Worms around plate edges

Hello,

I have been having this problem where worms seem not to like the entire plate and always stay around the edges of the plate where the bacteria is less and eaten very fast due to all worms hanging around there.

Sometimes, all of a sudden, I come back the next day to find most worm dead. Other times, no worm is dead, but just around the edges.

I got rid of all old OP50 batches, made fresh ones. No worms are dying, but just around the edges. I even put streptomycin in one batch, and another without. I still see this happening (hanging around the edges).

Any suggestions on what I can do. Because my worms never used to do that and have been doing that for around 2 months.

PS: it is not limited to N2, I have tried with around 10 strains.

Note: I do notice that my OP50 are growing faster when I incubate them. Is it possible that the bacterial lawn is so thick/too concentrated for my worms?

Thanks a lot!
Faten

dear faten,

“I have been having this problem where worms seem not to like the entire plate and always stay around the edges of the plate where the bacteria is less and eaten
very fast due to all worms hanging around there.”

and

"Because my worms never used to do that and have been doing that for around 2 months.
PS: it is not limited to N2, I have tried with around 10 strains. "

it is normal for worms to accumulate at the edge of the lawn. that is where the food is thickest. some strains exaggerate this behavior (e.g., CB4856),
but sounds like you are observing regular worm behavior.

"Sometimes, all of a sudden, I come back the next day to find most worm dead. "

it’s unlikely that all of the worms are dead. to paraphrase the 911 operator in the world’s funniest joke “okay, let’s not panic, first make sure they are dead”.
but, as steve would point out, we need more details. why do you think the are dead? is there still food on the plate? does your incubator spike up to high temperatures?

More about the deaths of the worms might be helpful. Assuming no horrible contamination, the most common reason for worms to suddenly die on a plate is if moisture causes an airtight seal and the worms die of anoxia. This has fairly consistent effects on the posture and appearance of the dead worms, and moreover it’s pretty hard to miss when the lid must be pried off of the plate.

Hey :slight_smile:

Thank you for your help. It seems that the worms stay on the edges even when no food is there. "its like they are telling me I would rather starve than eat this food :’( "

Here are a couple of pictures for the dead worms. They are dead for sure as some had larvae hatched within them, did not respond to touch or M9. Even the larvae are dead. (not all larvae but they occur in like patches).
It becomes worst every day.

I did notice when I dilute my bacteria much more, the worms are okay. Could it be that the bacteria is producing more toxins than the worms could handle (perhaps It is more concentrated than it should).

Thank you for your feedback always.

Best,
Faten

OK, assuming we don’t have to get “Bones” McCoy in to confirm ‘he’s dead Faten’, then I would say the following information might help us track down the source of your problems.

  1. Do you incubate your plates with worms in a room or in an incubator and have you checked the temperature profile of either over a 24 hour period?

  2. How did you start your new OP50 cultures, from frozen stocks or from a plate?

  3. What does the bacterial lawn look like after an O/N incubation, is it normal, overgrown, patchy, shows secondary colonies, a different colour etc?

  4. You say that the worms stay in the areas were there is no food (do you see clear agar in these areas then?), what about areas where there is food?

  5. What does 'my OP50 are growing faster mean? Have you measured the OD600?

  6. How do you spread the OP50 on your plate?

  7. How did you transfer the worms on to these plates?

  8. What do your plates smell like?

  9. have you incubated your NGM plates without OP50?

Answers on a postcard…

i like hillel’s hypoxia hypothesis. if you don’t use vented plates and you have a lot of bacteria using up the oxygen that would do it. get some vented plates.
meanwhile you could try incubating the plates upright in a humidified box, or make some holes in the plate rim or something.

Thank you very much for your kind responses!!

