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Invisiblekrock
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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
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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
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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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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
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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


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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:


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InvisibleLadysKnight
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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
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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
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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
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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
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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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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
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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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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
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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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InvisiblePastywhyteMDiscord
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26792888 - 06/29/20 06:25 PM (3 years, 6 months ago)

I’m dealing with a yeasty vendor syringe myself. Yeast is growing pretty slow though so I’m thinking it will be pretty easy to grab some clean myc in a day or two.


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26794607 - 06/30/20 01:44 PM (3 years, 6 months ago)

Quote:

I’m dealing with a yeasty vendor syringe myself. Yeast is growing pretty slow though so I’m thinking it will be pretty easy to grab some clean myc in a day or two.




Great! Please share some pictures of your agar, would love compare the initial growth to what I got on my plates.
What I experienced with the yeast on my plates is that it initially grows very quickly and then just stops growing. It does seem that it really inhibits germination when close by spores.


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26794642 - 06/30/20 02:05 PM (3 years, 6 months ago)

Agar update!

So I am pretty sure the plate I thought to be mycelium is in actuality mold :puke:

1st observation)
It is 3 days since I transferred this growth from A4 plate and it grows way too quickly. The three pieces have now covered the whole plate:


2nd observation) In the following picture.
To the right: A 7 month old plate of king oyster mycelium (healthy culture that I have fruited).
At the bottom: The growth that I now believe to be mold. It is grey compared to the king oyster mycelium.
At the top: The bacteria infused growth that I now believe to be mycelium.


It's sad that I am most likely growing mold here.
However this made me shift my focus to my other transfers. They grow much slower and also the hyphae seem whiter and more condensed, less airborne and seems to grow in circular sectors. Even when they are growing together with bacteria.



Also I found another plate, A1 to be exhibiting the same kind of growth as those I transferred. But much cleaner looking. Doesn't seem like it has bacteria. Has many circular sectors.


So yeah I am doing more transfers today. But I will stop propagating what I now believe to be mold.


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OfflineVernsMB
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26794677 - 06/30/20 02:24 PM (3 years, 6 months ago)

if you use a stainless straw you can make nice small circular transfers.  Place agar inverted so mycellium is sandwiched and if you have healthy mycellium it will quickly out grow contaims onto clean agar.  Transfer again and then you should see contaim free cultures, assuming no airborne critters make it into your petris.

I made mistake of leaving window open morning before I did a large volume of transfers.  Even with SAB and misting with Alcohol I managed to pickup bacteria/yeast in many of my petris...Starting to suspect it was from supplier as infections appear where mycellium liquid was injected.


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OfflineC12ShroomMan
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26794779 - 06/30/20 02:58 PM (3 years, 6 months ago)

Quote:

krock said:
Agar update!

So I am pretty sure the plate I thought to be mycelium is in actuality mold :puke:

1st observation)
It is 3 days since I transferred this growth from A4 plate and it grows way too quickly. The three pieces have now covered the whole plate:






I wouldn't call that mold until the spores show up in the middle.
All my mycelium has grown faster than mold.
1 transfer per plate would of made this easier :smile:


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InvisiblePastywhyteMDiscord
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: C12ShroomMan]
    #26794818 - 06/30/20 03:16 PM (3 years, 6 months ago)

I’m not convinced that first plate is mold. However if it is cubes they are weak and lack vigor.


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26794904 - 06/30/20 04:04 PM (3 years, 6 months ago)

Haha, now I doubt myself again.

I just came back from transferring pieces from the different smaller growth areas. Used a small piece for each plate.

I didn't transfer anything from the "mold" plate. I will keep it around and see if it pins. Maybe it is in fact not mold. I hope so. Because it's certainly much faster than the other myc-like grow areas.


