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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Agar problems. Help needed.
    #28058228 - 11/18/22 10:29 PM (2 months, 10 days ago)

First, a brief explanation of the problem:

I can't seem to get past 2 or 3 transfers on agar without the mycelium showing signs of weakness or infection. My working theory is that there must be some bacteria or some other infection riding along with each transfer.

Some background:

I've been growing in the same place for about 2 years now (not my first grow). Last year, I began to notice some issues with (possible) verticillium. Here's some pics of what I mean:





There were more symptoms present on different varieties (bubble-like growths, deformed fruits, etc), but these are the only pics I have right now to illustrate.

Hastily, and after reading too much here about 'enmeshed mold', I panicked a bit, stopped growing, and eventually diched all of my cultures. This was probably a mistake because I had a B+ and Chitwan strain that grew consistently well despite other varieties showing problems. I was under the impression that even fruits that show no signs deformity might still be infected, so the best way to get rid of it would be to start completely over from scratch. So here I am now, trying to get some mycelium that's healthy.

Current problem:

Started a bunch of new strains from spores. Typical crapshoot with suspect looking growth, bacteria, etc. I also obtained live cultures from a sponsor in Canada that looked very nice. No matter which strain I am transferring, however, the same problems seem to be occurring- very slow growth, disorganized and aerial mycelium, and possible bacteria.

Here is a GT x PE (goldmember) variety transferred from a live culture plate:

first transfer (after 10 days):



2nd transfer (after 5 days):



This is the most puzzling because, assuming the original pate was clean, and my process was okay, the growth looks much worse after the 2nd transfer. Agar formula for these plates is 5g LME, .5-1g NY, and 10g agar.


Here are some pics of possibly bacterial plates after a couple transfers from MS. Usually the 1st transfer looks okay, then by the 2nd transfer I get this:









Some 1st transfers that look okay:






I've tried water agar, agar sandwich, and josex pokes with varying results. One of my agar sandwiched PE cultures looks okay after being transferred away from the sandwich (T3), but others persist in having the wet spot around the wedge and disorganized growth. Can post pics of whatever else if need be.

I'm really getting frustrated with the progress here, and could really use some help. Apologies for the long post.


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"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


Edited by Phaedo (11/24/22 03:23 AM)


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Invisibleaintlifegrand
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Posts: 114
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28058274 - 11/18/22 10:56 PM (2 months, 10 days ago)

What changed when the problem started?  Maybe try new agar, bottled water, no pours?

If it is some kind of pathogen you're going to have to find resistant genetics


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: aintlifegrand]
    #28058486 - 11/19/22 12:53 AM (2 months, 10 days ago)

Thanks for the reply,

I'm planning on doing some grain soak agar and some PDA tomorrow, make some transfers, and see what happens.

Any ideas what the wet spots around the agar wedges might be?


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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Invisibleaintlifegrand
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Registered: 11/16/22
Posts: 114
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28058601 - 11/19/22 01:49 AM (2 months, 10 days ago)

No idea condensation?

I think sandman420 is who you want to talk to about mycopests


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: aintlifegrand]
    #28066036 - 11/23/22 10:22 PM (2 months, 5 days ago)

Updating this thread because I'm still pretty confused as to what's going on with my plates.

Here's that same goldmember T2 plate after 10 days, now showing a satellite colony:


Here's some more weirdness:



And some okay looking plates (still very slow growth):




So, do I have 'hidden contams', 'enmeshed mold', or is it just cube myc being weird?

Contams seem likely to me based on the slow growth and at least one visible satellite colony, but I would really like to hear from some experienced cultivators what they think. I suspect verticillium (or some kind of white mold), which has been an issue for me in the past. But I really can't believe it could be getting into almost all my plates. Transfers were taken in front of a flow hood. The filter has been in use for 2+ years. Agar formula is either grain soak (full strength and 50% dilution) or MEYA at 1% malt and 1g yeast.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073251 - 11/29/22 12:44 AM (2 months, 9 hours ago)

I don't know if I should be updating this thread or posting somewhere else, but here we go with another update.


I put some of the better looking plates to grain and this is how they looked before a shake:




and 24hrs after the shake:



I know my millet is a bit too hydrated. Got a bit distracted when simmering the grains and overdid it.

What do you guys think?


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073256 - 11/29/22 12:47 AM (2 months, 9 hours ago)

I'm still getting really inconsistent growth on agar.

