Originally posted on WormAtlas by “madhura”, May 31, 2004
All C.elegans community,
I want to see single worm under the microscope. I need to do single worm mounting as I do not get many males in the mutant I’m studying. Is there any standardized protocol for single worm mounting?
And, is there any way to prevent males going up the walls of the petridish? Since there are only 1 or 2 I get each time from RNAi, I cannot afford to loose them.
Thanks
Madhura
Originally posted on WormAtlas by “robyn lints”, June 01, 2004
Depends what kind of microscopy you want to do. Are you trying to look at unfixed animals or do you need to treat them in some way? If you are trying to do antibody staining then a freeze-crack type procedure, in which individual worms can be picked onto to poly-lysine coated slides, is one way of making sure you don’t lose rare animals (see http://www.wormatlas.org/anatmeth/anatmeth.htm). If you are just looking at morphology or gfp in unfixed animals then picking an individual worm from the plate directly into a drop of M9 on an agar or agarose pad (5%) will do it. As to preventing males from crawling up the walls, I can only suggest that you make sure that your OP50 is restricted to the center of the plate and doesn’t extend to the walls and that your plates don’t accumulate too much condensation. Why is your frequency of males low? Do you have him-5 or him-8 in your mutant background (these mutations increase the frequency of males to ca.30% in an otherwise wild type background). Or is it that you already have a “him” in the background and the number of males is low because of your mutation?
If you want to do time-lapse imaging of the whole worm (especially for fluorescence microscopy) you might want to anesthetize your worm to prevent movement. Here is the protocol we have had success with: (1) anesthetize the worm(s) for imaging- make up fresh a mixture of 1mg/mL Tricane (ethyl 3-aminobenzoate methanesulfonate salt) and 0.1 mg/mL of tetramisole hydrochloride (TMHC) in M9. Place worms into a pool of anesthetic (a depression slide works well for this) for 15-30 minutes, or until worms stop moving. (2) Transfer the anesthetized worms to an agarose pad (set up an agarose pad by sandwiching a drop of melted 1% agarose between two glass slides) and (3) carefully place a coverslip on top. Worms can be recovered (if you are careful) from anesthetic by flushing under the coverslip with a small amount of M9.