Alternate cleaning techniques

Model Organism: Worms Scientific Discussion
1

Hi,

I have a few transgenic lines with GFP on the extrachromosomal array. It’s been very difficult to clean the plates by bleaching as the worms tend to lose the array every time i bleach. Are there any alternate methods that I could use to clean my worm plates?

Thanks

Sudhanva

2

Any decontamination method is going to be subject to loss of unstable extrachromosomal arrays, so I suspect this is not a technique issue.

First, decontaminate multiple parents, all selected for presence of the srray, Then, if your GFP is visible in the L1 I would select GFP animals crawling out of the bleach. If only visible after L1, then I would use brute force: pick many L1 animals from the bleach decontamination, then screen for GFP later. Unless your array transmits at very low frequency, you should recover.

Or maybe I’m missing something here?

3

It’s theoretically possible to clean a strain by letting the larvae crawl around on an antibiotic plate (or a series of antibiotic plates) in the absence of food for many hours (you want to give them a good chance to empty their gut, and to get a good dose of antibiotic). I’ve always been in favor of the bleach method, though, and if it doesn’t work at first simply bleach more animals next time, as Reiner says. Bleaching protocols vary; the one I use and recommend is adapted from the one in Michael Koelle’s protocol collection, with the slight variations of using a bit more NaOH (2M, not 1.5M), using a bit more gravid worms, using a smaller volume initially (<10 ul), and reapplying more solution each time the spot dries until nothing or almost nothing but eggs are visible there.

4

Sudhanva,

I use a brute force approach when bleaching. I grow up a plate or plates of worms until they contain many gravid adults and eggs. Then:

  1. Wash worms off in 1ml of M9.
  2. Add 250ul of 25% bleach (in water), and another 250ul 2M NaOH
  3. Leave for 5 minutes, then spin at 1500rpm for 30 secs
  4. Remove supernatant and resuspend in 100ul of M9.
  5. Put suspension on edge of seeded plate and leave overnight for eggs to hatch and larvae to crawl to food
  6. Pick worms onto fresh plate next day

I often do 2 plates at once, and they’re crawling with L1s the next day.

I’ve never had much luck with the antibiotic treatment, but I’ve only ever done it on seeded plates. Maybe I’ll try it on unseeded as Hillel suggests

5

I’ve never met a strain I couldn’t decon with bleach, though paralyzed Uncs can be quite touchy. It’s usually just a matter of numbers, especially with a low transmitting array.

Another important tip for bleach cleaning: I always pick the L1s off within 18 hours of bleaching. If I wait a second day, often enough I’ll see some contamination return. It persists in the gut I suppose, so down but not out.

6

sometimes I try this “antibiotic bath” for single or several gravid worms:

  1. Drop antibiotic solution (I use Amp/Strep/Neo/Nystatin mixture) on a new plate near the lawn
    2.pick your worm and place it into the drop “bathtub”.
    3.after 2~3h, seek your worms and remove it to new clear plate.
    4.pick out the laid eggs from new plate and remove to another new plate, then the progeny would be cleaned.