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InvisibleNOS4A2
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results of poorly prepared agar
    #19169325 - 11/21/13 10:33 AM (10 years, 3 months ago)

I have a question as to the possible results of agar that isn't quite perfect ignoring any obvious conversation about contams.
I've recently witnessed what appears to be mycelia growing from a spore print innoc which has a very fuzzy nature. It is definitely very white like mushroom mycelia and radiating out from a center point, yet there is not any growth that I would refer to as even remotely rhizomorphic. It is soft and fine with 50% becoming fuzzy tufts which have a bit more substance. When it was time to transfer it was hard to grab a chunk because it was so wispy and sometimes would even slide rite off the agar chunk and seem to dissolve into thin air like cotton candy.

So I've been studying up with the TMC and read a vague comment about the effects of environment on initial mycelial growth. Fuzzy can be a result of over moist conditions which some of these plates had a good amount of condensation. But I also wonder if the actual mixture of the agar can affect the properties of young mycelia. The mixture I'm talking about had a slightly grainy appearance after it set up making me think maybe the nutrient ratio was too high. Lastly, I wonder if what I'm seeing is just a result of old spores. And I wonder if the growth might improve?
Anyone's experiences with agar and less than vigorous growth would help me narrow down what has to be corrected.

TIA


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OfflineOregonMushys
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19169337 - 11/21/13 10:36 AM (10 years, 3 months ago)

Did it look like this? Sounds like you got cobweb mold, though i wouldn't be able to tell unless you post pics.


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InvisiblebodhisattaMDiscordReddit
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Re: results of poorly prepared agar [Re: OregonMushys] * 1
    #19169352 - 11/21/13 10:40 AM (10 years, 3 months ago)


Mushroom


Cobweb.

Pictures will help


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: bodhisatta]
    #19169934 - 11/21/13 12:45 PM (10 years, 3 months ago)

It definitely doesn't look like Oregon's pic.
It did look like the second pic above only it did not grow as consistant all the way around. There are no upward threads as it lays relatively flat but there were just as many areas that had denser clumps. I'm really sorry I don't have a pic, I will try to post one tomorrow.
At first I thought cobweb but after hearing that cobweb has a more grey color and is more 'hairy', I was betting on weak strains of mush.
dangit lol

pics 2morow


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InvisiblebodhisattaMDiscordReddit
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19170253 - 11/21/13 02:03 PM (10 years, 3 months ago)

Could be tomentose mycelium. The mycelium can switch between tomentose and rhizomorphic but generally you'll want to use rhizomorphic if it shows up to do transfers.


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OfflineMrcloudy
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Re: results of poorly prepared agar [Re: bodhisatta]
    #19170628 - 11/21/13 03:25 PM (10 years, 3 months ago)

How fast is it growing? Too much nutrients will slow and thicken the growth making rhizomorphic mycelium hard to see.


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Invisibleblindingleaf
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Re: results of poorly prepared agar [Re: Mrcloudy]
    #19171522 - 11/21/13 05:58 PM (10 years, 3 months ago)

this is what come of my plates look like at first... but not from a spore solution, these are from a clone...



sometimes they will look like that from an ms but only if I accidentally let them grow out to the edge of the plate


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: blindingleaf]
    #19174441 - 11/22/13 10:30 AM (10 years, 3 months ago)

Ok managed to get some pics. I hope it's just the agar. Everything that's been done on this mixture has been VERY slow to grow and in most cases contam but in these two plates, they sat for a good two weeks with no signs of life yet everything else that contammed did so relatively quick, hence why this was expected to be good.
The last pic is of an older plate that was forgotten about that was made at a different time but with the same agar with spore syringe where the solution rolled around inside the moist plate spreading around spores. It's also a different strain. It looks more like contam to me. Looks like I posted in wrong forum. lol
I don't mind getting moved if an Admin choses to do so...
OK pics







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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19174497 - 11/22/13 10:49 AM (10 years, 3 months ago)

Quote:

this is what come of my plates look like at first... but not from a spore solution, these are from a clone...



