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OfflineTGS
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Separating mycelium from contams fine points
    #26456483 - 01/28/20 09:03 AM (4 years, 20 days ago)

Familiar story, I started with syringes, had some early success and thought I knew what I was doing. Then lost tub after tub to trich. I’ve gone to agar from clone, print, and colonized grain with better results but still seeing trich after 1-2 flushes.

I’m seeing that new agar dishes, uncolonized grain, and coir in the bucket stay clean, so it must be the spawn. I’ve scoured the threads so I know in theory what I need to do. I’m developing a more discerning eye but could use another opinion on this.

The pic is a quarter sized circle from a 6th transfer off a colonized grain. It’s hard to photograph but there is a 2mm wide halo around what looks like good mycelium. If you look through the mag it is possible to see that there are very fine filaments in that halo, or at least that is what my eye thinks.

Is this just the leading edge of the mycelium, or a depletion zone of nutrients and food coloring, or a contam running just ahead of the myc?  If it is a contam, how am I ever going to separate it being at the leading edge?





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Offlinetrubblesome
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26456494 - 01/28/20 09:14 AM (4 years, 19 days ago)

without being able to see the morphology under a scope it's hard to tell if it's a contam or the leading edge of myc burrowing under the surface. If you're nervous about that you might try delicately lifting just the smallest amount of clean looking myc from the top and putting that to a new plate.


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OfflineA.k.aM
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Re: Separating mycelium from contams fine points [Re: trubblesome]
    #26456520 - 01/28/20 09:39 AM (4 years, 19 days ago)

Aren’t there techniques for contams meshed in?

I’ve heard hot pours which I have no idea what that is yet, and putting a clean piece of agar ontop of the myc so it climbs up and over leaving the contam on the plate.

That might not be how it actually works I’m just trying to remember stuff I’ve read before.


I’ve been having clear edges lately too but it seems to be linked to softer agar. I think when you can see the myc strands in there when you hold it to light it’s probably clean.


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Edited by A.k.a (01/28/20 09:40 AM)


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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: A.k.a]
    #26456856 - 01/28/20 01:20 PM (4 years, 19 days ago)

Ok I think when it gets close to the edge I will attempt to lift a sample of just the myc off the top and just the fine stuff from the halo. See if I can tease them apart and figure out what they are.


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Offlinefeldman114
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26456863 - 01/28/20 01:25 PM (4 years, 19 days ago)

It was my understanding that contams grow much faster than myc. So unless that’s another fungus piggybacking on the cube culture, it should be clean, no?
Either that or the wispy growth will speed away from the visibly thick myc.


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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: feldman114]
    #26467684 - 02/03/20 10:37 PM (4 years, 13 days ago)

Update I just lost the tub from the prior plate to trich. Can anyone Id that particular contam as Trich?

I lifted a little piece off the interior and went to a new plate. Still too early to see if we left the contam behind. I chose not to culture the outer ring separately.  But I’m not putting this to grain unless I see it run without that second phase.


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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26467692 - 02/03/20 10:42 PM (4 years, 13 days ago)

Inquiring further are there cultures that are just too intertwined that you never get them clean?  At what point is it better to give up and swipe a new  plate off print?


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Re: Separating mycelium from contams fine points [Re: A.k.a]
    #26467820 - 02/04/20 01:46 AM (4 years, 13 days ago)

Quote:

A.k.a said:
Aren’t there techniques for contams meshed in?

I’ve heard hot pours which I have no idea what that is yet, and putting a clean piece of agar ontop of the myc so it climbs up and over leaving the contam on the plate.

That might not be how it actually works I’m just trying to remember stuff I’ve read before.


I’ve been having clear edges lately too but it seems to be linked to softer agar. I think when you can see the myc strands in there when you hold it to light it’s probably clean.



Hot pour is pouring hot agar over a plate thats already has growth. As far as I know it only works for bacteria because bacteria wont grow up through the agar but mushroom mycelium can. The heat is supposed to sort of stunt the bacteria while giving the mycellium a surface to grow up through. Im not sure if it works for other contaminates. If mold is meshed into a culture I feel like the best action would be to start fresh from spores and transfer earlier before the mold has a chance to get meshed into the culture. If its happening constantly then maybe its best to start from a different print. If it still hapoens its time do do some deep cleaning and reevaluate ones sterile technique.
Or atleast thats how I see it.