1. The incubator(s) temperature is more or less constant (+/-1 degrees C)
2. I started OP50 from frozen pellet 
3. Bacterial lawn does not have different morphologies or colors, nor fungi. It does look thick, no patches
4. The no food "edge" area were clear. The worms stayed there despite having a lot of food from the center and around it. Rarely was a worm passing over food region (if any). I do see traces that the worms did move over, but worms stayed in the clear edge areas.
5. I didn't measure OD600. but my bacterial pellet has been double the size I normally used to have before despite incubating the same amount of time and even less @ same temperature and RPM.
6. Dilute OP50 in M9, transfer onto center of plate, spread with sterile spreader uniformly around the plate
7. By chunking or by pipet/m9
8. Plates don't have a strong unpleasant smell. Just as I was first introduced to them 3 years ago.
9. Yes, at around 20C. I can try to incubate plates at 25C without food as well. But those food free plates never showed any type of growth on them without food. Even after a month at 20C. 
10. I will definitely try to control for oxygen and moisture, to see if this improves the conditions. And will make new OP50 from a new stock

Thanks again, all the best

Faten

I’m not really sure I like the anoxia hypothesis in this case - the image is pixelated but I don’t recognize the rigid posture I associate with anoxia; in any case, anoxia is typically unmistakable as it takes considerable force to pry the lid off of the vapor-locked plate. You don’t need to purchase vented plates to test this; you can take a heated inoculation loop and cut a notch out of the rim of the plate.

My best guess is that the bacteria being used is not in fact OP50. This would account for the unusual bacterial growth rate/density, and could account for the lethality. I’ve never really heard of anyone bothering to genotype their OP50 (in case of confusion and uncertainty, it’s easier to make more from frozen stocks). Still, if you want, you could check the uracil mutation, either phenotypically or molecularly. More simply, I think SteveH mentions smelling the plates above, as contaminants often have different smells; also, colors. But you say both seem normal.

You say you started “from a frozen pellet”, which is a bit ambiguous. In general, best practice is to start cultures for spotting plates with picking a single streaked colony - it may not in fact be the perfect colony, but at least it’s not a contaminated mixture.

on balance, the most likely explanation for:

  1. Your dead worms and other worms heading for the hills.

  2. Rapid growth of the bacterial culture.

3 No deaths when you dilute your culture before spreading onto plates.

is that, as Hillel suggested, you simply don’t have OP50 (or just OP50) in your starter cultures.

Like the man said, go back to a glycerol stock, streak a plate, pick a single colony and start a new OP50 culture.

Don’t forget to tell us what happens…all of this advice is wasted if we never find out what was really the problem.

‘from a frozen pellet’ :-[ or from a glycerol stock :wink: ?

Will get right on to doing that! and definitely will update.

Can’t thank you enough for all the troubleshooting and ideas! Best worm community!

Hello :slight_smile:

Just wanted to share my updates. After streaking for isolation from the many OP50 batches I had, all the colonies looked exactly the same and the cultures were pure. Worms still looked fishy.
However, other events were taking place at the same time. This problem started appearing around the same time I got some new worms from another lab. I originally thought my bacteria were killing the worms, but now I think it is the other way around.
I also learned that this lab had a big contamination problem recently.
My guess is that this contamination transferred among plates within the same incubator. Other observations that support that is that my worms were fine after plating for a day or two, and suddenly death :’(
happened for several plates on more than one occasion. This might be due to acquired contamination…

I managed to get rid of contamination with the new worms (synchronized with bleach). But also I will from now on start my ‘wormies’ food from single bacterial colonies.

Thank you very much for all you wonderful and kind feedback and time. I am really happy to be part of this worm community.
Best,
Faten

***"I originally thought my bacteria were killing the worms, but now I think it is the other way around. "
By the other way around I mean: bacteria from the new worm plates were somehow killing and initiating this contamination and spreading even (perhaps). Because my food seems fine.

dear faten,

I think I’m in the same situation as you.
My worm also died suddenly two or three days after bleach, or without bleach. I would like to know if you have finally determined the cause of this contamination, or what measures have you taken to get rid of it.
I hope you can give me some advice!

Thanks a lot.
Hlucky