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.
