bacillus

Model Organism: Worms Methods & Reagents
1

Hi all:

Does anyone know how to get rid of bacillus contamination? I tried bleaching and serial transfering but both don’t work. I’m the only one working on nematodes in my lab, any tips or suggestions will be very helpful for me!

Thanks in advance :slight_smile:

2

You can simply add antibiotics to your NGM. Some labs always add strep to their NGM and seed the plates with OP50-1 (which is strep resistant). To just clean a strain and passage it, you don’t even have to worry about killing off your ordinary OP50. Just add your antibiotics of choice and just seed with 5x concentrated OP50 so you still end up with a sufficiently thick lawn. Combine this as necessary with bleaching and serial transfers, and you should be able to get rid of your contamination problems.

Out of curiosity, how do you know it’s bacillus? I never bother to classify a contaminant, but I do wonder…

3

Thanks for reply :slight_smile:

I’m not 100% sure it is bacillus, my colleague told me so. She worked on nematodes before, and had the same problem (and lost a whole set of treatment because of bacillus, that’s why I’m so nervous about it). She told me the terrible smell is the icon of bacillus - every time I open a contiminated plate, I want to get everything out of my stomach…

4

Hi again,

Sorry if this sounds a bit stupid, but I wonder in which form I should add streptomycin? I found this (http://www.ipmscientific.com/enriched_ngm_agar_formula.htm) component table, do I need to dissolve it into certain amount of water? I guess it’s not okay if I autoclave it…

Thanks :slight_smile:

5

Hi,
You mentioned bleaching and serial transfer, but I wasn’t sure if you had combined them. I apologize if this sounds very basic, but here’s my method which has worked for all the contamination I’ve ever encountered.

Toward the end of your day, bleach worms and place a small aliquot of the bleached eggs toward the periphery of a fresh plate, well away from the food. The next morning, take the hatched larvae that have crawled away from the area the bleach was spotted onto, and move them to a fresh plate. I take the worms that have crawled the furthest from where they hatched. I’ve found that carefully separating the hatched worms from the bleach droplet area as soon as possible prevents their re-contaminating themselves on anything that came through the bleach solution alive.

If you have already tried this, or If this fails, then I agree you might need to try antibiotic.

Hope this isn’t too basic, but I wish someone had given me some of this highly routine, yet helpful info early on in my worm career.

Good luck!

6

Hi!

Thanks for reply :slight_smile: It’s very helpful! I’ve tried bleaching and serial transfer but not at the same time. Actually, now it’s toward the end of my day and I just bleached the worms! So I transfer them tomorrow morning and that should work. I think I’m kind of lucky to have this problem before I start my experiments, not after :stuck_out_tongue:

Thanks again!

7

I use a method similar to cashewbeast, except I bleach the worms directly on the plate. I first pick 5-10 gravid worms to an NGM plate with no food and let them crawl around for a bit. While that is going on, I prepare bleach / NaOH solution as you normally would, and then pipet a 10-15 ul drop onto an NGM plate close to the edge and away from the food. I then pick up the worms by sliding the pick under them pancake-flipper style and transfer them to the drop of bleach / NaOH. The worms are broken down by the bleach / NaOH, which then gets absorbed by the NGM. Sometimes the worm bodies don’t completely break down right away, but that is ok. They will be gone by morning. I then transfer the worms to a new plate immediately the next morning, picking worms the farthest from the bleach / NaOH spot as cahshewbeast suggests. I always save the bleach plate for a few days to see if bacteria grow back in the bleach spot. If not, you’re in the clear for sure. If you eventually see bacteria, you just have to check the plate you transfered to carefully to make sure contaminating bacteria didn’t go along for the ride. To make checking for contamination easier, I usually place the worms on the new plate away from the food and circle that spot with a Sharpie.

This method almost always works for me and eliminates the need to obtain a large volume of worms to vortex in the traditional bleaching process.

8

Hi,

Thanks northwood and everyone :slight_smile: These tips really help me a lot!

9

Oh my !!!

You have no idea how much better I feel hearing I’m not alone! I’ve just been hitting my head against the wall for the last couple months wondering where the @#$@ I’m getting this Bacillus contamination from.

I’m starting to wonder if it’s happening somewhere between my bleaching and feeding my worms onto OP50. Does anyone know if bleach kills Bacillus spores? I’m wondering if it’s something “stupid” like my bleach is old and although it’s killing E.coli it’s leaving behind the Bacillus… Not sure I understand why the Bacillus would take over the OP50 on the plates that I feed the larvae with though.

And I know it’s Bacillus because I’ve done DGGE on this contamination.

Anyways, trying brand new bleach this time… :stuck_out_tongue:

10

My colleague told me there is bleaching-resistant bacillus (spores are very resistant), so bleaching alone doesn’t work. and if I don’t get anything wrong, bacillus grows faster than OP50…

I’ve tried all the new methods provided here (I really appreciate you guys :D), but very unluckily, yesterday I found my contamination source - it is LB, and that’s why I never succeed in decontamination :frowning: The best solution so far is to bleach the worms and seed them onto strepto plates, starve them a bit if necessary, and then transfer them to ordinary OP50 plates.

Now I’m ordering new OP50 and hopefully don’t get contamination in the future. I’ll report back my results :slight_smile:

11

Thanks for all of the advice everyone. I’m also the only one in my lab working with C. elegans and have been struggling with an unknown contamination issue. Hopefully this will help clear it up once and for all.