Thoughts on the breeding project

[ian]

Well across the social mediasphere enough people are interested to make a go of this which means I need to do something. Somethings actually happening Reg! ( 400 quatloos for anyone who gets that reference and a bonus to anyone who gets the other two )

Yes I'm an actual professional scientist but physarum isn't my focus of research ( it's microscopy, see what I did there? ) so this is on my own time and nothing to do with my department - that bit's important as it's a legal disclaimer. Iff ( for lovers of formal logic ) it look like this is going somewhere there are definitely grants that could be applied for but that's a long way down the road. 

I want to keep techniques as low tech as possible and postage down to a minimum - I could get petris & tubes from work to send out but I'd rather not so I need ideas.

This all hinges on raising mature slimes from spores, we need plans for improvised/scavenged culture vessels and other kit. Our huge advantage over any other life form is physarum can be dried down at pretty much any stage of its life cycle and stored at room temp so we have incredible archiving potential and can backcross through many generations.

Basic physarum sex - spores are made by meiosis. Spores hatch into amoebae which are haploid ( one set of chromosomes ), amoebae eat bacteria and can divide and multiply ( by division - yes it's confusing if you do maths ) as any respectable amoeba should. With crowding and stress amoebae fuse and become diploid ( two sets of chromosomes ) and become a mature slime mould.

I've tried two methods - paper and agar in both I'm letting rotting food provide bacteria to feed the amoebal stage.

Paper is a doddle - add spores to kitchen towel with a few oats and basically let it rot in the dark, occasionally topping up with an oat and water. It's reliable >90% success but it takes 8-16 weeks which is effing slow and you've a shedload of real mould to deal with. Reused plastic food tubs and bog roll/kitchen towel are all you need.

Agar - add one oat and some spores to 1ml ( ish ) of water and leave for a week, mix two such cultures and add to multiple agar dishes. Colonies crop up 2-5 weeks later with less contamination.

So - questions for people in the real world - how easy/cheap is agar to get hold of, is the paper method too slow, what about small containers for a few ml of liquid ( I use microtubes ) ?

What we need it to maximise the number of mature slimes raised - selective breeding is very much a numbers game, the more offspring, the more chance to hit upon interesting features.

Ultimately I want this to be a community project I want to coordinate and help but I don't want to be 'in charge' - some sort of github with forks and branches would be amazing.

[Evan Brown]

I was actually planning on trying a breeding project, if you're willing to provide guidance given your background I'm sure a standard protocol that everyone could follow wouldn't be too difficult to come up with.  In the "real world" agar is a bit tricky to ff, I'm told Asian grocery stores carry it, but they never know what I'm talking about.  A few dollars online isn't too bad though, I did manage to find a kit with 3 Petri dishes and 3g agar for $10 at a local school supply store. The paper method is a bit slow, the liquid method however sounds promising and straigtforward.  Most Home Depot stores here in the US that I know of have free water test kits with a centrifuge tube in them, I like to use those for various experiments, could be an option for some I suppose.

[ian]

There's things like alginate as well, I might investigate the vegan jelly options. I wonder if paper's slow because this is a surface area effect. They will fuse when the population reaches a critical level, agar is basically a 2d surface whereas paper is a tangled mass of fibres. Smaller paper might just do the job.

I'm raising spores at the moment - I have few ready but I think they need a couple of weeks dry before they'll hatch. 

[Evan Brown]

From what I know most vegan jelly is essentially agar.  I believe aliginate sets up similar to silicone doesnt it?  so it might not work too well, but I'm sure it's worth a shot. I think vegan jelly is probably the most widely available option, if it works I'm wondering if you could do some sort of bacteria broth on an agar slant inside a microcentrifuge tube.  I'm pretty scientifically minded myself with access to most basic resources so I've been looking into the idea of culturing e. Coli to feed them, that seems to be standard procedure from what I can find.

[ian]

I'm trying to keep to low tech methods but I think the anything that works is right approach is needed here - I want to encourage DIY but lab methods are also fine. If someone can get microtubes they can probably get agar - I could easily supply both but I don't want to end up paying for postage and explaining to customs, spores and sclerotia fit nicely in flat inoffensive envelopes. An oatflake in 1-2ml of water generates many tasty bacteria for our amoebal chums.

Trying a large scale spore batch at the mo, I'll have two small batches that should be ok to post next week.

As for posting spores, tiny fine black dust, bit of an arse to package so I'm wondering, smudge onto paper? Seems the simplest solution and makes it easy to measure small quantities.

Current plan - get what I have ready out there in the next two weeks, a quick note of "here's what we've tried that works", ask people to write up/in with what they've tried.

In the background - start being systematic with my stuff - try paper with no nutrient, varying  paper disc sizes, agar size etc.  Take samples into work so I can check on viability and survival rates if they're left hanging around, maybe even get the haemocytometer out to count ( maybe, perhaps, I hate the damned things )

[Evan Brown]

I like the idea of a slime mold spore "print" of sorts, but what about spores on dry oats? Then they can be re hydrated by the recipient ready to start growing. I bet you could even put it all in a small zip top bag and use that as a container with the added bonus of fitting neatly in an envelope. If you want I'd certainly be interested in testing some of them out for you :D

[ian]

My first thought was oats but it excludes trying other methods for spores and might affect viability. Zip locks though - as long as they get reused might be a great plan 

[Evan Brown]

I didn't consider that, the spore print might be the best option in that case. 1x2" ziplock bags can be had for around a penny each online and at some shops, and reusing them would probably be ideal since they'll have spores and bacteria festering in them from previous uses so it might actually work better I'd think.

[ian]

Here we go - six tubes - three each of two strains, one of each with a paper strip of spores, two by dipping oats in spores. Forceps and a lighter for sterilising them. I set this up last night, spores start to hatch after around 24h. I'll stick them under the scope at work tomorrow and if I've got cells ( they'll be swarmers rather than amoebae with this method ), I'll start posting spores.

I'll try dotting out these cultures onto paper and agar of various sizes and see what we can do to optimise. I'm hoping smaller containers will mean faster plasmodia formation, being able to screen fifty to 100 at a time is the level I'd like to be at.

I might try more spore picking for controlled crosses, takes a long time at home but if I use the lab microscopes....

[Evan Brown]

Nice! Sounds like a good simplified setup.  I'm waiting on a 100 pack of 1.5ml centrifuge tubes and a 72 position tray from eBay that I intend to use for my breeding project.  I have a clone I took of my slime in a Petri dish that should be ready to start receiving light exposure in the next couple days or so.

[ian]

Spores are viable - I have swarmers so much pipetting tonight!