance on single-worm measurements in host-microbe association, as well as protocols for increasing accuracy and throughput in single-worm disruption. These protocols are designed to facilitate mechanical disruption of large numbers of individual C. elegans for quantification of viable bacterial load, while providing better repeatability and lower effort per sample than pestle-based disruption of individual worms. A recommended gut-purging step, where worms are permitted to feed on heat-killed E. coli prior to the preparation for disruption, is included to minimize contributions from recently ingested and other transient (non-adhered) bacteria. These protocols include a cold-paralysis method for cleaning the cuticle with a low-concentration surface bleach treatment; surface bleaching can be used as a preparatory step in single-worm disruption or as a method for preparing live, externally germ-free worms. This surface-bleaching method is sufficient to remove a wide range of external microbes, and cold treatment provides an alternative to conventional levamisole-based paralysis; while levamisole will be preferred for cold-sensitive experiments, cold paralysis minimizes contributions to hazardous waste streams and allows rapid resumption of normal activity. While the full protocol describes a laboratory experiment where worms are colonized with known bacteria, the procedures for cleaning worms and single-worm disruption can readily be applied to worms isolated from wild samples or colonized in microcosm experiments. The protocols described here produces live bacteria extracted from the worm intestine, suitable for plating and quantification of colony forming units (CFUs) in individual worms; for sequencing-based intestinal community analysis, subsequent cell lysis and nucleic acid extraction steps should be added to these protocols.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well flat-bottom polypropylene plates, 300 uL</td>
			<td>Evergreen Labware</td>
			<td>290-8350-03F</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate sealing mat, silicon, square wells (AxyMat)</td>
			<td>Axygen</td>
			<td>AM-2ML-SQ</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates, 2 mL, square wells</td>
			<td>Axygen</td>
			<td>P-2ML-SQ-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well polypropylene plate lids</td>
			<td>Evergreen Labware</td>
			<td>290-8020-03L</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>443570050</td>
			<td></td>
		</tr>
		<tr>
			<td>Bead mill adapter set for 96-well plates</td>
			<td>QIAGEN</td>
			<td>119900</td>
			<td>Adapter plates for use with two 96-well plates on the TissueLyser II</td>
		</tr>
		<tr>
			<td>Bead mill tissue homogenizer (TissueLyser II)</td>
			<td>QIAGEN</td>
			<td>85300</td>
			<td>Mechanical homogenizer for medium to high-throughput sample disruption</td>
		</tr>
		<tr>
			<td>BioSorter</td>
			<td>Union Biometrica</td>
			<td>By quotation</td>
			<td>Large object sorter equipped with a 250 micron focus for C. elegans</td>
		</tr>
		<tr>
			<td>Bleach, commercial, 8.25% sodium hypochlorite</td>
			<td>Clorox</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Breathe-Easy 96-well gas permeable sealing membrane</td>
			<td>Diversified Biotech</td>
			<td>BEM-1</td>
			<td>Multiwell plate gas permeable polyurethane membranes. Thin sealing film is permeable to O2, CO2, and water vapors and is UV transparent down to 300 nm. Sterile, 100/box.</td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Fisher Scientific</td>
			<td>AC423525000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>VWR</td>
			<td>AAA11470-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Citric acid monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124910010</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) sulfate pentahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC197722500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 6765 LSE Mini Microcentrifuge</td>
			<td>Corning</td>
			<td>&#160;COR-6765</td>
			<td></td>
		</tr>
		<tr>
			<td>Disodium EDTA</td>
			<td>Fisher Scientific</td>
			<td>409971000</td>
			<td></td>