sometimes they will look like that from an ms but only if I accidentally let them grow out to the edge of the plate




this is encouraging and looks very similar. The plates in the pics I shared are not real clear because they are a PP5 container that isn't actually intended to be used as petris. A test showed they can be PC'd but they suck in ever-so-slightly due to being a flexible plastic to begin with which isn't that wonderful. They are free however, and therefore expendable...:smile:

You can see from the pics these plates have been transferred and one of the things they were transferred to was 3 quart jars of WBS spawn -to see what comes of them in another media. Everything is rather iffy since the cultivator hasn't done this type of work in ages. The spawn seemed a little too sticky which will be addressed next go round. It will be interesting to see if anything even happens. Sterile procedure is being refined with every maneuver as well. So hopefully...with some patience...


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19185498 - 11/25/13 05:20 AM (10 years, 2 months ago)

Just for the record, a few days after the transfer to WBS and the growth looks totally different. Definitely NOT cobweb.
Perhaps it was tomentose as described earlier. I'm surprised at the limited comment about inadequate agar. Seems like everyone would have experience with it. Perhaps most people would rather just throw things away when in doubt rather than investigate further?

After the transfer was made renewed growth in the original agar plate was also noted which brings to mind FAE. I've seen where people have asserted that the air inside a plate is plenty. After this I beg to differ. two wraps of Glad wrap was being employed to seal the 'plates' which I think was too constrictive. Or perhaps these particular containers seal all too well even without the sealing layer which would explain why they suck in a little when PC'ed.
So in conclusion I believe the agar was mixed too rich and to a certain extent FAE was inadequate to show rihzopmorphic growth.


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Offlinekrypto2000
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19185808 - 11/25/13 08:15 AM (10 years, 2 months ago)

That looks like mycilium on your plates to me, whether there's hidden contams or not I don't know. It's probably just tomentose myc though, even with proper agar prep going from ms-agar still tends to start off with a lot of growth like you see there. Do a few transfers and you should start to get some healthy rhizo growth.


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: krypto2000]
    #19186259 - 11/25/13 10:24 AM (10 years, 2 months ago)

'That looks like mycilium on your plates to me, whether there's hidden contams or not I don't know. It's probably just tomentose myc though, even with proper agar prep going from ms-agar still tends to start off with a lot of growth like you see there. Do a few transfers and you should start to get some healthy rhizo growth. '

thanks for the encouragement


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InvisibleFrankHorrigan
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19186539 - 11/25/13 11:33 AM (10 years, 2 months ago)

What is your agar recipe?

I get shitty growth when my agar is too rich or not mixed well enough when poured.

If those pics are all you have after two weeks, you did right to transfer to new plates.

Honestly though, it's hard to say if that is mold or not, it looks suspect to my eyes.


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: FrankHorrigan]
    #19186701 - 11/25/13 12:04 PM (10 years, 2 months ago)

Thank you FH.
It was first try at agar and I'm pretty certain it was the culprit. It was derived from a basic PDA recipe which was scaled down to do a small batch. Possible there was too much P in my DA. It also called for nutrient yeast which I used a small amount of but perhaps that caused my problem?

Would you be so kind as to check out my question in the Contam forum. It's not directly related to this but I've been hoping you'd chime in since I think you have my answer.
http://www.shroomery.org/forums/showflat.php/Number/19185572


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19186724 - 11/25/13 12:09 PM (10 years, 2 months ago)

I still don't know what to think about that last picture. Looks similar but from totally different innoc. Same agar though. Perhaps some transferring is in order for that as well.


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Offlinekrypto2000
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19187002 - 11/25/13 01:22 PM (10 years, 2 months ago)

I actually didn't look at that pic, it does look like it could turn green in a few days, looks kinda curdy or something which often happens with trich*.


Edited by krypto2000 (11/25/13 01:24 PM)


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: krypto2000]
    #19195173 - 11/27/13 05:59 AM (10 years, 2 months ago)

Believe it or not that plate looks like it's forming knots. And it's was thrown out in a garage in 20*-30* outdoor temps for the last week or more!

