How to raise plasmodia from spores

[ian]

The results are coming in from my first trials and it looks like all the other methods aren't necessary.This has a 70% hit rate, faster development would be nice but this works.

Some repetition here just to keep the thread comprehensive. I'll be setting up more batches soon and I'll make this a pictorial guide when I do.

The aim here is to get away from the lab methods that require things like media and autoclaves. This method does need agar but that's easily bought as vegan gelatin and does not need to be sterile.

Spores - Leave physarum in the light and it'll make spores after a week or so, restricting food and having something older than a week from sclerotium speeds things up. Spores must be allowed to sit dry for 2 - 3 weeks before they'll hatch.

Germinating spores - they'll germinate in water, I use plain tap water that's sat around overnight. The resulting amoebae need bacteria to feed off. Labs use lawns of E.coli, we'll use whatever crap is living on our oats.

I've crushed my sporangia and dipped oats in the powder, adding a sporangium directly to the water or using a piece of paper dipped in spores also seem to be fine.

I left them five days - just so I could check under microscope that I had hatching, overnight just for the oat to break up should be fine.

Agar 1 - 2% plain agar in water, I tried mini petri dishes, multi well plates and large petris, physarum doesn't care but I found standard ( 90mm, they don;t need to be sterile and you can reuse them ) dishes the most convenient for access.

Cut strips out of your agar to make small roughly rectangular islands around 2cm square.

Shake your decaying oats/spore mix and add one or two drops to each square ( that's about 200 microlitres ).

Leave it warm and dark ( the literature says temp is key - around 24C or higher speeds things up. Mine were at twenty ), this is going to take a few weeks but check weekly. Magnification - even a hand lens will be a big help - you'll spot clear patches where amoeba are feeding.

Young plasmodia - hard to spot, little pigmentation but you'll know it when you see it.

One they start appearing start sectioning up the agar to isolate individual colonies ( or let them fight it out ). Half an damp oat planted next to each one will provide food but they'll take a week or more to fully colonise it

They're really slow to get going, expect a fortnight for them to reach cm size then things pic up a bit and you can start subculturing.

Next steps - well I've about 25 young plasmodia to deal with - I don't know how many will make it, I'll start making spores for distribution.

[RyChu]

11/10 Amazing Work...

[ian]

Thanks 

Spent a couple of hours slicing out young plasmodia last night - mm cubes of agar onto paper to minimalise cannibalism. 

I was wrong - a supplement did help - mushroom extract ( 10g mushroom boiled 100ml water 3mins strained) one drop added the week after inoculation gave a massive boost to the number of plasmodia

[ian]

One of the new colony trays - the rips of filter paper will hopefully make it easier to move these around - wet filter paper doesn't cut well. JM2 is (Japan x Mazie) selfed on the assumption that outcrossing followed by inbreeding is a sensible way to look for recessive traits and that recessive traits are worth looking for. JM is the only controlled cross I've done - picking single spores and culturing is isolation is a massive pain in the bum

[ian]

One of the excised colonies after 24 hours - they survive cutting out. The colonies on paper were from multi well plates - too small to use the agar slicing method I used in larger petris - I won't be repeating the multi wells. 

Tiny dot left and slightly down of centre - new plasmodium approx 100 microns, wasn't visible 12 hours before. As far as I understand it the pock marks on the agar are plaques created by feeding amoebae 

24 hours after offering a new colony an oat - it's just visible on the bottom right edge

The same oat four days later - growth is slow at this stage

Young plasmodium - maybe 48 hours, TL of fifteen minutes showing it's already streaming.

[RyChu]

So are you suggesting the oats be left out for a little bit when you say "Labs use lawns of E.coli, we'll use whatever crap is living on our oats"?

[ian]

Oh no, grains are are already crawling with bacteria, it'll be lactic acid and enterobacteria mostly 

[ian]

Growing away slowly - it seems to take around 4 weeks for them to really pick up. This does work with non sterile conditions but I'm going to start dry toasting the oats - there's a heck of a lot of pin mould and it might not be bothering the slime but it's a massive pain in the arse. 

I've also learnt paper is a bad move for small plasmodia - they grow on it fine but there's bugger all water in reserve on a 2cm disc of paper so they dry out easily even when sealed - the water condenses on the petri lid leaving the disc dry. Got around fifty colonies on agar now though and some variation is showing up.

[ian]

I'm starting to wonder what the hell I was thinking - moving cultures took a couple of hours yesterday but hey, I got to binge Marilyn Manson. All babies now into their own 9cm petri dishes on water agar so I don't have to worry about watering.  I think paper strip on top of an agar bed will be the way to do the next one - paper is very handy for moving delicate plasmodia around.

Here's one of the nursery dishes - you can see a little variation in colour but there's far more variation in growth rate so I might have to change selection criteria ( which will be a pita to characterise properly ). On each paper strip there's the tiny snip of agar from the mating dish ( the one with the amoebae on it ) and the one oat it was originally fed. They then got moved onto agar squares and now as some of them are starting to explore they need moving to their own dishes.

Here's one that did what it was supposed to - move off the paper so I can remove it. You can see just how much contamination there is - the dishes had cm deep rolls of actual moulds growing across them but it's not bothering the slimes ( really cheesing me off though )

All set up, as soon as the fresh oats are colonised I'll remove the old agar - this is the 'Let the slime outrun the contaminants' strategy.

Here's one that's a week ahead from a paper test - very active explorer here.

[RyChu]

When you say water agar, do you mean agar slices sitting in a pool of water or just standard agar?  

[ian]

2% agar made up in water. The percentage probably doesn't t matter that much but 2% is easier to pick up in forceps than 1