Placing a dissecting scope into laminar hood?

Model Organism: Worms Methods & Reagents
1

Hello wormers,

As many of you, I routinely do worm picking using a dissecting scope. In my plates I am looking at worms and bacteria and since these are longevity experiments they might last for several days. My problem is that I am experiencing lot of cross-contamination, sometimes I spot fungi growing on plates, sometimes other nasty things, and I end up throwing away 1/4 of my plates. Right now I am wearing gloves+coat+mouth mask, I sterilize the bench and the scope with ethanol or diluted bleach, keep a burner next to the scope, wash my gloves with ethanol inbetween switching plates. Nothing… these nasty bugs keep growing :slight_smile:

In several threads in this forum people found successful adding drugs like nystatin, perhaps combined with antibiotics but I’d rather not to do that. Instead, I was thinking of placing the dissecting scope inside a clean bench.

Has anybody done that? Do you have a class of products you’d feel to recommend me? Do you have reasons not to do that?

I have basically two concerns:

  1. Vibrations. The bench needs to be stable.
  2. The screen should not be in the way. Because I should be able to look inside the oculars of the scope.

Wormers. I am ready for your wisdom,

Ll.

2

Quick question before exploring picking in a laminar hood (never heard anyone doing this): you’re sure that the contamination is coming from picking? So unseeded plates are ok? And then seeded plates without worms are ok? Have you bleached the worms to clean them (they could be transferring contaminants from plate to plate). I know some folks who seed plates in a laminar hood, but never anyone who’s set up a scope in one.

3

Thanks for the answer. Unseeded assay plates are fine so are plates with worms w/o picking. Reagents are also OK. More in details, what I do is:

  • Synchronized L1 larvae complete development into adulthood in seeded NGM+OP50 plates
  • Adults worms are incubated in S-medium + kanamycin for 24 h to remove E. coli.
  • Sterile worms are then plated on 6-well plate with SK media (= peptone) + E. coli and P. aeruginosa.
  • 6-well plates are then parafilmed and incubated at 25 C.

Once a day, I measure the number of alive worms. An experiment typically lasts 10 days. On a ~1/3 of my plates, I spot fungus at various days. I am assuming spores comes from air.

I am not an experience wormer though: you guys never had this problem?

@JordanWard: when you asked me “have you bleached the worm”, you mean if I perform a sort of external wash with low-concentrated bleach? Can you link me a protocol for that?

Ll.
Ll.

4

I don’t know what can be done to solve contamination issues, but I do know that gloves, a lab coat, and a mask aren’t necessary. That doesn’t prove they’re not helpful in a marginal way, but I and probably a lot of other people have managed large numbers of plates with minimal contamination issues without them.

The main things are pretty obvious: keep your plate area clean, and don’t leave old contaminated plates next to newer clean ones. If you’re having problems with mites, get rid of any plates that might have them (or at least move them elsewhere), clean thoroughly, and use flame: ethanol doesn’t bother mites (I don’t know about low bleach concentrations). Seal up cracks in and near the bench where mites might be living. There are a couple of things that can dissuade or stop mites: they apparently can’t traverse a film of mineral oil (they either avoid it or get stuck, I’m not clear), although that makes a terrible mess, and they don’t seem to like traveling on exposed metal and can’t burrow through it like they can parafilm, so you can lay down aluminum foil to deter them (you can also flame aluminum foil) and can wrap plates in aluminum foil before placing them in contaminated incubators.

Regarding sources of contamination beyond your immediate benchtop, I don’t know what you can do. Obviously, you can try to control mess and dust over your bench, but only to an extent. It is however my experience from having less fastidious baymates or even having some of my own old plates lying around that even the visually dirtiest plates are not a significant source of mold infection beyond a couple of feet (laterally - downward from shelves might be a different matter).

Regarding using a hood, I’d be a bit worried about the higher flow rate. I’d be especially worried it would tend to rapidly dry plates left in the hood, but even while picking I could see it being a problem, as it might make fungus spores travel more easily.

A couple of more points:
“bleaching the worm” usually means dissolving worms in bleach (where “bleach” means various compositions of sodium or potassium hydroxide and sodium hypochlorite) to recover their eggs. It is often the only way to eliminate bacterial or yeast contamination. Completely dissolving the mothers is not usually necessary to eliminate fungus: you can mark the spot on the plate onto which you transfer worms from a fungus-infected plates, and after the worms move away kill everything at that spot with strong sodium hydroxide or other “bleach”.

If you have minor fungus contamination and spot it early, you may be able to kill it by direct application of a few microliters of “bleach” without harming the worm (elsewhere on the plate). This isn’t usually best practice or even successful in the longer term, though.

5

Hi Llewlyn,

We had similar issues with mold spores in the air affecting our experiments. We ended up buying an AirClean PCR workstation (see picture) that we use for picking worms during sensitive experiments. It cost about $1600.

http://i.imgur.com/vbLWLte.jpg

6

Are you looking into that scope through the perspex, at a distance? Do you open up the chamber completely to use the scope?

7

Thanks for that peteUW. I am also interested in what HillelSchwartz asked you. Could you also comment about vibrations? And more in general: are you happy with that thing? :slight_smile:

Ll.

8

I agree with Jordan that make sure it’s new contamination, not from the old plates or prep you are getting the worms from. Assuming it’s not that, I would try adding nystatin to your multi-well plates. We’ve had issues with 24-well plates getting contaminated, particularly around the edges where air inflow can happen. I think it’s worse because the condensation seems to be worse in this type of plates, allowing spores some place to settle. Maybe if the plates were inverted or placed in a HEPA hood after drying it shouldn’t be as bad. I don’t think it has anything to do with the picking since it’s apparent just after sitting on the bench for a few days. Nystatin does help clear it up without all the other stuff, although I would prefer if we didn’t have to add it.

-Kevin.

9

Just to be sure we are on the same page about it:

  • A newly poured 6-well plate is parafilmed, incubated at 25 C, and monitored for 10 days (time scale of the experiment).
  • After having plated worms for the assay, 75 uL of M9WB buffer (from 4 mL) are spread using beads into an agar plate and incubated at 25 C for 10 dd.
  • After having plated worms for the assay, a few (~30) remaining worms are moved to an NGM plates, parafilmed and incubated at 25 C for 10 dd.

Results: the three controls are negative but fungi grow occasionally on the 6-well plates in which I do worm picking on various days.

Just to re-stress it: I sterilize scope and bench using diluted bleach, wash gloves with ethanol and wear a mask.

I conclude contamination comes from the air. Please tell me whether you think this is reasonable.

Ll.

10

I don’t think the gloves, mask, etc. are really helping you. It’s in the air, not on you (hopefully). I would check around the lab to see where the mold is coming from and settling. I’m a big fan of just using soap and water to suds the bench area first (not bleach), followed by a spray of 70% ethanol (works better than 100%). Make sure the bunsen burner is on and everything is autoclaved (beads, buffers, etc.). Make sure you’re flaming your pick will well, and be careful where you’re setting the lid of the 6-well plate and minimize off time. Beyond that, consider using nystatin. Such long incubations (e.g. aging assays) can be difficult to make through without a few plates getting fungal contamination.

11

Thanks for your help Kevin.

I think I’ll drop the mask :slight_smile:
Also: I’ll try to add a bit of nystatin.

Ll.