Hand dissection of this elegan's intestine is a powerful yet simple technique that enables the investigation of various aspects of post biology by isolating tissue in cell-specific populations. This fine-scale tissue dissection technique is easy to perform and utilizes standard laboratory equipment, making it a robust and accessible method for many. Begin by pulling the 4 inches long and 1.2 millimeter outer diameter standard glass capillary into an injection needle shape using a needle puller.
Prepare at least 5 50 and 100 micrometer micro capillary pipettes. Use a micro forge to forge the micro capillary pipettes to either 50 micrometers or 100 micrometers by ticking 3 or 5 tick marks respectively as indicated by the micro forge ocular ruler under the 5X objective lens. Then affix a micro capillary pipette to the mouth aspirator tube.
Prepare one M9 bath and one dissection buffer bath by adding 2 milliliters of M9 in a 35 millimeter diameter sterile Petri dish and 2 milliliters of dissection buffer to the other 35 millimeter diameter sterile Petri dish. Finally, add 100 microliters of working bovine serum albumin or BSA solution to each bath, and mix it by swirling. To prepare the dissection array add 150 microliters of working levamisole solution to the first well of the two well concavity slide and 150 microliters of dissection buffer to the second well.
Add 20 microliters of working BSA solution to each well. To hand dissect the caenorhabditis elegans CL2122 worm intestine, move 20 adult worms from the nematode growth medium or NGM plate into the M9 bath using a worm pick. Then move all 20 worms from the M9 bath to the dissection buffer bath.
Next move batches of 10 worms from the dissection buffer bath into the well containing levamisole solution. Once the worm movement slows down quickly move them from the levamisole well to the well containing the dissection buffer. Allow the worms to begin moving a bit in the dissection buffer well before starting the dissection.
Under a fluorescent dissecting scope, make one cut just behind the pharynx or in front of the rectum using a hypodermic needle to produce an equal number of anterior mid half and mid posterior half sections of the intestine. While waiting for about one minute for the intestines to maximally extrude from the body, add 50 microliters of chelation buffer to the well to help reduce RNA degradation. To facilitate intestine extrusion use the 100 micrometer micro capillary pipette attached to a mouth aspirator and draw the worm in and out of the pipette.
Once an intestine is sufficiently extruded use the 27 gauge hypodermic needle to cut it away from the rest of the body and any remaining gonad. Aspirate the intestines section using the micro capillary pipette and transfer it to the micro centrifuge tube containing nucleic acid isolation reagent. Keep the isolated intestines on ice and repeat the procedure for the remaining intestines.
This method demonstrated the successful dissection and isolation of the intestines. The method also reported RNA isolation and microbial surveillance from isolated intestines. As CL2122 worms harbored the intestine specific metal two promoter fused to GFP, the intestine was observed as a green glow under the fluorescent dissecting scope.
The dissection of the adult worm displayed an extruded intestine after making a primary incision just behind the pharynx. The successful dissection of an intestinal segment appeared free from visible contaminants such as debris from the gonad or carcass. In contrast, unsuccessful dissection displayed the gonad and carcass attached to the intestinal segment.
The RNA yields from hand dissection were more efficient as the final sample of 60 total intestine sections yielded roughly 15 nanograms of high quality total RNA. The worm disaggregation and FACS isolation of intestinal cells was less efficient as these required hundreds of thousands of intestinal cells to obtain commensurate quantities of total RNA. The total microbial genomic DNA amplification using a pan-bacterial detection assay showed that the final sample of 40 total intestine sections from the anterior, mid, and mid posterior regions yielded the low but detectable quantity of around 0.009 picograms of total microbial DNA.
The most important thing to remember when performing this procedure is to not over-paralyze the worms prior to dissection as this can inhibit maximal intestine extrusion.
