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OfflineTranscendent Other
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My first succesful grow on agar. Seeking feedback!
    #26945518 - 09/20/20 04:57 AM (3 years, 4 months ago)

Finally! After a world of faffing and failing I finally managed to grow out some agar!

In the pictures you'll see some button mushroom clones that I did on two different agar mixes; both MEA, but one with nutritional yeast (the fainter, light yellow one) and one without (the brown and orange one).

I had a few questions I wanted to ask, and of course welcome any other feedback you might have :smile:

1. I'm curious to know why the mycelium on the MEA without the nutritional yeast seems to be stronger? If you look at the image you may notice that the rhizomorphic hyphae are more defined than on the other plate. Not only that, the other plate has several patches of tomentose growth on it (and also some primordia!). Could this be to do with button mushrooms favouring agar without the yeast?

2. What is the brown discolouration, and should I be concerned? The mycelium looks healthy, so if theres any contam it's certainly not visible...

3. I'm about to use these two plates to do some transfers, and was curious as to how you 'isolate' strains. Is it possible to isolate phenotypes by eye (I.e. look at hyphae and establish what it might represent in regards to phenotype, and those slice this out for transfer), or is it a case of slicing out random sections that seem to show strong mycelial growth, transferring, and repeating until you have an isolate? (and then presumably growing this into mushrooms to establish what you've isolated?).

4. A slightly different note; when sterilising my plates in my PC I ended up with some moisture inside the plates, and also between them (making the stick and difficult to lift when pouring!). I've tried to avoid this before by wrapping them in tinfoil; whilst it was an improvement it didn't make a huge difference. What would you recommend, or should I not worry too much about the moisture?


Thanks!






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OfflineForresterM
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Re: My first succesful grow on agar. Seeking feedback! [Re: Transcendent Other]
    #26945562 - 09/20/20 06:19 AM (3 years, 4 months ago)

1. Mycelium tends to grow "stronger looking" when there's less nutrients as it's trying harder to find nutrients, but that's just a simple explanation and not my particular field of expertise.

2. I would be weary of the brown area as the mycelium seems to be avoiding it (see pic2, 1 o'clock).  Sometimes brown is normal, but that seems suspicious to me.  I would take another transfer away from that.


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OfflineChapuco
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Re: My first succesful grow on agar. Seeking feedback! [Re: Forrester]
    #26946242 - 09/20/20 01:46 PM (3 years, 4 months ago)

I'd reccomend you using grain boil water, i had issues using MEA. The rec is 500ml GBW and 10 gr agar agar. Hope that help you


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Edited by Chapuco (10/07/20 11:11 AM)


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OfflineFlufferNutter
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Re: My first succesful grow on agar. Seeking feedback! [Re: Chapuco]
    #26946279 - 09/20/20 02:13 PM (3 years, 4 months ago)

For condensation issues that arise with no pour plates, there's a couple strategies that I was taught and have worked for me:

  First is to be sure you're not making them too thick. I went and bought a 60ml syringe to make it much easier and more accurate. I also let them cool a bit with the lids off before you PC. That helps a bit, too. Definitely wrap the lids with foil to prevent anything coming in/going out.
 
The best thing you can do to combat condensation is to let the PC cool as SLOWLY AS POSSIBLE. I mean SLOWWWW. I will let my PC cool to the point that it's not going to burn you, and then I wrap in a heavy blanket, something that's not going to melt, obviously.

  I'd let it sit like until its room temp, or until I'm ready to crack it open and get to work. This wont completely stop it, but for the most part it cuts it down to nominal levels.

  Store your plates upside down. They dont need to be put in the fridge unless you have a culture you're trying to save/stall. Just keep them in something cool and dry.

  Storing upside down gives any condensation that forms a place to go other than your culture. Then you can carefully wipe it dry in the SAB.

Hope that helps! Carry on!


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OfflineFlufferNutter
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Condensation [Re: FlufferNutter]
    #26946461 - 09/20/20 04:08 PM (3 years, 4 months ago)

This has gotten me thinking about other ways to accomplish this other than tying up the PC. Honestly, I'd never thought to find another way. I always just cooked plates in the evening or on the last run of "PC day". I'm in the process of getting things back up and moving after a long hiatus,so Ive got the agar, motivation, and I'm not backed up with tons of projects (yet).
 
  Plus, I have an instant pot here which should make it easier to experiment this without the full time investment of a PC run.  I dont have to use the plates, just cool them down with minimum condensation. Agar is cheap, and the instant pot will make it easy to repeat.
 
  I know this question came up all the time on the forum I was on years ago, and I've seen it a bunch here too. At any rate, I'd like to figure it out. Thanks for the inspiration TO!


