TITLE: High-throughput assessment of changes in the Caenorhabditis elegans gut microbiome
AUTHORS: Fan Zhang, Jessica Weckhorst, Adrien Assie, Anastasia Khodakova, Mario Loeza Cabrera, Daniela Vidal, Christopher Ayoub, Buck Samuel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The gut microbiome is an important driver of host physiology and development. Altered abundance or membership of this microbe community can influence host health and disease progression, including the determination of host lifespan and healthspan. Here, we describe a robust pipeline to measure the microbiome abundance and composition in the </span><span style = "font-style:italic;">C. elegans </span><span>gut that can be applied to examine the role of the microbiome on host aging processes.</span></div></div>

[STEPS]
?. [High-throughput Colonization Assay]
Wash worms from bacterial lawns with M9 buffer +  Triton X to a sterilized 2 ml 96-well deep plate.Use  M9+ Triton X to wash worms from a 12-well plateUse M9+ Triton X to wash worms from a 24-well plate
1000 µl
600 µl
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?. [High-throughput Colonization Assay]
Bring each well to  total with M9 + Triton X using a multichannel pipette
1.8 mL
?. [High-throughput Colonization Assay]
Centrifuge deep well plate at  to pellet down worms, then remove liquid by using an aspirating manifold. Alternatively worms can be allowed to settle in the deep well plate by gravity without centrifugation to separate adults from progeny.
[at 300g]
?. [High-throughput Colonization Assay]
Repeat steps 2-3 5 times with M9 to further remove bacteria in liquid.
?. [High-throughput Colonization Assay]
Bring the volume in each well down to using the aspirating manifold.
100 µl
?. [High-throughput Colonization Assay]
Add  of levamisole in 1X M9 + 0.01% Triton X to each well and allow the worms to paralyze for . Confirm paralysis via microscopy before proceeding to the next step.
100 µl
?. [High-throughput Colonization Assay]
Add of 4% bleach solution (2:1 Bleach:5M NaOH) in M9 to each well for
200 µl
This will further eliminate bacteria in liquid and attached on worm cuticle.
?. [High-throughput Colonization Assay]
Repeat step 2-3 2 times with 1X M9 +   Triton X to remove bleach and levamisole.
?. [Optional]
Save  of supernatant from the last wash to use as a control to assess the background bacterial residual before host lysis.
100 µl
?. [High-throughput Colonization Assay]
Bring liquid volume in each well to 1X M9 +   Triton X, mix and transfer  of the mixture to a flat bottom 96 well plate (Costar 3370, Corning).
1 mL
100 µl
?. [High-throughput Colonization Assay]
Take bright field images for each well for later analysis of worm population size, life stages, and individual length.
?. [High-throughput Colonization Assay]
Aspirate remaining liquid volume down to and add 1.0 mm sterilized garnet beads (Biospect) to each well (enough to cover well bottom).
300 µl
?. [High-throughput Colonization Assay]
Seal the plate with a sterile plastic mat and lyse worms in Mixer Mill (Restch) or TissueLyser at 25 Hz speed for 5 min.
[Bead beating]
?. [High-throughput Colonization Assay]
Mix by pipetting and transfer of cell lysate to a 96-well plate prepared with  1x PBS per well (1:10 dilution) and mix
20 µl
180 µl
?. [High-throughput Colonization Assay]
Transfer of the dilution from step 14 to  LB in 96 well flat bottom for liquid OD growth assay.
20 µl
180 µl
?. [DNA extraction and 16S rRNA PCR]
Centrifuge the deep-well plate at 4000g for 10 mins to pellet worms and bacteria, then bring the remaining liquid volume down to
190 µl
[Centrifugation at 4000g]
?. [DNA extraction and 16S rRNA PCR]
Freeze the lysate in -80 °C freezer overnight.DNA extraction and 16S rRNA PCR
[Freezing]
?. [DNA extraction and 16S rRNA PCR]
Thaw the plate and add 0.1 mm sterile zirconia/silica beads (BioSpec products) to each well (enough to cover well bottom) and bead-beat in Mixer Mill (Restch) or TissueLyser for 5 mins to further lyse bacterial cells.
[Shaking/Lysis]
?. [Calculate CFU based on liquid growth curve]
Fill a flat-bottom 96-well plate with  of sterile filtered PBS. Pipette  of the microbiome mixture used in the assay into four replicate wells in column 1 of the plate (e.g., A1, B1, C1, and D1). For controls, pipet  of LB in the remaining wells of column 1.