		</tr>
		<tr>
			<td>DL 1,4 Dithiothreitol, 99+%, for mol biology, DNAse, RNAse and Protease free, ACROS Organics</td>
			<td>Fisher Scientific</td>
			<td>327190010</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 1.5 mL microcentrifuge tubes, natural</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf 5424R microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5406000640</td>
			<td>24-place refrigerated benchtop microcentrifuge</td>
		</tr>
		<tr>
			<td>Eppendorf 5810R centrifuge with rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22627040</td>
			<td>3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates</td>
		</tr>
		<tr>
			<td>Eppendorf plate bucket (x2), for Rotor S-4-104</td>
			<td>Eppendorf</td>
			<td>22638930</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Fisher Scientific</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, 2.7 mm</td>
			<td>Life Science Products</td>
			<td>LS-79127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads, acid-washed, 425-600 &#181;m</td>
			<td>Sigma</td>
			<td>G877-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass plating beads</td>
			<td>VWR</td>
			<td>76005-124</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>VWR</td>
			<td>BDH7204-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>423731000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes pellet pestle motor</td>
			<td>DWK Life Sciences</td>
			<td>749540-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimble Kontes polypropylene pellet pestles and microtubes, 0.5 mL</td>
			<td>DWK Life Sciences</td>
			<td>749520-0590</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica DMi8 motorized inverted microscope with motorized stage</td>
			<td>Leica</td>
			<td>11889113</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica LAS X Premium software</td>
			<td>Leica</td>
			<td>11640687</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC124900010</td>
			<td></td>
		</tr>
		<tr>
			<td>Manganese(II) chloride tetrahydrate</td>
			<td>VWR</td>
			<td>470301-706</td>
			<td></td>
		</tr>
		<tr>
			<td>PARAFILM M flexible laboratory sealing film</td>
			<td>Amcor</td>
			<td>PM996</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>Fisher Scientific</td>
			<td>BP1420-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 10 cm</td>
			<td>VWR</td>
			<td>25384-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, round, 6 cm</td>
			<td>VWR</td>
			<td>25384-092</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes, square, 10 x 10 cm</td>
			<td>VWR</td>
			<td>10799-140</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-buffered saline (1X PBS)</td>
			<td>Gold Bio</td>
			<td>P-271-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene autoclave tray, shallow</td>
			<td>Fisher Scientific</td>
			<td>13-361-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>AC134062500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP363-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP362-1</td>
			<td></td>
		</tr>
		<tr>
			<td>R 4.1.3/RStudio 2022.02.0 build 443</td>
			<td>R Foundation</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Scoop-type laboratory spatula, metal</td>
			<td>VWR</td>
			<td>470149-438</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon carbide 36 grit</td>
			<td>MJR Tumblers</td>
			<td>n/a</td>
			<td>Black extra coarse silicon carbide grit. Available in 0.5-5 lb sizes from this vendor.</td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate</td>
			<td>Fisher Scientific</td>
			<td>BP166-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>VWR</td>
			<td>BDH7247-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic anhydrous</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodum chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>AC419760010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-potassium citrate monohydrate</td>
			<td>Fisher Scientific</td>
			<td>AC611755000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc sulfate heptahydrate</td>
			<td>Fisher Scientific</td>
			<td>AC205982500</td>
			<td></td>
		</tr>
	</tbody>
</table>

