So I’m a 9th grader doing a science project on C. Elegans and I want to run a chemotaxis assay.
I found this protocol (http://www.babec.org/node/96), but its severely lacking, as it is a student version of the protocol, and as such,
doesn’t include materials and things that the protocol assumes a teacher set up beforehand. Getting access to the teacher version of
the protocol requires you to email them, which I did to no avail.
Part of the protocol also confuses me. It says you need to synchronize the worms in module 2, which from my understanding means
you “synchronize” the worms to be the same age, so age won’t be a factor in the results of your assay. However, it is not clear whether
you start with worms or worm eggs. Again, the student version of the protocol doesn’t include the materials list, and it assumes a lot of context
has been explained by a teacher.
I guess what I’m really asking for is some advice on what protocols to use. I have searched extensively for a concise, clear, protocol but have come up
empty handed, so I’m going to ask you guys if you have any experience running a chemotaxis assay on C. Elegans, or any experience with culturing and
synchronizing C. Elegans in general.
Any information on where to get worm eggs (if that’s what you need) and supplies in general would be appreciated as well. I know Carolina.com carries
C. Elegans strains, but an email to their customer support found their worms to be age-unsynchronized.
welcome to the forum and to the world of C. elegans. Yep, it’s always so that teachers have access to the inner sanctum of knowledge (teacher protocol with useful details) and use this knowledge to mesmerise their students…
To your general questions: Synchronisation is indeed a way of achieving a level playing field for your experiments. On any one plate containing C. elegans there will be worms at various stages of development. because you are never sure beforehand that worms of different ‘ages’ will respond in the same way in your experiment, you have to age synchronise them.
The easiest way of doing this is to isolate the eggs from the worms and let all the eggs hatch without food. You end up with a more or less age sychronised plate of worms (so-called L1 worms). These worms can then be used for your experiments as they are all ‘more or less’ the same age.
In terms of you setting up a chemotaxis assay, well perhaps the first thing to do would be to find out if there is anyone nearby at a university who is working with C. elegans. I am sure they would be willing to give you the reagents you need for your project and even possibly some friendly advice.
It’s not that the protocols are difficult, but making up the solutions, pouring plates and obtaining worms may or may not be that easy for you at the school?
It may well be the case that you can do some things yourself and ask local researchers for other items that are not so easy to find.
Let us know where you are based and we can see if there is anyone that might be able to help. We can also advise on protocols, which chemoattractant might be easiest and other general details.
The site you link does seem to assume that a lot of things will be set up by and explained by the instructor, and I don’t see where it explains those things to the instructor.
Still, as Steve says, even if you have complete protocols I think you might find some parts challenging without help - access to an autoclave, to a constant-temperature incubator, a dissecting scope, etcetera. And then there are some expenses that aren’t going to scale well. Take the making of a worm pick, for example: typically you’d use an inch to an inch-and-a-half of platinum wire, which is about $4 or $5 worth, so not incredibly expensive - but I don’t think you can easily buy the wire in such small quantities, you typically buy it three feet at a time. And even if you can get all the equipment and reagents, I think you’ll learn to handle C. elegans much more quickly if you can find someone nearby who can show you around the organism, and probably also learn a lot from discussing your project with such a person.
Thanks for the responses guys, realllyyy appreciate it. <3
Steve, I would really appreciate it if you’d give me a quick rundown of the easiest chemotaxis protocols. All I really need is to run a simple chemotaxis
assay on C. Elegans using a proven substance like salt or alcohol. As long as it gives me a measurable chemotaxis index, it should be fine, the main
issue is how easy and costly the protocol is.
Also, where do you get eggs? The wormbook website says the worms come on NGM plates with food on them, I didn’t see any mention of eggs. (I assume
you can only synchronize eggs and not grown adults, that seems pretty intuitive)
Hey nichwch, the real costs come with what Steve and Hillel mentioned. I doubt your high school would have the proper equipment and materials for you to readily do the worm work. Have you checked out to see if there are any worm labs near you? As Steve suggests, it would be useful to talk to someone to discuss your experimental design, arrange for usage of facilities (likely necessary), etc.
but it is basic, no Chemotaxis Index, just what scares worms away and what draws them closer kind of thing.
Even then, you need the worms, you need agar plates containing worm food (bacteria), you need agar plates for the assay AND you need a helping hand to work with worms (this is not to say you are not a good scientist, but everyone has learned (almost everything) that they know from someone else.
These basic requirements, in turn, need a friendly advisor and laboratory facilities in the background, have a look at 1, 2 & 3 on this page from the Silencing Genomes Project which give you the basics on keeping, handling and experimenting with worms:
The thing is, even very basic science experiments normally require laboratory facilities and supplies.
