Mechanical Disruption of Individual Worms to Assess Variability in Host-Bacteria Interactions

Abstract

Source: Taylor, M. N. et al. Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions. J. Vis. Exp. (2022)

This video demonstrates the quantification of intestinal bacterial load variability in individual Caenorhabditis elegans through mechanical disruption. It outlines the steps for sample preparation, mechanical disruption, serial dilution, and colony counting to assess differences in intestinal bacterial load among individual worms.

Protocol

1. Separate worms into a deep 96-well plate with silicon carbide grit for mechanical disruption. Prepare the 96-well disruption plate as follows.
   1. Obtain a sterile 2 mL deep-well 96-well plate and a matching silicon 96-well plate cover.
      NOTE: It is important to use plates that are compatible with the 96-well adaptors for the tissue disruptor. Tiny differences in external dimensions make the difference between a plate that can be removed from the adaptors and one that cannot.
   2. Using a sterile scoop spatula, add a small amount of sterile 36-grit silicon carbide to each well of the plate that will receive a worm. Use enough grit to barely cover the bottom of the well (about 0.2 g per well). Excessive material will make it difficult to get a pipette tip to the bottom of the well when retrieving the contents.
   3. Add 180 µL of M9 worm buffer to each well.
   4. Label the columns or rows to indicate where each sample will go, then cover the plate loosely with the silicon 96-well plate cover.
2. Transfer individual worms to the 96-well plate for disruption.
   1. Move permeabilized worms carefully to a small (35 or 60 mm) Petri dish containing sufficient M9 worm buffer supplemented with 0.01% Triton X-100 (M9TX-01) to fill the dish to a depth of ~1 cm.
      NOTE: If a large number of worms are present, it may not be feasible to transfer the entire sample as the liquid will become crowded, and it will be difficult to pipette individual worms.
   2. Using a dissecting microscope or other low-magnification device, pipette off individual worms in 20 µL volumes and transfer these worms to individual wells of the 96-well plate.
      NOTE: It is best to harvest only freshly killed worms. Avoid worms with a rigid, linear shape, as these worms may have been dead for some time. Try to take worms that are curved or S-shaped, with normal gross physiology and an intact gut.
   3. After transferring each volume, make sure that the selected worm was actually ejected into the well. To do this, pipette up 20 µL of M9TX-01 from a clear area of the Petri dish and release the full volume back into the dish; this will normally eject the worm if it is stuck to the pipette. If the worm was stuck, remove 20 µL from the well and try the transfer again.
   4. Once all worms have been transferred, cover the 96-well plate with a sheet of commercially available flexible paper-backed sealing film (2 x 2 squares), making sure that the paper-backed side of the sealing film is facing down onto the sample wells. Be careful not to stretch the sealing film too thin, or it will be very difficult to remove later.
   5. Place the silicon sealing mat lightly on top of the flexible sealing film; do not press the cover down into the wells at this time.
   6. Move the plate to 4 °C to chill for 30-60 min. This will prevent overheating during disruption, which can damage the samples.
      NOTE: This is a breakpoint in the protocol. In most cases, the plate can be left at 4°C for up to 4 h before grinding. Do not leave the worms overnight, as this will change the bacterial counts.
3. Load 96-well plates onto a tissue disruptor to break up worm tissues and release intestinal bacteria.
   NOTE: (Optional) If using an odd number of 96-well plates for digests, it is necessary to prepare a counterweight before proceeding. Use an empty deep 96-well plate and fill wells with water until it weighs the same as the first plate.
   1. Press the silicon sealing mat down firmly into the wells to create a seal, making sure the lid lies flat across the entire surface of the plate.
      NOTE: If the flexible sealing film is too thick after stretching, it will be difficult to secure the silicon lid such that it lies flat in all wells. This will result in an insufficient seal and well-to-well contamination during shaking.
   2. Secure plates in the tissue disruptor using the 96-well plate adaptors. Shake plates for 1 min at 30 Hz, then rotate plates 180° and shake again for 1 min. This will help ensure an even disruption in all wells of the plate.
   3. Tap plates firmly on the bench two or three times to dislodge any grit from the flexible sealing film.
   4. Using a large centrifuge with two 96-well plate adaptors, spin the plates down at 2400 x g for 2 min to gather all material to the bottom of the wells.
   5. Remove the silicon lid and carefully pull off the flexible sealing film.
      NOTE: If the flexible sealing film sticks in any of the wells, use a 200 µL pipette tip to remove it. This is common when the flexible sealing film is stretched too thin.
4. Serially dilute worm digest samples in 300 µL in 96-well plates.
   1. Using a multi-well pipettor set to 200 µL, pipette up and down several times slowly and carefully to re-mix the contents of the wells, then draw off as much of the liquid as possible. Transfer this liquid to the top rows of the 96-well plates prepared for serial dilution of disrupted worm samples.
   2. Using a 96-well pipettor set to 20 µL, remove this volume of liquid from the top row and dispense into row B. Pipette up and down 8-10x to mix. Discard tips.
   3. Repeat step 4.2, starting from the 0.1x samples in row B to create 0.01x dilution samples in row C.
   4. Repeat step 4.2 again, going from row C to row D.
   5. Plate onto solid agar for bacterial quantification. For mono-colonized worms, it is generally sufficient to plate 10-20 µL drops of each dilution [1x-0.001x] on agar plates. For multi-species colonization, plate each dilution separately by pipetting 100 µL onto a 10 cm agar plate; spread immediately using glass plating beads.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well plate sealing mat, silicon, square wells (AxyMat)	Axygen	AM-2ML-SQ
96-well plates, 2 mL, square wells	Axygen	P-2ML-SQ-C-S
96-well polypropylene plate lids	Evergreen Labware	290-8020-03L
Agar	Fisher Scientific	443570050
Bead mill adapter set for 96-well plates	QIAGEN	119900	Adapter plates for use with two 96-well plates on the TissueLyser II
Bead mill tissue homogenizer (TissueLyser II)	QIAGEN	85300	Mechanical homogenizer for medium to high-throughput sample disruption
Breathe-Easy 96-well gas permeable sealing membrane	Diversified Biotech	BEM-1	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.
Eppendorf 5810R centrifuge with rotor S-4-104	Eppendorf	22627040	3L benchtop centrifuge with adaptors for 15-50 mL tubes and plates
Eppendorf plate bucket (x2), for Rotor S-4-104	Eppendorf	22638930
Petri dishes, round, 10 cm	VWR	25384-094