Picking and Mounting C. elegans

One of the very first things I learnt in the lab was to pick and mount C. elegans for my future experiments. This technique is fundamntal in C. elegans research, especially in a developmental biology lab where observations of the worm embryos under a microscope is crucial to identify changes in cell morphology.

Picking worms

The process of moving worms from one location on a plate to another or transfering worms to a new plate is picking. Here, I used a fine platinum wire to handle the worms (the pick). In preparation of picking, the platinum wire is sterilised by briefly holding it over a flame. Due to the high thermal conductivity of the wire, it cools rapidly and can be used on the worms almost immediately!

When picking the worms, the bottom layer of the pick is coated in E. coli (OP50) using the outskirts of the bacteria lawn. This is where the E. coli is the driest and therefore best for sticking to the worms. Under the microscope, the pick can then tap onto the worms that we want to pick, and the thin bacterial layer acts as an adhesive to pick the worm up on the bottom of the pick. This worm can then be transferred to another region on the plate (such as a region with no bacteria) or to a completley new plate. This was done by gently resting the tip of the pick onto the agar and allowing the worm to crawl away.

I first picked L4 C. elegans, these worms are in the final larval stage, and roughly within 24 hours, these worms will start producing embryos, essential for developmental biologists. To identify these worms, I had to spot the vulva, a clear triangular structure on the side of the worm.

L4 C. elegans, identified by vulva (arrow)

Initially, when I first picked the worms, I had some difficulty in getting the worms to stick to the underside of the wire, however wiht practice I slowly began getting better, ensuring enough bacteria was coating the pick to stick to the worms. This technique also requires a steady hand, given the size of the worms (~1mm length), and them being highly mobile. Eventually, I found it easier to pick worms by using my other hand to stabilise the one that was picking the worm.

Mounting worms

Despite my difficulty in picking the worms, mounting them was much more harder!

I was first taught how to prepare an agarose layer to immobolise the worms for observation. This was achieved by placing 2-3 drops of 3% agarose on a slide and pressed down. This step was faced with many difficulties as I found it hard to place the correct amount of agarose down on the slide. Too much caused the agarose to overflow and too little didn't fully cover the slide. Furthermore, I found it hard to work fast enough so the agarose didn't crack when pressing down, however with enough practice I got better.

Then, 6 ul of M9 was placed on a coverslip and early adult worms were picked from a sample plate and suspended in this liquid. once suspended, the worm was then cut using 2 needle tips, cutting the worm 1/3 and 2/3rds of the way down the worm (rouhgly where the spermetheca is). This allowed the early embryos to be released when the worm was cut. Again, this step was faced with many difficulties, given the worm's increase in mobility when suspended in M9. This step started getting easier with more and more practice however still remains one of the hardest steps when mounting C. elegans. The agarose drop was then added to the cut worms and visualised under the microscope under DIC microscopy.

Observation of C. elegans embryos using DIC microscopy.

Learning this technique allowed me to develop my fine motor control and although challenging at first, it slowly started becoming easier (after several attempts!).