Bottom line, don’t be disheartened by all this…just look at it as a challenge to find someone in the vicinity that might be able to help get you started.
first of all, i completely agree that you would be best off meeting up with someone who actually works with c. elegans (or some similar nematode).
but, if that is not an option, or if you are just really stubborn about doing it yourself….
i see you can buy pre-poured NGM plates from teknova (about $35, definitely cheaper than an autoclave).
and, wow, worms are free from the silencing genomes site!
instead of platinum wire for a pick you can use a piece of nylon monofilament fishing line cut off beveled with a razor blade (sterilize by dipping in ethanol).
i think the hardest part is getting a dissecting scope with decent substage illumination, but i suppose you don’t need high fidelity optics for chemotaxis expt.
i’m not sure about sodium azide, but you can either buy air bags on the black market (bad idea) or ask your chemistry teacher to procure a little bit for you.
to stage worms, you can just suspend them in buffer in a small test tube and the biggest ones will sink down to the bottom in 30-60 seconds. repeat that a couple of times and you will have only adults.
wow, ‘Rough Science’ meets worm research…C. elegans on a shoestring.
I’ve never tried it (will do tonight), but I wonder if you can see worms on a plate if you place the plate on an overhead projector?? (For those under 30, long ago some of us used these devices for communicating information in presentations).
yep, that’s also an alternative, but I thought I would be very clever and switch the OHP on and OFF :o
Nichwch, we are ready to help you…you just need to let us know where you are based. I know the US is big, but are you actually in NC? I ask because, for example, there are people at Chapel Hill who work with C. elegans.
I’ve never built or used one myself, but I’ve seen a worm tracker based on the one used by Aravi Samuel’s lab; it consisted of a ring-light consisting of cheaply available LEDs glued into a cardboard ring (admittedly, powered by an old electrophoresis power supply - something an established lab is likely to have lying around unused and so available for free, but I don’t know how much a suitable power supply would cost), and an old lab camera pointing down at the plate in the middle of the ring. In this day of cheap cellphones with multi-megapixel cameras, I suspect you could rig something up to collect images for calculating chemotaxis indices for a small amount of money (actually building a proper system for automated analysis of the images might be a slightly taller order).
Mind you, you’d still need the plates, and some ability to move worms around; maybe a powerful magnifying glass could suffice, but I suspect a dissecting scope would help a lot. And a constant-temperature incubator would be nice, though failing that you can buy a temperature datalogger cheaply and at least know what your plates experienced.
Any information on where to get worm eggs (if that's what you need) and supplies in general would be appreciated as well. I know Carolina.com carries
C. Elegans strains, but an email to their customer support found their worms to be age-unsynchronized.
Synchronizing is something you'd have to do for yourself; you can get very roughly staged animals by letting animals settle in buffer as lmu1 suggests, or you can let animals of roughly similar age lay eggs for a couple of hours and then pick them off, or you can bleach gravid ("pregnant") mothers to dissolve them and recover their eggs, which you can then let hatch on food for a rough synchronization or let hatch in the absence of food so they enter developmental arrest, so that you can then start them growing all at once for a more precise synchronization. Yet other methods are available; a lot of this is in the links above, or can be found fairly easily.
You can get N2 worms from Carolina.com as you say, but presumably you can also contact the CGC, the standard stock center. They are a bit more expensive (because of a one-time registration fee), but then you can order any of a vast array of mutant worms for use in your assays - and I’d trust them more than a vendor I’m not familiar with. Or, better yet, you can contact any nearby worm lab, and they will probably be happy to set you up with wild-type worms and likely with mutants for free.
OK. Whilst I am in complete agreement that High School Science projects should be more than Ed Murrow’s ‘wires and lights in a box’, there is a danger that we make the simple complicated here.
Let’s think about your 101 Chemotaxis Expt nichwch and what the options are:
1. I’m going to do it myself option.
a. You would need a supply of agar (so-called NGM) plates for your experiments.
To make the plates, you would need to beg, steal or borrow the following:
Some sterile 10cm diameter plastic Petridishes
A large pressure cooker
agar
Peptone
NaCl
1moldm-3 MgSO4 solution
1moldm-3 CaCl2 solution
1M K+ phosphate buffer
cholesterol
BOTTOM LINE: For the few plates you need for your project, well I think lmu’s suggestion of pre-poured plates from Teknova is much easier.
b. You need a supply of worms (the standard worms are called N2).
Write to a lab and ask them for a plate containing N2 worms.
To get a bacterial culture, scape a bit of bacterial lawn off the plate you receive and mix it in some bacterial growth medium (I would ask the same people that are willing to send you a plate if they could also send you a small amount [10mL] of bacterial growth medium, it’s called LB medium).