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26796337 - 07/01/20 06:33 AM (3 years, 6 months ago)

Update on the first transfers


A4T1 Day 4:



B2T1 (small growth transfer) Day 5:



B2T1 (big growth transfer) Day 5:



I still think the B2 transfers look more like mycelium than the A4 transfers. I transferred a piece from the A4T1 plate to a new plate today so we can inspect the growth more closely in a few days.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock] * 1
    #26796786 - 07/01/20 11:02 AM (3 years, 6 months ago)

You are letting the plates grow too much before transferring, in the phase you're at its good to be ready to transfer asap before other things than cube (if it is cube) get the chance to develop. Transfer as soon as you have enough to do so. Keep the original plates if possible so you may know if its mold (if it changes color etc)


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26797035 - 07/01/20 01:35 PM (3 years, 6 months ago)

Quote:

You are letting the plates grow too much before transferring



You're right. I let the A4T1 plate grow too much. Mostly because I was unsure if it is mold or not.
For the B2T1 plates I couldn't transfer them before yesterday as the hyphae growing past the bacteria patch was not big enough to cut at my current skill level. I remember the suggestion to use hot pour or sandwich method, but seeing as the hyphae started outgrowing the bacteria I think it was better to wait a bit.

Quote:

Transfer as soon as you have enough to do so. Keep the original plates if possible so you may know if its mold



I didn't think about that! It makes perfect sense to inspect the parent plate for changes instead of the newly transferred one.

Again thank you for the advice, it helps a lot!


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InvisiblePastywhyteMDiscord
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26797137 - 07/01/20 02:19 PM (3 years, 6 months ago)

Not the best pic but you can see the mycelium popping up in the centre of the yeast colony. I’m going to try and grab a piece of it. This yeast is really slow growing in these temps so the cubes should able to be isolated.



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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26797169 - 07/01/20 02:34 PM (3 years, 6 months ago)

Thank you for posting your yeasty agar! How did you apply the spore solution? Streak or drop?

I find it interesting that the mycelium is able to grow from the center of the colonies on your plate.

On my plates, the growth that I assume to be mycelium is only germinating when it is spread far enough apart from a yeast colony.
Although my solution is also pretty old, which might explain the low germ rate as well.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26797369 - 07/01/20 04:12 PM (3 years, 6 months ago)

Most yeasts are pretty weak compared to cubes. Doesn’t really surprise me to be honest. I just dropped right off the syringe. No swab.


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26798567 - 07/02/20 05:26 AM (3 years, 6 months ago)

Agar update

I've taken pictures of all the plates and their transfers. I see that a lot of the new transfers still have bacteria so I am most likely going to attempt a hot pour soon.


    B2

    Streak plate, day 9:


    T1 plates day 6:


    T2 plates day 2:








    A4

    Streak plate, day 9:


    T1 day 6:


    T2 day 1:





    A1

    Streak plate, day 9:


    T1 day 2:




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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26801159 - 07/03/20 09:52 AM (3 years, 6 months ago)

My agar is just finished cooking in the PC.  Now I am waiting for it to cool down enough to pour.

I will hot pour all the T2 plates in probably 15 min. And also make at least 10 more plates for future transfers.

This is what the T2 plates look like right now:



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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26801297 - 07/03/20 11:22 AM (3 years, 6 months ago)

I'm really impressed-- and amazed you got something out of such a shitty storage situation.

:cheers:


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Invisiblekrock
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26801320 - 07/03/20 11:36 AM (3 years, 6 months ago)

Quote:

I'm really impressed-- and amazed you got something out of such a shitty storage situation.
:cheers:



Thanks man! I just came back from pouring and wrapping 30 plates as well as hot pouring the T2 plates.
Heres hoping for that the myc is cubensis :cheers:


Edited by krock (07/03/20 11:37 AM)


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26802636 - 07/04/20 01:34 AM (3 years, 6 months ago)

I like these! But again I urge you to not wait too long to take T3, and take as small transfers as possible. Hmm one question, why isn't your agar clear? Whats your recipe?


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26802675 - 07/04/20 01:57 AM (3 years, 6 months ago)

Quote:

Pastywhyte said:
Not the best pic but you can see the mycelium popping up in the centre of the yeast colony. I’m going to try and grab a piece of it. This yeast is really slow growing in these temps so the cubes should able to be isolated.






What temp slows bacteria? Hot or cold? Isn't it summer for you? Where u keep your plates?


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Offlineredhandmat
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: 3some]
    #26802693 - 07/04/20 02:14 AM (3 years, 6 months ago)

Quote:

3some said:
Quote:

Pastywhyte said:
Not the best pic but you can see the mycelium popping up in the centre of the yeast colony. I’m going to try and grab a piece of it. This yeast is really slow growing in these temps so the cubes should able to be isolated.






What temp slows bacteria? Hot or cold? Isn't it summer for you? Where u keep your plates?