using single-worm data to quantify heterogeneity in caenorhabditis elegans-bacterial interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Bleach sterilization of live worms 
Surface-bleached worms are effectively free of external bacteria until motility returns and excretion resumes. Under the conditions used here, rapid extinction of bacteria in buffer is observed (Figure 1A-C, Supplementary Figure 2, Video 1) without disturbing the gut-associated bacteria in cold-paralyzed worms (Figure 1D-F, Video 2). Thes...

Watch this Scientific Journal Video about Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions at JoVE.com

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

This protocol demonstrates mechanical disruption of sea elegans. In a 96 well format allowing medium throughput quantification of bacterial load in individual worms. This technique increases the speed and uniformity of mechanical disruption as compared to Pasel based methods allowing researchers to easily produce larger data sets of high quality single worm measurements.This technique involves many moving parts and it's easy to lose your place. Make sure that you have all materials, labeled tubes, buffers and plates set up before beginning. Demonstrating the procedure will be Megan Taylor a research specialist lead from my laboratory.Begin by re suspending the worm sample in one milliliter of M nine TX zero one in a micro centrifuge tube. Collect the adult worms by centrifugation and discard the supernatant. Using the same centrifugation parameters.Rinse the worms twice with one milliliter of M9TX01 and once with one milliliter of M9 worm buffer, to minimize the external bacteria. Then, re suspend each sample in one milliliter of S medium plus two x heat killed OP50 in a culture tube. Incubate the tubes at 25 degrees Celsius for 20 to 30 minutes to pass the non-ad adhered bacteria from the gut.After incubation, rinse the purged worms twice with one milliliter of cold M9TX01 and discard the super natant. Put the tubes on ice for 10 minutes to paralyze the worms. Then add one milliliter of ice cold M nine worm buffer with unscented bleach to each tube.Incubate the tubes on ice for a minimum of 10 minutes to kill external bacteria. Discard the bleach buffer and return the tubes to the ice to prevent the worms from pumping. Next, add one milliliter of cold M9TX01 to each tube and centrifuge for five seconds.In a mini centrifuge. Return the tubes to ice and remove the supernatant. Repeat this step once.For chemical permeabilization of worm cuticle. Transfer the tubes containing the worms in 20 microliters of buffer to a room temperature tube rack. Then add 100 microliters of SDS/DTT solution to each warm sample.Incubate the tubes for up to eight minutes on the bench to partially break down the cuticle of the adult worms. At this point, the worms will die and settle at the bottom of the tube. After incubation, carefully discard the SDS/DTT supernatant.Then, add one milliliter of M9TX01 to each tube and centrifuge briefly to pellet the worms. Discard the supernatant and re suspend the worms in one milliliter M nine worm buffer with 0.1%Triton x-100. To prepare for mechanical disruption of worms, obtain a sterile two milliliter deep well 96 well plate and a silicone plate cover.Use a sterile scoop spatula to add a small amount of sterile 36 grid silicone carbide to each well such that the grid barely covers the bottom of the well. Add 180 microliters of M nine worm buffer to each well. Label the columns and rows and then cover the plate loosely with the silicone plate cover.Next, transfer the permeabilized worms to a small Petri dish with M9TX01 filled up to a depth of one centimeter. Using a dissecting microscope pipette out individual worms in 20 microliter volumes and transfer them to individual wells of the 96 well plate to eject any worms stuck to the pipette, aspirate 20 microliters of M9TX01 from a clear area of the Petri dish and release it back into the dish. Once all worms are transferred cover the 96 well plate with a sheet of flexible ceiling film with the paper backed side facing down onto the sample wells.Place the silicone ceiling mat lightly on top of the flexible ceiling film without pressing the cover down into the wells. Place the plate at four degrees Celsius for 30 to 60 minutes to prevent overheating during disruption. After chilling the plate press the silicone ceiling mat down firmly into the wells to seal them.Then secure plates in the tissue disruptor. Shake the plates for one minute at 30 hertz. Then rotate the plates by 180 degrees and repeat shaking for one minute.Tap the plates firmly on the bench two or three times to dislodge any grit from the flexible ceiling film. Centrifuge the plate set 2, 400 times G for two minutes to collect the samples at the bottom of the wells. Then remove the silicone lid and pull off the ceiling film.To prepare tenfold serial dilution of the worm digests, fill 180 microliters of one X PBS in rows B to D of a 96 well plate. Using a multi-well pipetter set to 200 microliters, slowly mix the worm digest by pipetting. Transfer the maximum amount of the liquid to row A of the 96 well plate containing PBS.Using a multichannel pipette, remove 20 microliters from the top row and dispense into row B.Followed by mixing. Repeat this step for row B to C and then row C to D to get a 1000 fold dilution For bacterial quantification of mono colonized worms. Plate 10 to 20 microliters of each dilution on solid agar plates.For multi-species colonization, plate 100 microliters of each dilution on 10 centimeter agar plates. Using this protocol, effective external sanitization was observed after surface bleaching of cold paralyzed worms as seen by the decline of external bacteria in buffer. Without affecting the gut associated bacteria.Manual disruption of worms resulted in more heterogeneity. Silicone carbide grit was ideal for disruption with or without Tri Andex. In the 96 well method, large glass beads were not suitable for the 96 well technique.Small glass beads produced consistent results but clogged the pipe at tip. Worms colonized on the same pool of bacteria showed high variation in intestinal load when observed for individual bacteria and multi-species bacterial communities. Worms colonized with bacteria expressing GFP, showed heterogeneity in individual worm measurements.For this GFP expressing bacterial strain comparing the colonization of wild type Bristol Len two worms. To DAF two IGF mutants showed that the DAF 16 mutant supports larger populations of bacteria, while DAF two is resistant to colonization. This heterogeneity was characteristic and consistent over different runs of the same experiment.Re sampling of data to simulate batch digests was found to alter the distribution as compared to individual worm data. The batch extrapolated CFU per worm was centered around the arithmetic mean of the individual data leading to loss of biological variation. Instead of proceeding to digest live surface bleach and rinse worms can be used for further experiments.Movement will resume quickly once worms are removed from the cold.

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Biology

1.9K visualizações.  Emory University. Este protocolo demonstra a interrupção mecânica dos elegans marinhos. Em um formato de poço 96 permitindo quantificação média de rendimento de carga bacteriana em vermes individuais. Esta técnica aumenta a velocidade e a uniformidade da interrupção mecânica em comparação com os métodos baseados em Pasel, permitindo que os pesquisadores produzam facilmente conjuntos de dados maiores de medições de vermes únicos de alta qualidade.Esta técnica envolve muitas partes móve...