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OfflineTranscendent Other
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Re: My first succesful grow on agar. Seeking feedback! [Re: FlufferNutter]
    #26950407 - 09/23/20 02:35 AM (3 years, 4 months ago)

Quote:

Forrester said:
1. Mycelium tends to grow "stronger looking" when there's less nutrients as it's trying harder to find nutrients...

2. I would be weary of the brown area as the mycelium seems to be avoiding it...




1. Ah I see, that makes sense!

2. Well it may be hard to see in that image, but the brown does actually more-or-less cover the entire underside of the mycelium. That said, i'm still going to try to avoid it as much as I can.


Quote:

Chapuco said:
I'd reccomend you using grain boil water...




Cheers, i'll give that a crack too.


Quote:

FlufferNutter said:
...I went and bought a 60ml syringe to make it much easier and more accurate. I also let them cool a bit with the lids off before you PC...
 
The best thing you can do to combat condensation is to let the PC cool as SLOWLY AS POSSIBLE..

  Store your plates upside down...




Sorry i'm confused, in your method are you pouring the plates before sterilising the agar in the PC?

RE PC Cool; okay cool, that makes sense. Though I may have to rethink my method, as i've been putting my plates and bottle of agar in at the same time, then opening and doing my pours as soon as everything is cool enough.

I'll experiment with your upside down method. I did managed to dodge getting too much of the condensation on the agar, but obviously the more control over this the better.

Yep, it certainly helps! It's all good stuff! :smile:


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OfflineGan
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Re: My first succesful grow on agar. Seeking feedback! [Re: Transcendent Other]
    #26950497 - 09/23/20 05:10 AM (3 years, 4 months ago)

Are they no pours? If so, you can just dump the water out once inside your SAB. Just make sure there's not enough that it is sloshing around on the agar surface. As that will spread contams around and make it harder to grab some healthy growth.

Also, use whatever nutes you want. MEA works perfectly fine. BRF agar works perfectly fine. Dog food agar works fine grain water agar works fine. Dont get caught up on the recipe too much. Just use what's easiest to find for your area. Normally if people have a problem with a particular recipe, it means they did something wrong. I've used MEA (as have many others on here) for years for probably 90% of my plates with no issues.


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Offlineseand04
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Re: My first succesful grow on agar. Seeking feedback! [Re: Gan]
    #26950731 - 09/23/20 09:10 AM (3 years, 4 months ago)

I find that a lil condensation dosent really effect mine. But like FlufferNutter said, let that PC cool down all the way.


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OfflineTranscendent Other
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Re: My first succesful grow on agar. Seeking feedback! [Re: seand04]
    #26957103 - 09/27/20 07:19 AM (3 years, 3 months ago)

Quote:

Gan said:
Are they no pours? If so, you can just dump the water out once inside your SAB. Just make sure there's not enough that it is sloshing around on the agar surface. As that will spread contams around and make it harder to grab some healthy growth.

Also, use whatever nutes you want. MEA works perfectly fine. BRF agar works perfectly fine. Dog food agar works fine grain water agar works fine. Dont get caught up on the recipe too much. Just use what's easiest to find for your area. Normally if people have a problem with a particular recipe, it means they did something wrong. I've used MEA (as have many others on here) for years for probably 90% of my plates with no issues.




I'm not gonna lie... I have no idea what no pours are! :blush:

Thanks for the tips - I have 8 dishes on the go; 4 clones from my 'yeast free' mix, and although its only been a week the mycelium is growing, and it doesn't look too hungry either!
Also there isn't excessive moisture, and obviously not enough for it to be an issue. And to be honest, this batch has a contaminated plate but its actually the first one i've ever had contaminate.

Quote:

seand04 said:
I find that a lil condensation dosent really effect mine. But like FlufferNutter said, let that PC cool down all the way.




Cheers but, i'll bare that in mind :smile:


In regards to one of the questions in my first post that hasn't been answered yet, how exactly do isolates work? Do I literally just clone parts of the mycelium that 'looks strong', and keep cloning until I visibly have an isolated strain? (so its a bit of a guessing game until you get there). Or is it possible to look at the behaviour of the hyphae, i.e. the way in which they are branching out, and identify what phenotype it represents, and thus deliberately select it for transferring and cloning out?
My curious mind is going! I also realise this is either a silly newb question, or i'm getting ahead of myself haha. Either way i'd love your thoughts!

Thanks.


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Offlineseand04
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Re: My first succesful grow on agar. Seeking feedback! [Re: Transcendent Other]
    #26957970 - 09/27/20 08:07 PM (3 years, 3 months ago)

What I do is make a agar plate from spore print or syringe.  Take off the best looking growth transfer that to a clean plate, grow it, spawn it,then make a live tissue transfer onto agar. An isolate from a live tissue sample. Hopefully this helps


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