Cold slows bacteria, and cold enough slows everything down.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26802816 - 07/04/20 05:08 AM (3 years, 6 months ago)

Wow, I just woke up and all of the T2 plates already broke through the hot pour layer!
I will transfer them ASAP.



Quote:

why isn't your agar clear? Whats your recipe?



My agar is clear. It is just photographed on a black background. Recipe: 10g Malt extract 10g agar agar 500 ml water. Very standard MEA.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26802959 - 07/04/20 07:32 AM (3 years, 6 months ago)

Ah I guess it was condensation then. IMO Its looking good mate. Looks like you probably have some myc. The step is now to get a couple of clean looking plates aimed for grains :smile:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26803299 - 07/04/20 10:23 AM (3 years, 6 months ago)

Sweet! I really hope they are the mycelium I am looking for :laugh:


I was thinking of making 5 brf pucks in half pint jars so that I could do a test run from the T3 plates. Do you think it's easier to test them on grain?

Then take a print from whatever fruits to restart the gene pool before putting it in a shoebox?

I am unsure if the genetics I have currently are any good, would this be a good idea? Or should I just try growing what I have?

I could also clone one of the fruits and use that as a base?


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26803337 - 07/04/20 10:46 AM (3 years, 6 months ago)

Quote:

krock said:
Sweet! I really hope they are the mycelium I am looking for :laugh:


I was thinking of making 5 brf pucks in half pint jars so that I could do a test run from the T3 plates. Do you think it's easier to test them on grain?

Then take a print from whatever fruits to restart the gene pool before putting it in a shoebox?

I am unsure if the genetics I have currently are any good, would this be a good idea? Or should I just try growing what I have?

I could also clone one of the fruits and use that as a base?




Fo BRF you would need to wait longer and how are you doing to inoculate BRF with agar? (need syringe no?)

As long as you "save" a clean plate you dont have to worry about not doing a shoebox. You have many options once you get a few clean plates. You could take it to grains (depending on the amount of agar, temp, cleanliness) and have fully colonized jars within 2 weeks (I just had jars finish by 8 days). And then just do a few separate boxes and you'll get shrooms to clone, to print, and to eat :wink:

You could also top fruit your grains if you want quick shrooms and your jars have some contams still. But either way, get yourself the nice and clean plates first.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26803451 - 07/04/20 11:52 AM (3 years, 6 months ago)

I thought I could inoculate BRF with agar, the same way as with grains. I searched a bit and someone suggest squirting a small agar sample with a sterile water syringe to BRF.

But I see your point. BRF is probably slower. I think doing grains sounds like a good idea once my plates are very clean! I have all the necessary supplies. Thanks for the input :smile:


I'm really looking forward to testing the cultures :laugh:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26803478 - 07/04/20 12:06 PM (3 years, 6 months ago)

Looks like the myc on my yeasty spore inoculated plates have over come the yeast and will be in clean territory, enough to get a transfer. I’m gonna transfer soon, will post a pic. Most yeasts are pretty weak and stall in cool temps. Should have some plates ready by T3. Was cool in some spots it looked like the mycelium chose to grown through the agar under the yeast. Then shot up at the edge of the colony. Then helped more mycelium leap the yeast. I wish I took more pics but I’ve been busy.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26803492 - 07/04/20 12:17 PM (3 years, 6 months ago)

Quote:

Pastywhyte said:
Looks like the myc on my yeasty spore inoculated plates have over come the yeast and will be in clean territory, enough to get a transfer. I’m gonna transfer soon, will post a pic. Most yeasts are pretty weak and stall in cool temps. Should have some plates ready by T3. Was cool in some spots it looked like the mycelium chose to grown through the agar under the yeast. Then shot up at the edge of the colony. Then helped more mycelium leap the yeast. I wish I took more pics but I’ve been busy.




Wow Pasty that sounds pretty awesome actually!


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26803681 - 07/04/20 02:18 PM (3 years, 6 months ago)

Quote:

Looks like the myc on my yeasty spore inoculated plates have over come the yeast and will be in clean territory, enough to get a transfer. I’m gonna transfer soon, will post a pic. Most yeasts are pretty weak and stall in cool temps. Should have some plates ready by T3. Was cool in some spots it looked like the mycelium chose to grown through the agar under the yeast. Then shot up at the edge of the colony. Then helped more mycelium leap the yeast. I wish I took more pics but I’ve been busy.