More agar was made last night. Unfortunately the sugar burnt in the pan and then it boiled over leaving a nice thick layer of agar on the cook top to peel off later, lol. Multitasking while making agar is stupid. When the volume was brought back to 1liter with water I was concerned about it being too thin. It was used to cover(hot!) some contams on a couple previous MS plates which haven't germinated yet and also filled a few empty jars(ghetto plates). The rest was tossed away. Surprisingly the agar set up nice and has a clean look to it. Curious to see what happens with the contam plates. Hoping it burned out the contam agent and spores will germ. It's a long shot but what the hay.
I didn't think agar would be such a trial. It's basically just cooking. I did read a recipe where they suggest boiling the potatoes separate but simultaneously from the agar. Then at the end of the boil adding the sugar(dextrose) to the agar and mixing with the strained potato water. I believe not actually boiling the sugar will be easier. Things tend to homogenize thoroughly while PC'ing anyway.
this is the recipe:
PDA(MDA)
250g potato (or 25g malt)
20g  dextrose
20g  agar

nutritional yeast and other additives will be excluded until a decent simple mix can be made consistently. Practice makes perfect.


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19248935 - 12/09/13 06:40 AM (10 years, 2 months ago)

New question:
Thinking agar prep is being done all wrong...
What I thought was sugar burning in the bottom of the pot was actually the agar. Made a new batch of PDA only this time the potatoes were boiled while simultaneously boiling the agar in another pot. Both for 30 min. Then after straining the potatoes the sugar was added to the simmering broth and allowed to mix for a minute and then broth and agar was combined in flask and mixed. This time there was no multitasking going on and 100% attention given yet it was amazing how the one time back was turned the pots boiled over! Dang it. And agar also formed a burnt layer on the bottom and left little dark colored bits in solution. Very unacceptable.
Is the agar being heated up too much? I'm thinking maybe it needs to be separated from the heat like a water bath? Maybe it doesn't even need to be "cooked" for a period of time? Can it just be heated up at the end to a liquid consistency mix with the broth?
It kept wanting to form a skin while it was boiling too. Perhaps too much agar is being used?


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InvisiblebodhisattaMDiscordReddit
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Re: results of poorly prepared agar [Re: NOS4A2]
    #19249452 - 12/09/13 10:35 AM (10 years, 2 months ago)

Quote:

NOS4A2 said:
New question:
Thinking agar prep is being done all wrong...
What I thought was sugar burning in the bottom of the pot was actually the agar. Made a new batch of PDA only this time the potatoes were boiled while simultaneously boiling the agar in another pot. Both for 30 min. Then after straining the potatoes the sugar was added to the simmering broth and allowed to mix for a minute and then broth and agar was combined in flask and mixed. This time there was no multitasking going on and 100% attention given yet it was amazing how the one time back was turned the pots boiled over! Dang it. And agar also formed a burnt layer on the bottom and left little dark colored bits in solution. Very unacceptable.
Is the agar being heated up too much? I'm thinking maybe it needs to be separated from the heat like a water bath? Maybe it doesn't even need to be "cooked" for a period of time? Can it just be heated up at the end to a liquid consistency mix with the broth?
It kept wanting to form a skin while it was boiling too. Perhaps too much agar is being used?




it's not rocket sci. 500ml h2o 10g nutrient I like LME and 9-10g agar. Use hot water mix together strain if you want to then put it into a bottle and PC it or pour it into your no pour jars and PC it.


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InvisibleNOS4A2
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Re: results of poorly prepared agar [Re: bodhisatta]
    #19250073 - 12/09/13 01:10 PM (10 years, 2 months ago)

well, duh. So simmering or boiling the agar itself isn't necessary. I've read some misleading recipe's which tell you to basically cook them both for some allotted time, yet it always seemed to turn out fine no matter how badly I burnt the agar to the bottom of the pan. I just know it could be better and I hate scraping burtn shit off the bottom of pans, hence why I asked.
OK, no more cooking shit! lol


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