Sometimes they look pretty good, but then I transfer and it turns out like this:



that came from this plate:




What does this look like to you??

And what do you think would be causing this? (satellite colonies are from moving the wedge back to the center of the plate):



--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


Edited by Phaedo (11/29/22 12:48 AM)


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OfflineSmellyhobbit
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073319 - 11/29/22 01:23 AM (2 months, 8 hours ago)

Well your grains look fine. This looks to me like an agar issue, not a culture issue. Could you tell me in grams and ml what your recipe looks like, and how long do you cook it?


--------------------
Grow more shrooms :mushroom:

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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Smellyhobbit]
    #28073343 - 11/29/22 01:35 AM (2 months, 8 hours ago)

Thanks for the reply!

I am definitely leaning towards it being an agar formula problem, but what doesn't make sense to me is sometimes the myc likes what it's eating and sometimes it doesn't. I've never had myc be so finnicky. I used to simply switch back and forth between MEA and PDA at full strength (2%) without ever seeing this kind of thing for years.

The agar with charcoal was 5g malt, 10g agar powder, 1g nutritional yeast, and 500ml water (distilled or tap).

I also made a batch of grain soak (well really grain boil) agar with millet water at both 50% dilution and full strength. The millet was boiled/simmered, strained, the water was collected and strained again through a coffee filter. Both diluted GW or full strength were mixed with 10g agar and 500ml water. The last pic, with the see-through agar, is GWA.

All media was cooked at 15psi for 20min +10-15m venting time.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073349 - 11/29/22 01:39 AM (2 months, 8 hours ago)

Is it possible that somehow the agar I have stored has changed over time? I've been using the same agar powder for almost 3 years now. Could that be a factor?

The same is true for the malt powder.

I've also used a different type of malt in the past. The kind that comes in a jar and is partially hydrated into a syrup. It has worked well before. I'm thinking of doing a batch with that stuff once I get through these plates I already poured.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflineSmellyhobbit
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073375 - 11/29/22 01:49 AM (2 months, 8 hours ago)

Man I don’t know that all sounds right. Some of your growth reminds me of water agar growth. Then some of it looks like it’s struggling. I know some people say charcoal powder can give you aberrant growth, but I can’t say for sure since I haven’t tried it.

This is a head scratcher.


--------------------
Grow more shrooms :mushroom:

A Love Letter to New Growers


πŸ…ƒ πŸ„΄ πŸ„° πŸ„ΌΒ  Β  πŸ„² πŸ„» πŸ„Έ πŸ„½ πŸ„Ά πŸ…† πŸ… πŸ„° πŸ„Ώ


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Smellyhobbit]
    #28073392 - 11/29/22 01:58 AM (2 months, 7 hours ago)

Quote:

Smellyhobbit said:

This is a head scratcher.




Trust me, I know. I've been struggling to find out the problem for too long now. I can't seem to figure it out.

In the OP, I mention verticillium because my last attempts from spore basically went exactly the same way. Weird unexplained growth patterns, really inconsistent on agar, with some plates looking good and others bad. Then I went to grain with the best looking plates. Everything looked fine until fruiting when I noticed what looked like symptoms of verticillium or lecanicillium fungicola to be more precise.

I was convinced that I had a parasite like infection and ditched everything, went to new suppliers for spores, and yet the problem persists. I guess I will know for sure once I get fruits from these jars.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28073535 - 11/29/22 03:39 AM (2 months, 6 hours ago)

Here's some pics of what I mean about the vert infection (aside from the pics of split stipes in OP):





--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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Invisiblesandman420
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28074119 - 11/29/22 04:36 PM (1 month, 30 days ago)

What type of clean workspace are you using? DIY laminar hood, SAB, FFU?

What about the source of grain and it's prep? Doesn't explain cultures but curious anyway.


--------------------
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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: sandman420]
    #28074129 - 11/29/22 04:50 PM (1 month, 30 days ago)

Sandman! You're the guy I would really like to discuss this issue with, thank you for chiming in.

I have a home-made 2x4 laminar flow hood. I believe the filter is 99.9999% effective at .03 microns. Can't really remember tbh, I had a good amount of help putting it together/sourcing the materials. I suck at building stuff and doing math lol.