That sounds really cool! Please post pics when you can :laugh:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26803773 - 07/04/20 03:10 PM (3 years, 6 months ago)

Here’s the best I can do without firing up the flowhood. Some of the colonies were totally overtaken. Now this yeast isn’t dead, just trapped enough so I can make a transfer.



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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26805664 - 07/05/20 05:21 PM (3 years, 6 months ago)

On the subject of mycelium overcoming yeast;
I just noticed that a few of my initial streak plates are exhibiting what looks like mycelium from some of the yeast colonies. Almost like the cube spores germinated inside the yeast colonies.
Quite interesting to look at. Does this resemble the initial growth you saw, Pasty?




Here is an update on my T3 transfers I did yesterday from the hot poured T2 plates. I scraped off a thin layer of agar that had surface growth on it.

I had to photograph the plates with back light since the growth is very transparent at the moment (is this something to be concerned of?).

With black background the growth is almost invisible right now:


Here the plates are backlit:


Growth seems cleaner for most plates. Though B2T3X and B2T3Y still look a bit bacterial, not really sure yet.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26806705 - 07/06/20 11:02 AM (3 years, 6 months ago)

Another agar update. T3's have started to be a bit more visible now. Also the bacteria contamination on the B2T3X and B2T3Y has become more obvious now. I guess I need to hot pour them a couple more times before they are clean.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26810275 - 07/08/20 04:12 AM (3 years, 6 months ago)

yo, it is day 4 of my T3 plates and here they are. I think B2T3X2 and B2T3Y2 are starting to look a lot like proper mycelium. What do you guys think?



I'm going to transfer all the T3's to new plates today because I going away for a few days soon. Bigger chance of clean plates if I do another transfer right? I will try out my newly acquired metal straw tool that I bent for better sterility: maybe I can get some nice looking transfers :smile:

Unfortunately I don't think I will be able to do a hot pour today, so that'll be when I'm back again.
Also when I come back again I will prep some grain to test grow the non-bacterial plates.

Also here is an update on the T2 plates (day 8):

Looks like the B2T2X2 and B2T2Y2 are going tomentose? Not sure why, but seems promising in regards to having proper mycelium.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26810435 - 07/08/20 06:33 AM (3 years, 6 months ago)

Looks like your diligence has paid off!!

:vibin:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26811198 - 07/08/20 03:35 PM (3 years, 6 months ago)

Dude those T3s look really good. Well done mate. I dont think you really need to keep on transferring, but you have nothing to lose by doing it. Probably will have more rhizo (good) growth with subsequent transfers.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26811308 - 07/08/20 04:41 PM (3 years, 6 months ago)

My T1 transfers look clean and leaping off great. Yeast can suck it.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26820609 - 07/13/20 07:44 AM (3 years, 6 months ago)

Quote:

Looks like your diligence has paid off!!



Quote:

Dude those T3s look really good. Well done mate. I dont think you really need to keep on transferring, but you have nothing to lose by doing it. Probably will have more rhizo (good) growth with subsequent transfers.




Thank you guys!!

I just put 7 half pint jars with oat grain into the PC. Excited to test out the plates I have :laugh:


Quote:

My T1 transfers look clean and leaping off great. Yeast can suck it.



Cool, pasty! I had some of the streak plates that got some kind of similar growth to what you described earlier where myc grows from the colony.
does this look similar to you?


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26820663 - 07/13/20 08:52 AM (3 years, 6 months ago)

No those look like just yeast. In mine very obvious rhizomorphs sprung up out of the centre of the colony and reached towards the colony that grew under the yeast and surfaced outside the perimeter. Together they bridged the yeast and trapped it allowing me to get a clean sample from the leading edge.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26820767 - 07/13/20 10:07 AM (3 years, 6 months ago)

Quote:

Pastywhyte said:
No those look like just yeast. In mine very obvious rhizomorphs sprung up out of the centre of the colony and reached towards the colony that grew under the yeast and surfaced outside the perimeter. Together they bridged the yeast and trapped it allowing me to get a clean sample from the leading edge.




They were waging war on eachother. I guess with your help the myc won out :laugh:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26835702 - 07/21/20 08:58 AM (3 years, 6 months ago)

Heya people!
Grain jars have been growing for 1 week now. I think the growth looks a bit weird, any comments on these jars?

