This article describes a method for quantifying intestinal bacterial loads in C. elegans using mechanical disruption techniques. The process involves preparing worms, disrupting their tissues, and quantifying the released bacteria through serial dilution and plating.
Begin with age-matched, sterile, freshly killed adult C. elegans worms with permeabilized cuticles, suspended in buffer.
These worms are infected with bacteria in their intestinal tracts.
Take a deep-well plate containing abrasive particles in buffer and transfer a single worm into each well.
Cover the plate with sealing film and a lid.
Incubate the plate at a low temperature to prevent overheating during tissue disruption.
Press the lid to seal the wells.
Using a tissue disruptor, mechanically shake the plate. The abrasive particles disrupt worm tissues, releasing intestinal bacteria.
Tap the plate and centrifuge it to pellet the contents.
Mix the contents to evenly distribute the bacteria, then transfer them to fresh wells.
Serially dilute the bacterial suspension in buffer.
Plate the dilutions onto agar plates and incubate to allow colony formation.
Count the colonies to quantify variation in intestinal bacterial loads across individual worms.
To prepare for mechanical disruption of worms, obtain a sterile two-milliliter deep well 96-well plate and a silicon plate cover.
Use a sterile scoop spatula to add a small amount of sterile 36 grid silicon carbide to each well such that the grid barely covers the bottom of the well. Add 180 microliters of M9 worm buffer to each well. Label the columns and rows, and then cover the plate loosely with the silicon plate cover.
Next, transfer the permeabilized worms to a small Petri dish with M9TX-01 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 M9TX-01 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 sealing film with the paper-backed side facing down onto the sample wells.
Place the silicon sealing mat lightly on top of the flexible sealing 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 silicon sealing 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 sealing film. Centrifuge the plates at 2,400 times g for two minutes to collect the samples at the bottom of the wells. Then remove the silicon lid and pull off the sealing 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.
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