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InvisibleLadysKnight
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26468053 - 02/04/20 07:57 AM (4 years, 13 days ago)

Quote:

TGS said:
Familiar story, I started with syringes, had some early success and thought I knew what I was doing. Then lost tub after tub to trich. I’ve gone to agar from clone, print, and colonized grain with better results but still seeing trich after 1-2 flushes.

I’m seeing that new agar dishes, uncolonized grain, and coir in the bucket stay clean, so it must be the spawn. I’ve scoured the threads so I know in theory what I need to do. I’m developing a more discerning eye but could use another opinion on this.

The pic is a quarter sized circle from a 6th transfer off a colonized grain. It’s hard to photograph but there is a 2mm wide halo around what looks like good mycelium. If you look through the mag it is possible to see that there are very fine filaments in that halo, or at least that is what my eye thinks.

Is this just the leading edge of the mycelium, or a depletion zone of nutrients and food coloring, or a contam running just ahead of the myc?  If it is a contam, how am I ever going to separate it being at the leading edge?








I've been fighting a mold in my cultures as well that meets most of your descriptions. A Halo of filaments extending just ahead of the culture. Yours looks milky, but mine is totally clear. It can only be seen when holding a light at the right angle. I've had a couple plates where the mold is growing as a satellite by itself. That was when I knew I wasn't crazy.

I've had some success slowing it down with black tea agar.


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OfflineLikeMyc
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Re: Separating mycelium from contams fine points [Re: LadysKnight]
    #26516286 - 03/04/20 10:57 AM (3 years, 11 months ago)

Maybe it would help when placing the mycelium onto a new plate, to streak that small sample across the agar in an attempt to create satellite growth, like LadysKnight mentioned, to also better visualize and extract clean myc.

I agree with Gizmo1. Sometimes we have to revisit the basics to make sure our technique hasn't morphed over the years. I used to certify medical staff in sterile technique and bloodborne pathogen cleaning, and as simple as it may seem - trust me that with a decade of experience I have to revisit and so do others to cover every detail to pass the course and at periodically State scrutiny.

Seems you've been at it for a while so maybe it's time to invest in a microscope if you haven't done so already?

I would love to know what becomes of the myc off that plate.
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OfflineLikeMyc
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Re: Separating mycelium from contams fine points [Re: LikeMyc]
    #26516314 - 03/04/20 11:20 AM (3 years, 11 months ago)

One question I forgot to ask.
Has the halo just appeared at that stage of mycelium growth or does the mycelium always appear to have a halo regardless of how big it is? If it always appears to to have the Halo and the mycelium grows beyond the previous size Halo then it is most likely the leading edge of the mycelium.


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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: LikeMyc]
    #26622226 - 04/23/20 12:20 PM (3 years, 9 months ago)

It’s hard to tell through a mini round. I didn’t really watch it through a progression but that’s a good idea. I have another shot that’s kind of a smoking gun. The halo outrunning a spot of known contam. This tells me that the halo effect can be non-mycelium.



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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26622246 - 04/23/20 12:27 PM (3 years, 9 months ago)

Given what I can read off the label this either got tossed or I tried to process it and it failed in the tub with trich.

Here are a couple more much better pics that I could take in the open because this one got tossed.

Ignoring the white spot of contams on the edge can anyone comment on whether the finer filaments in between the rhizo growth are good mycelium branching off or contams intertwined with the myc? 

Close up it looks ok to me but given my recent failure rate I need all the help I can get.




Edited by TGS (04/23/20 12:30 PM)


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OfflinePinkStormtrooper
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26622265 - 04/23/20 12:38 PM (3 years, 9 months ago)

i have not much help other than to let you know you are not alone


i watched it speed around the whole plate super fast, in this pic its almost 100% around the sample. my stupid phone reflection got right in the  way.

here it is under 10x


right where its about to touch each other

the only clue i have figured out so far it if the agar looks foggy its cashed. other samples the agar is still perfectly see thru.


