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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
4th attempt at cleaning up my yeasty spore solution
    #26781924 - 06/25/20 11:50 AM (3 years, 7 months ago)

I somehow managed to acquire a AM+ cubensis spore solution from Netherlands and brought it home to where I live. However I've had a lot of problems with it and zero germination yet.

1st attempt I used a normal MEA agar recipe: 10g malt extract, 10g agaragar and 500 ml water. And streaked two plates with PC'd bamboo skewers. 5 months later they got trichoderma that had found it's way from the outside.

2nd attemt I made some BRF pucks. But I used way too much water. There is some kind of growth on these that vaguely looked like mycelium, but not sure if it useful. Also one of the jars has contaminated recently.

3rd attempt I made classic BRF cakes. But there was no germination on these. Except one of the jars got triched, but the rest of my BRF jars are still without any growth even after 2 months time.

You can see my previous attempts with pictures in this thread: https://www.shroomery.org/forums/showflat.php/Number/26389054


This is my 4th attempt.

For my 4th attempt I went all in. I made 15 MEA plates.
This time I made a softer agar. 10 g Malt extract, 8.5 g agar agar and 500 ml water.

My plates are marked as "Shiitake" to avoid any trouble with LE.

So three days ago I went all in and inoculated 15 plates of agar. Most of the plates are streaked with flame sterilization in between streaks. Like I learned from c10's excellent agar guide.







The last plates here are experimental plates. On some of them I just dropped a few drops of spore solution and splooshed it around in there to see if it'd make any difference.


So as you can see. There is a lot of yeast growing on my plates.



Only one of my plates has anything else than yeast growing on it. That is a tiny speck of trich that has germinated in the middle. So maybe there is hope that some of the spores also might germinate.



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InvisiblecoAsTal
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26781944 - 06/25/20 12:05 PM (3 years, 7 months ago)

I'm not the most experienced agar user, but I'm not seeing any mycelium on those plates anywhere-- I think you'd be far better throwing all that away and starting over with a print from somewhere- are you using a SAB? Look in the marketplace for pointers on getting a print.

I would expect (from your prior thread) that it's possible that the bottle of alcohol you put the syringe liquid in was filthy with contamination, so yeah, it was likely ruined before you even started unfortunately.


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OfflineC12ShroomMan
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26782005 - 06/25/20 12:32 PM (3 years, 7 months ago)

Quote:



My plates are marked as "Shiitake" to avoid any trouble with LE.






lol, GOT EM!

Not looking good, you should see myc at the same time. looks like contams already own those plates.

I started with spore syringes too and they suck for this.
I got a print, looped, and saw good growth within a day


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26782106 - 06/25/20 01:13 PM (3 years, 7 months ago)

The customs in my country are hardcore. That's why I am still trying to salvage this. I'll give the plates another 14 days. Maybe I am lucky and I get some mycelium.

I don't have enough posts to enter the marketplace yet. So I am trying to get a bit more active here. Hopefully I'll find someone who can send me a print domestically so I don't need to worry about customs. If not I will order a paper print for extra stealth and hope for the best.

This is my last attempt on this spore solution (Probably). 

I use a SAB and am pretty confident in my sterile technique, it's like you say the bottle probably was contaminated as fuck. I wish I could go back in time and smack myself. At the time my knowledge was very limited and I thought the 40% alcohol would help sanitize the bottle, but I was wrong.


Edited by krock (07/02/20 01:43 PM)


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26783646 - 06/26/20 04:35 AM (3 years, 6 months ago)

I am pretty sure the B2 plate has mycelium!!




So there are three growth areas besides the obvious yeast infection (smells like yeast).

Top left kind of looks like mycelium.

Middle right looks very much like tomentose mycelium. It's easier to see on the other pictures.

Bottom left seems like some nasty black mold.



So the big patch on the right looks very much like fluffy mycelium to me.




What do you guys think? Could this be it?


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Offlineredhandmat
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Registered: 05/09/19
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26783724 - 06/26/20 05:40 AM (3 years, 6 months ago)

Yeah I think this could be it! Transfer all those growth you pointed out. As well as the third pic in the middle, and the first pc in the top-left. Those could also be some mold spores, I dont think so but they could. One thing for sure is that you also have bacterial growth in there.

But you have to transfer now before contams grow more. Just transfer everything, and be super clean about it. If you work inside a SAB and not a flow hood, then move your hands very gently. I would also advice you that if the transfers are successfull and you see some growth then use the sandwich or hotpour tek to sandwich the growth between two layers or agar. Once the myc breaks through the above layer then transfer it asap to a new plate. This should definitely increase your chances of cleaning this up.


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26783820 - 06/26/20 07:03 AM (3 years, 6 months ago)

Thank you for the tips! I will whip up some agar quickly as I didn't foresee results this quickly and thus did not prepare any blank plates.

The MEA plates I'm going to make now will be normal softness as I am pretty sure it's best for mycelium growth. So 250 ml water, 5 g malt extract and 5 g agar agar.

I'm hoping for the best!


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InvisiblecoAsTal
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26783863 - 06/26/20 07:30 AM (3 years, 6 months ago)

Quote:

redhandmat said:
But you have to transfer now before contams grow more.



