Science Buddies: "Ask an Expert"

I am trying to perform osmotic ring assay on C. elegans (wild-type, osm-, dpy-11)(four worms of the same type per ring), but it is pretty difficult. Here are my questions:

1) I am using Methylene blue (because I don't have Trypan blue) to dye my salt ring. Is using Methylene blue alright? I am just concerned about how it would affect my worms. Also I am not sure how much salt I should dissolve in the dye for a small population assay like mine. (four worms of the same type per ring)

2) I am struggling with transferring the C. elegans to my osmotic ring. I use bare toothpicks to transfer the worms. I don't scrape the toothpick on an E. coli first before picking the worms because I don't want to transfer the E. coli to my osmotic ring. I noticed when I accidentally do, the worms, especially osm-7, would just hang around the E. coli, and not cross the barrier. Do you guys have any recommendations on how to pick the worm? I thought of using eyelash, but I do not know how to do it.

3) The size of my barrier is the size of a lid of an Ultra Fine Point Sharpie. I want to know if it is okay to decrease the diameter of the ring so that I can pressure them to respond to the osmotic barrier.

4) I am also transferring worms in L4 and adult stages. I read on a website that I have to use young adults. Do I really have to transfer just young adults? I just want to measure the time they cross the barrier, so I am not really concerned about the stage of my worms. Will this have a significant effect on my work?

5) How many trials should I do? I am planning on doing the assay 7 times for each worm type. Is that enough?

Here are my sources, but they don't seem to answer my questions:
http://www.wormbook.org/chapters/www_be ... avior.html
http://groups.molbiosci.northwestern.ed ... idance.pdf
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346054/

Thank you so much for your time and for helping me!

Hi,
It is ok to use methylene blue instead of trypan blue. In fact there is evidence that MB can protect the worms from oxidative stress.

I really don't know how much salt you need to use but look for the usual formula and just scale it down for the volume you need.

Worm picking IS a challenge. There are a number of Youtube videos about how to this, so I would check these if you haven't already done so:

https://www.youtube.com/watch?v=eTQtkQm5hOw

Sometimes you have to do an experiment BEFORE you can do the experiment. If you want to know if the size of the ring is ok, then you will just have to try it. It is better to do a prelim test and find out that something doesn't work than to get all set up for the big experiment, only to discover that the method is faulty.

Worms at different life stages may react differently, so that is why it is recommended to pick subjects of approximately the same age. The worm population you use is one of your independent variables and you need to keep that as consistent as possible to get good statistical results.

I would think that 7 independent trials would certainly be sufficient for getting an accurate mean and standard deviation and doing statistical analyses. Another important factor is the number of worms per trial and this is determined by using an experimental power and sample size calculator: http://powerandsamplesize.com/

If you have more questions let me know.

Sybee

Hello Sybee,

Thank you for replying to my post. I can't believe I only read this now when I am already halfway through my experiment and close to the deadline. This is supposedly a high school experiment, but it was so advanced for me. I spent months trying to study C. elegans because it was my first time handling them, but I still have difficulty handling them even now. I did not have any resources to make the worm picking instruments so I just resorted to using toothpicks with OP50 in them.
I am now doing this experiment for 8 trials with 5 worms in each osmotic ring. I am using I didn't know that I have to calculate for the sample size I should have known better

I changed my scoring method for this experiment. Instead of trying to determine the number of C. elegans that crosses the ring, I counted the number of worms that "respond" to the ring. Based on my research, worms that avoid the ring for more than six times in a row are classified as normal while those that touch, stay or cross in the barrier are classified as defective. In my experiment, however, there were some worms that never approached the ring and only stayed in the middle. (Probably because of the minute amount of bacteria that I accidentally transferred there) But some worms were just moving in places where there are no bacteria and that are far from the ring. So I did not know where to classify them. Is their behavior considered a response? Are they defective? Or are they neither?

Also, I wonder if worms from contaminated plates can be used in assays like mine? My osm-7 plates have all been contaminated, but I have no choice but to use osm-7 worms. I don't have any supply nor the money to buy new worms I was expecting the osm-7 worms to cross the barrier but most of them were only "responding" to the ring. Some touched it, while a very few of them crossed. Is this what you would expect? Or are you expecting them to have a different response?

Indeed, my experiment has so many sources of error such as contamination, varying worm stages used, bacteria in the ring, etc. At this point, I am very worried about the accuracy of the data I collected. I do not know if my grader will mark me down for this, but it is not in the rubric. Should I explain my sources of error in detail so that my grader is aware of them? What do you think matters? The accuracy of the data or an honest and elaborate explanation of what exactly happened in the experiment? Do you have any recommendation for what I should discuss towards the end of my research paper/ experiment?

C. elegans are really fascinating. I spent so much time with them that they would even appear in my dreams! I just wish I have more time to study these worms, but the deadline is March 4 Sometimes I regret studying these creatures because of all the failures with my experiment. But it motivates me to study them in the future.

Thank you for your time.

Sincerely,
Cedric

Wow, Cedric! Thanks for the great post--I hope you have stopped dreaming about wriggling C elegans! They are cool critters, but I would rather dream of other things.

Don't be concerned about getting the 'correct' data because there is no such thing. What you saw is what you got. Of course, as you said there were many potential sources of systemic error in your experiments and it is very good that you plan to describe them in your write-up.

The contamination may or may not have affected the worms responses to osmotic differences. That remains to be seen. But I think it was smart of you to record data about the worms that approached or touched the rings rather than just those that crossed it.

I don't know if age is important. That is something you should research so that you can make an educated statement based on published data about whether or not you think age might have had an effect.

As to the relative avoidance or approach responses, I really can't say what happened. You need to do a sample size calculation to determine how many worms you would have had to test in order to get statistically accurate data. I think i sent you a link for that in a previous post.

Let me know when you have questions again because I want your experiment report to be as accurate as possible. The grading process should be given so that you know ahead of time what the grader is looking for. Just state your hypothesis, give the background introduction, experimental methods, results and discussion and you should do fine.

Good luck!

Sybee