My grain is white proso millet, sourced from a local feed store. I soak for 18-24hrs and simmer until roughly 4-6% of grains are burst, strain until no drops of water are visible coming from the grains. Honestly, I've been pushing the simmer for longer and longer lately because I like it when the kernels/seeds have a nice plump look to them after the PC, but I could probably dial it back for various reasons (easier to shake, less wet, etc).

For bags, I use no prep. 2:1 ratio seeds:water, let them soak up most of the water in the bag before loading into PC.

I PC at 15psi for 90mins plus 15-20 mins vent time. Might start going for 2hrs but I've never really thought grain prep was the problem.


As for the cultures, let's assume for now my problem is with verticillium. What would the most likely vector be in this case?


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


Edited by Phaedo (11/29/22 04:51 PM)


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: sandman420]
    #28074163 - 11/29/22 05:30 PM (1 month, 30 days ago)

Many of my problems are similar to the one's you discussed with manlord in this post: Re: Mold 98% of the time...?

I've gone through so many similar posts, discussions of mycoparasites, and anything remotely similar and haven't gotten anywhere with understanding my own problem.

I'm trying to remain optimistic and open to the possibility that I don't actually have lecanicillium/vert attacking literally everything no matter what the starting material is, but the mixed results aren't making that very easy.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflineDERRAYLD
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28074173 - 11/29/22 05:41 PM (1 month, 30 days ago)

Are you getting the same contamination with plates left open in front of the hood?


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OfflinePhaedo
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Registered: 11/10/22
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: DERRAYLD]
    #28074178 - 11/29/22 05:51 PM (1 month, 30 days ago)

Not at all.

I've done the same tests with plates as the above linked poster did with his. I also keep separate agar plates for cooling the scalpel between transfers. These never show signs of contams despite being open for most of the time I'm doing transfers.

I've even touched a plate of agar with my gloves on, wrapped the plate, and nothing grew. Not even bacteria.

At this point I'm just running with whatever looks good on agar, growing it out in order to see if the fruiting stage has problems or not. Don't know what else to do really as far as testing goes.

If the fruits don't seem infected with vert, would it be reasonable to assume that it's either my agar formula, or something else- maybe bacteria?


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflineDERRAYLD
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28074184 - 11/29/22 05:53 PM (1 month, 30 days ago)

Have you tried a different grain yet?


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OfflinePhaedo
unemployed philosopher


Registered: 11/10/22
Posts: 107
Last seen: 1 day, 8 hours
Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: DERRAYLD]
    #28074187 - 11/29/22 05:58 PM (1 month, 30 days ago)

Nope, I've always used millet. I could definitely give it a go with different grains, but do you think this would help me narrow down the source of contamination/problem?

Do my jars look suspect to you?


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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OfflineDERRAYLD
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: Phaedo]
    #28074192 - 11/29/22 06:08 PM (1 month, 30 days ago)

The reason I ask is purely based on my own experience with random issues popping up despite having clean plates.

Have you left control jars of millet to test for cleanliness after pc?

Give a larger grain a try with a boil to prep or similar and see if the additional cooking vs soaking changes your situation.


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OfflinePhaedo
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Registered: 11/10/22
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Re: Very slow, aerial/disorganized growth, possible bacteria, and agar problems. Help needed. [Re: DERRAYLD]
    #28074216 - 11/29/22 06:34 PM (1 month, 30 days ago)

I'm open to trying anything if it will get me closer to understanding the issue.

Thing is, a lot of my plates don't seem very clean to me. I know there are lot's of variables affecting growth patterns on agar, but I can't help jumping to conclusions about infected myc. I'm definitely still a noob when it comes to contamination. So much to learn there.

I just don't understand how it could be the same infection with almost every variety I'm running. Some are from MS, others are from live culture plates (I live in Canada, where it's sortof legal to sell live mycelium). That seems to point to an issue with the agar, not contams IMO. But then when I get to fruiting, there does seem to be evidence for lecanicillium.

On a side note, when I first noticed deformed fruits/ bubble-like growths, it wasn't really and issue with every culture I had going at the time. I just ditched the ones that consistently had problems. I could still run tubs and get good flushes off other varieties. My agar work always looked good at this stage too. But then something changed. Now it seems like any variety I have going is problematic on agar and then later shows signs of infection/deformity.


--------------------
"A thing really exists if it has any capacity at all, either to do something...or to have the smallest thing done to it...I'll take it as a definition that being amounts to nothing other than capacity"

Plato-Sophist 274e


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