:werd:
If you get good mycelium out of that batch I'll be very impressed-- that looks fishy


--------------------
I am certain of nothing but the holiness of the Heart's affections and the truth of Imagination--  John Keats

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Edited by coAsTal (06/26/20 07:42 AM)


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InvisiblecoAsTal
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26783876 - 06/26/20 07:40 AM (3 years, 6 months ago)

Also, you really should have extra plates of prepped agar on hand at all times, because you really never know when you're going to need/want to do a transfer, so why go through a multi-hour prep for new agar each time?
It's also nice (for a less experienced agar person like me) to let your new plates sit for a few days to ensure you have no contams.
I've been working hard on my SAB-fu and have had 3/25 plates show funk. 2 of those that showed were extras I made, so they never impacted my transfers :smile:
It's been very helpful to have agar ready that you know is 100% clean.


Also, should mention that you ought to never use that bamboo thing either-- make an inoculation loop from a paper clip or piece of wire, or use a blade, or tweezers, but that plant matter is absolutely not appropriate to use for agar work under any conditions.

:cheers:


--------------------
I am certain of nothing but the holiness of the Heart's affections and the truth of Imagination--  John Keats

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InvisibleLadysKnight
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Registered: 10/09/15
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26783886 - 06/26/20 07:47 AM (3 years, 6 months ago)

Quote:

krock said:
Thank you for the tips! I will whip up some agar quickly as I didn't foresee results this quickly and thus did not prepare any blank plates.

The MEA plates I'm going to make now will be normal softness as I am pretty sure it's best for mycelium growth. So 250 ml water, 5 g malt extract and 5 g agar agar.

I'm hoping for the best!




You got this!


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26783969 - 06/26/20 08:40 AM (3 years, 6 months ago)

Thank you all for the encouragement!
I just poured the agar. Now I am waiting for it to solidify.

Quote:

never use that bamboo thing 



Yep! It was a bad Idea and I don't do that anymore!
For the 15 plates I made earlier I streaked with a proper loop I made out of wire so that I could flame sterilize it between each streak.


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InvisiblecoAsTal
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26784015 - 06/26/20 09:02 AM (3 years, 6 months ago)

:goodluck:


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26787655 - 06/27/20 03:17 PM (3 years, 6 months ago)

The plates that I transferred to haven't done much yet. Very slight growth from the slices I cut. But still they are going at it atm. I will post pics of them when there is more to see.

However I just realized there is mycelium on a different plate also: A4!
The myc looks similar to the big patch that I transferred from B2. Which makes me think they are of similar origin; germinated Cubensis spores.

With backlight I am pretty sure those threads are hyphae.


Here is a closeup of the edge I took with a 10x lens I have:


And here is the plate with side light.


Same pic but with contrast edited (for easier view?) (at least the yeast looks pretty):


I'm going to transfer this growth today!

BTW: Does anyone know the chances that the mycelium I am seeing is Monokaryotic? Would it be a good idea to introduce the two different mycelium strains I have to each other?


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InvisibleThe Fresh PrintsS
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26787678 - 06/27/20 03:37 PM (3 years, 6 months ago)

That last pic is rad


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: The Fresh Prints]
    #26787775 - 06/27/20 04:41 PM (3 years, 6 months ago)

Quote:

That last pic is rad



Thanks! I also think it looks really nice.


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26792172 - 06/29/20 02:01 PM (3 years, 6 months ago)

Time for an update on the transfers!


This is from the big patch that was in the middle of B2, As you can see only one of the pieces dodged the yeast infection, but is growing rather slowly. Transferred about 3 days ago.



This is the smaller patch that was in the top left of B2. Not really sure if this is myc or not, but it is for certain bacterial. On the left it looks like some mycelium is outgrowing the bacteria. Transferred about 3 days ago.



Here is the plate I transferred 2 days ago from A4. Looks pretty clean to me. I think it still looks like mycelium.


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OfflineC12ShroomMan
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Registered: 01/19/20
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26792221 - 06/29/20 02:15 PM (3 years, 6 months ago)

Last plate looks good to me too. i would only do one per plate tho


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26792310 - 06/29/20 02:40 PM (3 years, 6 months ago)

Quote:

Last plate looks good to me too.



Cool! So you agree that it looks like mycelium as well? :smile:

Quote:

i would only do one per plate tho



On the next transfer I will put a single slice on the plates. But for T1 I wanted three pieces per plate so that I don't have to pour new plates all the time :P


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Offlineredhandmat
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Registered: 05/09/19
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26792356 - 06/29/20 03:01 PM (3 years, 6 months ago)

And try to take smaller pieces of agar. Dont know if its just my perception but those look a little larger than what you'd want when cleaning up culture (jst a tiny rice corn size). But that last plate may be something good. Im not 100% sure yet because the threads may be a little more disorganized than Im used to seeing but Im leaning toward myc definitely. Well done mate. You should take second transfers like now.


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Invisiblekrock
Krockodile

Registered: 12/18/19
Posts: 154
Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26792531 - 06/29/20 03:54 PM (3 years, 6 months ago)

Quote:

try to take smaller pieces of agar



Thanks for advice! I am trying to improve on taking smaller pieces, it's still hard for me. These pieces are about 4 times the size of a rice corn. So I need to work on that.
I read about D3's way of doing transfers with a metal tube so that the transfers are small and uniform. Looks like an interesting way of doing it.

Quote:

last plate may be something good



I sure hope so! I am taking 2nd transfers tomorrow.


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