This technique allows us to collect large scale worm populations to be used for multiple omics platforms with minimal manipulation. This technique allows us to collect mixed stage C.elegans populations across multiple omics experiments to obtain a holistic picture of each sample. For spot bleaching of gravid adults onto large-scale culture plates, flame a worm pick over a Bunsen burner and use the pick to scoop fresh E.coli from the edge of the bacterial lawn onto a large-scale culture plate.
Pick a single gravid adult from the fourth chunk plate for spot bleaching and add five microliters of freshly prepared alkaline hypochlorite solution into one corner of the large-scale culture plate away from the E.coli lawn. Place the gravid adult into the alkaline hypochlorite solution and tap the nematode to disrupt the cuticle and to release eggs. When a total of five adults have been placed evenly around the E.coli lawn in the same manner, place the lid back onto the plate.
To harvest samples from large-scale culture plates, pour 50 milliliters of M9 solution onto one large-scale culture plate surface and swirl the plate to ensure that the M9 covers the entire nematode growth medium agarose surface. Prime a sterile serological pipette with M9 and tilt the plate so that the M9 and worm population gather in one corner of the plate. Use the primed pipette to aspirate the worm suspension and add the worms to a 50 milliliter conical tube.
Place the tube on a rocker to disrupt any bacterial clumps and debris. When all of the worms have been collected from all three plates, transfer 15 milliliters of worms from each tube into each of three 15 milliliter conical tubes and sediment the worms in the tubes by centrifugation. Carefully aspirate the supernatants without disturbing the worm pellets and add 13 milliliters of worm suspension to each tube.
Invert the tubes to resuspend the pellets and to wash off as much bacteria and debris as possible and centrifuge the worms again. When each entire worm population has been collected, wash the worm pellets three times in 10 milliliters of fresh M9 solution per wash. After the last wash, resuspend each cleaned worm pellet in 10 milliliters of double distilled water.
For population size estimation, quickly dilute three 100 microliter aliquots of worm sample from each tube in 900 microliters of M9 solution per sample and use the aliquots to make serial dilutions. Place the stock worm sample suspensions on a rocker for continuous movement of the cultures while the aliquots are being counted and mix the worm dilutions until they are homogeneous. Add five microliters of solution from the first one to 10 worm sample to a glass microscope slide and count the worms by light microscopy.
If there are less than 50 worms in the sample, count the 1:100 and 1:1, 000 dilutions. If there are more than 50 worms, move to the next serial dilution. After counting each aliquot replicate of each dilution three times, average the dilution counts to determine the estimated population size of the worm.
Then split the worm samples into the appropriate experimental aliquots and flash freeze the samples in liquid nitrogen for minus 80 degree Celsius storage. To prepare a worm sample for large particle flow cytometry, dilute an approximately five times 10 to the fourth mixed stage worm aliquot to a final volume of 10 milliliters of M9 solution and add 200 microliters of a one milligram per milliliter E.coli and 1:50 dilution of 0.5 micromolar red fluorescent microsphere solution to the worm suspension. After a 20-minute incubation at room temperature with rocking, collect the worms and microspheres by centrifugation and wash the worms two times with fresh M9 solution to remove any excess bacteria and uninternalized microspheres.
After the second wash, resuspend the pellet in five milliliters of M9 solution. If the pellet looks clean, add five milliliters of M9 supplemented with 50 millimolar sodium azide to the worms and place the tube on a rocker to both straighten and euthanize the worms for accurate counting and sizing. For population distribution documentation, open the calibrated 384-well plate template and set the template to dispense 20 gated objects into four wells to obtain four technical replicates of each gated region for each of the 20 bar regions of the sample.
Load a 40 milliliter sample of worms onto the large particle flow cytometer and begin automatically sorting the sample while continuously stirring the sample to prevent settling and simultaneously dispensing objects from the sample into the calibrated 384-well plate. Once the entire sample has been sorted and the maximum number of gated regions have been dispensed into the 384-well plate, remove the sample from the cytometer and clean the instrument. The large-scale culture plate method was tested on 15 strains of C.Elegans, including a mixture of Caenorhabditis Genetics Center mutants and Caenorhabditis elegans Natural Diversity Resource wild strains.
In this analysis, the large-scale culture plate method yielded population sizes from approximately 94, 500 to 9, 290, 000. The mean population size within the reference strain PD1074 and across strains was approximately 2.4 million worms. No significant differences were found in estimated population sizes between C.elegans strains over the course of an average of 12.2 large-scale culture plate growth days.
PD1074 large-scale culture plates took between 10 to 14 days to grow to a full mixed stage population with a mean growth time of 12.2 days. The slowest growing strain grew for a maximum of 20 days and the fastest growing strain grew for a minimum of 10 days. In this sample distribution for PD1074, worms were measured from the L1 stage through gravid adult on a large particle flow cytometer.
Subsequent imaging and visualization of the variations in the population distribution across samples revealed that the pipeline generated a mixed stage population of C.elegans. It's important to accurately count the worms throughout the sample collection so that you obtain data that is comparable across growths and studies. This collection approach can be applied to RNA sequencing, genomics, and other omics analysis to expand our understanding of the complicated dynamics that occur within C.elegans populations.
This technique allows us to collect mixed staged C.elegans populations across multiple omics experiments to obtain a holistic picture of each sample.