180 µl
20 µl
20 µl
?. [Calculate CFU based on liquid growth curve]
Using a multichannel pipette, serially dilute the microbiome mixture (1:10) in columns 2-12 to create a dilution series for all wells of the plate. Change tips between each dilution/column.
?. [Calculate CFU based on liquid growth curve]
From the serial dilution plate, plate  of each dilution onto a rectangular LB plate using a multichannel pipette. Incubate at optimal growth temperature overnight. Count colonies for each of the wells where possible.
10 µl
?. [Calculate CFU based on liquid growth curve]
Prepare a growth plate by filling each well of a flat bottom 96-well plate with  of LB. Transfer 20 μL from the serial dilution plate to the corresponding well of the growth plate. Incubate at the optimal temperature with intermittent shaking and monitor OD600 regularly (e.g., every 15-30 min) until growth is observed in all wells.
180 µl
?. [Calculate CFU based on liquid growth curve]
Use a plotting software (Microsoft Excel, R or otherwise), to visualize growth curves by plotting OD600 reading vs time for each well on a single chart. Verify that the growth patterns are consistent between replicates and growth curves are ordered by the dilution factor.
?. [Calculate CFU based on liquid growth curve]
Identify the range of OD600 values within the linear portion of the growth curves. Use the linear range identified and the following equation to calculate the optical density Threshold: Threshold = \frac{(OD_{600max}  - OD_{600min})}{2} + OD_{600min}
?. [Calculate CFU based on liquid growth curve]
Using the only data points where the OD600 readings for each well fall between the linear portion, calculate the Slope and Intercept for each well
?. [Calculate CFU based on liquid growth curve]
To calculate the time at which each well reached the threshold (Y-axis of calibration curve), use the following equation for each well: Time = \frac{(Threshold - Intercept)}{Slope}
?. [Calculate CFU based on liquid growth curve]
Plot CFU values collected in Step 19 vs.Time of Threshold crossing for each well, and calculate the R squared value and exponential trendline equation. Use the obtained equation to calculate CFU from the respective threshold time of each sample.
?. [Calculate CFU based on liquid growth curve]
Determine colonization levels on a per animal basis for each sample using the following formula: CFU / animal = \frac{(CFU * Dilution factor)}{number  of animals}
?. [DNA extraction and 16S rRNA PCR]
Add of    proteinase K to each well.
10 µl
?. [DNA extraction and 16S rRNA PCR]
In a ThermoCycler run a program as follow:at at to desactivate the proteinase Kkeep on hold at
[step]
60 °C
[step]
95 °C
4 °C
?. [DNA extraction and 16S rRNA PCR]
Centrifuge the plate to pellet down cellular fractions.Transfer  of supernatant to a clean 96 well PCR plate.If using the plate again soon, place at , otherwise, store at
[at 4000g]
100 µl
4 °C
-80 °C
?. [DNA extraction and 16S rRNA PCR]
In a 5 mL microcentrifuge tube, mix: of 2x Taq Master Mix (1 tube) of 515F primer H2O
1.25 mL
100 µl
1 mL
?. [DNA extraction and 16S rRNA PCR]
Addof newly mixed master mix into each well of a new 96 well PCR plate
23 µl
?. [DNA extraction and 16S rRNA PCR]
Add  of reverse primer 806R to each well of the 96 well PCR plate.
1 µl
?. [DNA extraction and 16S rRNA PCR]
Add of the supernatant from the PCR plate prepared in step 23 as DNA template for the PCR.The remaining supernatant can be frozen at  for storage.
1 µl
-80 °C
?. [DNA extraction and 16S rRNA PCR]
Place the PCR plate in the ThermoCycler and run the following program  ABC1TemperatureTime, 96-wellRepeat294 °C3 min394 °C45 sx35450 °C60 sx35572 °C90 sx35672 °C10 min74 °Chold
ABC1TemperatureTime, 96-wellRepeat294 °C3 min394 °C45 sx35450 °C60 sx35572 °C90 sx35672 °C10 min74 °Chold
?. [DNA extraction and 16S rRNA PCR]
Verify PCR amplification results with an agarose gel. Prepare a 1.5% agarose gel. Mix 2 µL of loading dye with 4 µL of PCR product per sample and load 5 µL in the gel along with 1 kb ladder.
?. [DNA extraction and 16S rRNA PCR]
Purify PCR product using commercial PCR purification kit.
?. [DNA extraction and 16S rRNA PCR]
Quantify the intensity of PCR product band from each library (gel or fluorometry), normalize PCR product amount by volume and pool libraries together with an equivalent amount of PCR product into a single tube.
?. [DNA extraction and 16S rRNA PCR]
Submit samples for multiplex sequencing along with sequencing primer