Some help regarding growth and inoculation please!

[Ronnie]

Hey physarumnators!

I am a year 12 student who is experimenting with physarum polycephalum to make optimised networks (similar to the famous tokyo railway experiment) to solve urban design issues as a part of a design folio. However, I have had some trouble actually inoculating the physarum onto my design. If someone could tell me what I am doing wrong, or could improve that will be great!

This is how I am going about this:
 I place oat flakes on a 2% agar petri dish (non nutrient based) while following a map that I created. I have sterilised the plane rolled oats by placing them in the oven on 160 degrees celsius for 20 minutes. I place my oats using tweesers which I place in a 70% ethanol diggers methylated spirit solution, and then burn the tip of using a gas stove to get rid of the ethanol. I am doing so to get a sterile culture, which seems to be working 90% of the time. 

The main issue/concern for me is the way I transfer physarum cultures onto the plate. I have sourced my cultures from Southern Biological supply and allow them to grow on the culture petri dish by placing oat flakes around the petri dish which seems to be working great. there are two main techniques i have tried.

1) I have broken colonised oat flakes to about 2-4mm pieces and placed onto my desired petri dish to allow them to grow. This resulted in sort of linear growth - only one strand is growing and often leaves a whitish trail behind and does not form plasmodium/scavenging growth. this is also really slow!

2) I then tried to place a 2x2x2mm agar cube of physarum, face down onto the petri dish to allow it to colonise the plate. This was better, but the growth was quite tiny, and it is not really moving like normal. my cube might be too small? I also did not place it in direct contact to any oat flake, so maybe it needs some nutrition to actually start making those networks?

Also, the Physarum might be dehydrated? most websites say that the physarum does not need to be watered if it is being grown on a moist medium like agar, is this true? I have also been keeping it completely in a dark cupboard, without access to any sort of sunlight/indirect sunlight which I know can do many things to initiate things in the physarums life cycle. 

I have also come accross this paper, https://www.nature.com/articles/s41598-022-05439-w 

which is similar to my design - but when they place their inoculating piece onto their plate, they surround it with outfakes (which I have not tried). This is what I will be trying next

[Ronnie]

cube inoculation, as you can see, its leaving a white ish trail and not acting the best! (day 2 of innoculation)

and here is the innoculation from an oat flake, sort of linear growth? (day 4 of innoculation)

[ian]

Hi

I think you're not using a large enough innoculum - slimes essentially burn biomass to move around, if you're getting white trails, they're abandoned branches which suggests there's not enough plasmodium there to get it to the first few oats. They don't need watering on agar but it looks like your oats are dry - they won't hydrate well off the agar and physarum needs solutes to follow, it looks like it's moving away from the food in those pics so it's not sensing them.

Try the slime manifesto tinyurl.com/slimemanifesto for some bulk rearing tips.

BTW you can supply different grades of food for different levels of incentive - 10% powdered egg in agar beats an oat flake ( Dusstour group) 

[Ronnie]

Hey Ian,

This was heaps of help! The growth of the physarum has been a lot better when I followed your advice, but I was wondering if the networks it makes are influenced by the size of the oat flakes I place on the dish? ofcource, it would but I am wondering weather my oat flakes are too small on my petri dish to even maintain growth? Ill provide a photo- but places where there is smaller oat flakes have turned white ish again? As it is a map I am recreating, placing large oat flakes on a petri dish is a problem - but I can definitely try and create the map in a bigger Tupperware container and place full sized oat flakes if that is the problem. Let me know what you think! Also, i'm sort of worried that my physarum is reaching sporulation? might be totally wrong but rather than making sort of 2D networks its now lifting up? not too sure about it's life cycle as its kind of complicated but if you could help me with identification, it'll be great (near the top of the petri dish?). I definitely would benefit from the palsmodium state of the physarum the most! thanks so much, this forum is such a help especially since resources on this organism are very scarce.