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Contamination issues - looking for assistance and guidance.


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#21 sandman

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Posted 26 June 2020 - 12:13 PM

Just wait to fruit the cakes then cut a piece of the inner tissue of your best fruits on agar (cloning) and throw all of these plate away. It would be worthless against the nice clone that you will have in a few weeks anyway.


Edited by sandman, 26 June 2020 - 12:14 PM.


#22 2Ape2

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Posted 26 June 2020 - 07:32 PM

None of the agar growth is good mushroom mycylium that will lead to a clean transfer, I'm really sorry to inform you that is all contamination. There is nothing there. Stop stop he's already dead :( 

 

I think the actual agar itself may be bacterial based on the look and the other jars that were shot. It has that really opaque and  kinda grainy look. Is that just the pics? It may have been incorrectly sterilized or gotten bacterial during the pour or cool down. Usually agar is very "crisp" and transparent. The entire plate even the uncolonized parts look grey and sad like a russian alley or something. 

 

 

No reason to be sorry, I'm here to learn.   I admit, I have a lot of things in motion here and it is all brand new to me.  Straight forward feedback is appreciated.

 

So, the grayish hue is more due to the haze on the inside of the plate resulting from being removed from the 80 deg. spot and into a room that was about 12 degrees cooler.  All the plates did that pretty quickly.  Here are a couple of more pictures I just took without the temperature change.  You can see the actual color and clarity.  My recipe was 500 ml water, 7 g agar-agar, 5 g LME (dry), and 5 g potato flakes.  The starches from the potato flakes contributed to a cloudiness in the final product.  I PC'd at 15 psi for 40 min and poured as soon as I was able to handle it without burning my hands.  I did so in my SAB.  Fresh out of the PC it wasn't crystal clear.  It is still completely possible there are wholes in my technique resulting in contamination.  It doesn't look systemic to me but, again, I'm still new to this.  All that being said, the results started off strictly bacterial and whatever the white fuzz is didn't start until I was able to stabilize the temps 79-80F.  

 

I'm glad I made the decision to give the BRF jars a shot.  My intention was to hedge the bets and so far they are looking good.  They are pint jars I've been told to watch for them to stall.  They were PC'd for 90 minutes.  All I need are a few fruits and I will take an entirely different direction and chuck these plates.  

 

So you really think that mycelium is mold or something else?  

 

 

104933026_284052682649379_8353928499217142150_n.jpg 105708896_266221204485412_5814933327111641495_n.jpg

 


Edited by 2Ape2, 26 June 2020 - 07:38 PM.


#23 sandman

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Posted 26 June 2020 - 08:52 PM

Oh its potato agar that explains the look

 

The pics are a little blurry so that may be why it looks off to me. Try to really steady your hand when you snap the shots. Can you get a closer and better focused picture of the plates? I'm about 98% sure those plates are either contam infused mushroom mycelium or just straight contam mycelium. 


Edited by sandman, 26 June 2020 - 08:52 PM.


#24 sandman

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Posted 26 June 2020 - 08:56 PM

when your leading edge has no definition and its a big cloud that is an indication that it aint what you want. Clean cubensis mycelium will typically be fairly thick and bright with a defined edge even when tomentose. It may just be the blurry pic I dunno. Pic 1 looks like it has a better chance of having some actual cubensis on it but dish 2 I really don't think so. But dish 1 at the bottom/right has that really cloudy fading out undefined myclium that looks real bad.



#25 sandman

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Posted 27 June 2020 - 07:24 AM

Honestly I have to say that pouring agar is not the strong suit of a SAB.

 

There are many that do this, but if that's all I had I would be doing no-pour agar in some kind of small tupperwares or half pint wide mouths or some such with a filtered lid on them.

 

I sure wouldn't want to pour in a glovebox either that's for sure. You would have such a hard time picking plate lids and stacks up with those gloves lmao. 



#26 2Ape2

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Posted 27 June 2020 - 08:05 AM

I really appreciate the feedback.  I'll snap some pictures later today and get them up.  



#27 2Ape2

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Posted 27 June 2020 - 11:41 AM

Well, taking a closer look I'm pretty certain you're right.  Unless you think this is just all tomentose growth it is way to cottony to be what I should be looking for.  Here are some pics.  Man I was hoping!  LOL  So, I will likely just toss these and wait for some fruits to get some prints and to make clones.  This has been a great learning opportunity.  

 

75253009_2641376922787116_8236354112134563121_n.jpg 104809814_683414388881282_2966320930635831546_n.jpg

 

I'm guessing that this is, at best, target species attempting to grow through the contamination.  Still not a good, either way.  

 

105414588_845234269339266_2872269571647610036_n.jpg

 

 


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#28 sandman

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Posted 28 June 2020 - 05:29 AM

Hmm those pics are much better. It looks like it could be tomentose. That one little rhyzo does let you know there is the right stuff hiding somewhere at least. 

 

Don't wait for fruits to make PRINTS. Either wait for tiny pins and just pluck a pinhead off and literally lay on agar. Or wait for bigger fruits and rip open a big fruit and cut inner sterile tissue and lay that on agar. That is cloning. If you wait for the bigger fruit you can have a better idea of what the culture will behave like.

 

I mean do wait for fruits to make prints but that's just so you can have some prints for backups and trades etc. Not for cloning.


Edited by sandman, 28 June 2020 - 05:31 AM.


#29 2Ape2

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Posted 28 June 2020 - 08:47 AM

Thank you for the insight.  At this point I'm going to let the dish run its course and see what happens.  I see what you're saying with the clone and I will do that, both methods.  I'd been planning on grabbing a couple of spore prints as well.  



#30 2Ape2

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Posted 14 July 2020 - 08:11 PM

Things are actually looking really good.  Of the 6 T3 plates, 5 are tomentose and one is rhizomorphic.  If nothing else it is just fascinating.  Also, I made some more agar for the next set of transfers and to get ready to make some clones off my BRF cakes.

 

T3 that is rhizomorphic came from this spot on a T2 dish.  

Rhizome 3.png

 

Pics of the T3 rhizo growth -  blue as I took them through the lid of the plate/dish.

rhizome 2.png rhizome 1.png

 

 

Working on my agar game too - these look much better.  Just a basic MEA recipe.

plate.png






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