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Edited by PinkStormtrooper (04/23/20 12:39 PM)


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InvisibleHobbit GDF
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26622269 - 04/23/20 12:40 PM (3 years, 9 months ago)

The leading edge of myc usually is thin and hasn't thickened up like the main part of the culture. Right? Like I could take a transfer of the leading edge.  Or let's say in front of it where it looks like nothing.  That has microscopic MYC reaching further and fills out.
I dont know if you could call it contaminated or myc.
Like likemyc said. You need a microscope


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OfflineTGS
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Re: Separating mycelium from contams fine points [Re: Hobbit GDF]
    #26684575 - 05/20/20 11:39 PM (3 years, 8 months ago)

Another clue. Here is a plate I let go until it pinned. This was a 6 th transfer from a print. I can see both the pin and some little spots of green starting to show up. Meaning the contam and the mycelium could coexist for quite a while before one dominates.

I do not believe it is my sterile technique because I have done the occasional clone that didn’t take and while I didn’t get what I wanted nothing else showed up in there either for several weeks. 

So far having better luck isolating a clean culture from a clone than from a print. And way better than syringe. And just tossed one whole line. After enough green in a row I think it better to start over.



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Offlinerumfor69
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Re: Separating mycelium from contams fine points [Re: TGS]
    #26684868 - 05/21/20 05:21 AM (3 years, 8 months ago)

I would've taken one of those pins with some flammed tweezers to another dish.
Those were probably sterile in vitro pins. All spores are contaminated
to some extent so that's why clones are easier to get clean cultures with
Syringes are a suspension of the spores and the contams on the spores and
tend to be a little worse in that sense because they're liquid.


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InvisibleLadysKnight
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Re: Separating mycelium from contams fine points [Re: Hobbit GDF]
    #26684976 - 05/21/20 06:49 AM (3 years, 8 months ago)

Quote:

rumfor69 said:
I would've taken one of those pins with some flammed tweezers to another dish.
Those were probably sterile in vitro pins. All spores are contaminated
to some extent so that's why clones are easier to get clean cultures with
Syringes are a suspension of the spores and the contams on the spores and
tend to be a little worse in that sense because they're liquid.




In my case, I tried this with several pinning plates and cloned fruits. The halo mold followed.


Quote:

Hobbit GDF said:
The leading edge of myc usually is thin and hasn't thickened up like the main part of the culture. Right? Like I could take a transfer of the leading edge.  Or let's say in front of it where it looks like nothing.  That has microscopic MYC reaching further and fills out.
I dont know if you could call it contaminated or myc.
Like likemyc said. You need a microscope




In my experience, healthy myc on agar has a defined, pronounced border, no thin iffy halos. D3monic has great pics of great cultures, not one with any sort of halo.

I made around 30 shoeboxes with my haloed culture. About 50% contaminated before or during first flush. Maybe 10% had a second flush. Once I put the contaminated tubs outside, most shook it off and came back to life.


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InvisibleRoger Clemency
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Re: Separating mycelium from contams fine points [Re: LadysKnight] * 1
    #26685000 - 05/21/20 07:08 AM (3 years, 8 months ago)

It depends on your agar recipe and the culture. Those first pics way up seem fine to me. There is almost always going to be a visible halo of really thin myc. It’s the exploratory hyphae checking out the area before committing more resources to growing there.

That’s not to say you don’t have something else on those plates but the halo is not the indicator. Sometimes though you will notice chaotic myc in the halo area. I don’t have a pic right now but when looking close you’ll see nice uniform myc, with a uniform but thin halo out in front and then in one area all the sudden the filaments start crisscrossing and looking like scribbled spiderweb. That’s the kind of stuff that makes me nervous. Next time I will transfer just that chaotic shadow and see if it’s mold or possibly just the myc reacting to something else I can’t see.

In that more recent picture with the rhizo growth the right side is fine, you can see that it’s all the same pattern as the thicker myc but that left area does indeed look sketch to me.


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Offlinepoisoned
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Re: Separating mycelium from contams fine points [Re: LadysKnight]
    #26685023 - 05/21/20 07:20 AM (3 years, 8 months ago)

Halos are not necessarily a mold. They're almost guaranteed to happen on germination plates and early transfers.

If you want to test if halo is myc or mold, take transfer from a halo.


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