Scrape off a little of the bacterial culture on your plate (no worms!) with a sterile spatula (carefully heat it in a flame) and swirl in the LB medium.
Seal the tube and place in a waterbath set to 37 degrees overnight.
The agar plates you will use need to be spread with bacteria and incubated before the worms are put on them, so you need some way of culturing bacteria @ 37 degrees.
Put the plates in a Tuperware box, put the lid on and place in a waterbath set to 37 degrees overnight.
The worms on your original plate will develop quickly, lay eggs and die, so you need a way of culturing their offspring. The eggs hatch on the plate and after a few days what on the plate is mainly very small, very young worms.
To grow new adult worms (they are big enough to see by eye and to count using a mobile phone camera), cut out a piece of agar from such a plate and place it flipped over (so the worms are sandwiched between the new agar plate and the old agar piece) onto your new plate containing bacteria.
Gently move the piece of agar along the surface of the plate using a spatula or scalpel so that you smear the worms on the agar surface. Voila, you have a new plate with mainly young worms on it…good enough for your experiment.
After a couple of days at room temperature you will have big, adult worms on your plate. You can see them with the naked eye.
c. The Experiment
To keep things really simple but scientific, you could set up a chemotaxis expt as follows:
Make a template (a circle of 10cm diameter) with three 1cm diameter circles drawn so that their centres are at each of the three points of a equilateral triangle (the centre of the triangle is the centre of the big circle and the length of each side of the triangle is 5cm).
(Use this to mark the base of each plate in your experiment with the three circles). One circle will be where your worms start, one circle will contain the attractant or repellant and the third circle will contain something neutral (a control).
Add a drop of your attractant/repellant/neutral solution to the appropriate small circle. Allow to soak in.
Add your adult worms to the third small circle (you need to know how many are added, so photograph them). Getting the worms into the circle can be accomplished by washing them off a plate with a small amount (5mL) of water, letting them settle in a tubeand then pipetting a drop onto the plate.
Once the water containing the worms has been absorbed into the plate, the worms will be free to move and your experiment begins.
You need to set a time for the length of your assay, say 1 hour. You count the total number of worms on the plate and the the number reaching your two test circles after 1 hour. As the worms do not just sit at a circle and wait for 1 hour you need some way of keeping them there. Sodium azide immobilises the worms, but it is a poison and you will not be able to obtain it. An alternative method would be to remove the worms that reach each circle with a toothpick, but this is very laborious. Or you can photograph each of the test circles with your camera at regular intervals and get a rough average from that. It’s not as precise, but you’ll get your chemotaxis index.
Of course, others might have simpler, better alternatives that they are willing to communicate.
Sodium azide immobilises the worms, but it is a poison and you will not be able to obtain it.
I have no idea what sort of regulations might apply, but TekNova (the same company selling the NGM plates) lists 5% sodium azide solution and I don’t see on the page any prominently posted restrictions on its sale. 5% is ~0.8 M, which is probably close enough.
I hadn’t looked at the TekNova site, but the fact that no clear warning is given makes me even more cautious about suggesting the use of sodium azide. A 5% solution is potentially lethal.
Many universities and colleges have standard operating procedures that cover the use of compounds such a sodium azide. It’s toxicity is such that a 5% solution represents a considerable risk when not handled correctly. This risk is augmented when the user is untrained (and training regarding it’s use is (and should be) a standard procedure for new lab workers.
Type ‘5% sodium azide toxicity’ into Google and you will find plenty of SOPs for its use and disposal.
I just wouldn’t go there unless there was a competent, trained instructor in the background willing to take responsibility for overseeing its use in such a project.
If TekNova does not already have somewhere a warning about its sodium azide solutions, then it should place one now.
it is indeed potentially dangerous stuff. you could potentially get a nice explosion if you poured some down the drain (if you have lead or copper pipes).
the 5% solution probably wouldn’t kill you unless you swallowed a few big gulps (50 ml). maybe one could use a hot metal cylinder to create a large circular depression in the agar, then fill that with (non toxic) buffer to create a trap for worms that stray into the area.
Annals of Emergency Medicine
Volume 16, Issue 12, December 1987, Pages 1378–1380
Case report
Suicidal sodium azide ingestion
MD Jonathan Abrams*, , MD Rif S El-Mallakh†, MD Robert Meyer‡
Show more
doi:10.1016/S0196-0644(87)80423-7
There have been only two prev
i o u s l y r e p o r t e d fatal cases described.
Both, as ours, were due to
suicidal ingestion of sodium azide.
The quantities were unknown in both
previous cases and were estimated to
be 15 to 20 g in our patient. With an
approximate LDso of 35 mg/kg, i, lo the
average human lethal dose would be
expected to be about 2,500 mg. Death
was rapid in all cases , ranging from
one to 13 hours.