A4T4: B2T3X2: B2T3Y2:

For comparison, here is a jar that is not inoculated with anything:



Also the yeast plates I showed earlier have grown more myc kind of like things (is this really how yeast grows?)


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26846343 - 07/26/20 03:35 PM (3 years, 6 months ago)

Update, day 12 half pint jars




I gave the jars a smell before spawning them.

B2T3X2; I shook the jar 5 days ago. The myc didn't really grow much afterwards. When opened, it smelled yeasty. Which is kind of weird since I haven't seen any yeast on the agar plates since the first few transfers. The grains did not stick to each other either.

B2T3Y2; I didn't shake this jar and it is now 100%. When opened smelled slightly yeasty. It came out as a compact grain cake (as you would expect from colonized grain jar).

A4T4; It's hard to tell whether this jar is fully colonized or not because of it's grey mycelium. However it looked like each individual grain had some on it. When opened, the jar smells like wild mushrooms picked in the forest. I take that as a good sign.



I decided to spawn all of the jars to their own shoeboxes today with coir.
I found some cool 1.5 liter totes at the hardware store, perfect size for a half pint jar of grain.
I'm following the SFF shoebox assembly TEK



Fingers crossed that at least one of the shoeboxes produces fruits!


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26846373 - 07/26/20 03:51 PM (3 years, 6 months ago)

Have a bad feeling about your prospects based on what you've said above bud-- none of them sound clean, and I guess you'll know pretty quick if they contammed out.
:goodluck:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26846375 - 07/26/20 03:52 PM (3 years, 6 months ago)

Looking good, how much were those totes?
look ideal height but the shorter ones @ 1$ piece is hard to beat


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: C12ShroomMan]
    #26847128 - 07/27/20 04:25 AM (3 years, 5 months ago)

yeah I am also concerned about the cultures, I really hope this works out!


The totes aren't as cheap as you're probably used to. I got all three of them for a total of 12 usd (converted from my own currency).
I would love to buy totes at 1$ a piece!

The cheapest totes I can get around here are 5 liter ones that only have transparent lid, rest of the tote is opaque. Cost 2.75$.


Edited by krock (07/27/20 04:57 AM)


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26847562 - 07/27/20 10:13 AM (3 years, 5 months ago)

I like the SFF tek too! Fingers crossed mate.

The jars look a liiitle bit weird to me, but if you get at least one fruit then clone it and restart from there.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26847568 - 07/27/20 10:15 AM (3 years, 5 months ago)

Quote:

krock said:
yeah I am also concerned about the cultures, I really hope this works out!


The totes aren't as cheap as you're probably used to. I got all three of them for a total of 12 usd (converted from my own currency).
I would love to buy totes at 1$ a piece!

The cheapest totes I can get around here are 5 liter ones that only have transparent lid, rest of the tote is opaque. Cost 2.75$.




Any IKEA near you? IMO the best.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26848302 - 07/27/20 04:15 PM (3 years, 5 months ago)

Wouldn't taking a print from the fruit to restart the gene pool be a better option than cloning the fruit?

Ikea is a bit of a pain to get to because I don't have a car. I go there maybe once a year by bus, lol. Do you know any ikea-totes that are good and cheap?


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26848324 - 07/27/20 04:28 PM (3 years, 5 months ago)

1) you should ALWAYS get prints of a successful grow, just to make sure you always have resources.
But,
2) you clone good fruits because they've already run the gauntlet of natural selection and won-- they're proven winners. You get that on agar to easily replicate strong, proven mycelium.

Spore prints give you millions of combinations that may or may not be awesome. Nothing wrong with them at all! But clones give you a guarantee that the mycelium has what it takes to go the distance without having to sift through thousands of colonies in a MS culture.


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Edited by coAsTal (07/27/20 04:28 PM)


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26848420 - 07/27/20 05:28 PM (3 years, 5 months ago)

I agree it makes sense to do both a spore print and clone it! I will do both if I actually get some fruits :laugh:

When I wrote about restarting would be better, I was thinking more about what makes sense in terms of what is best for the future. The collection of strains that I have right now in the culture, even if they are able to fruit, are probably kind of shitty because of their origin. These strains just happened to be lucky enough to survive among the contamination. I also don't know how many strains are in there, it could be that there are very few strains and thus very little diversity. My reasoning is that restarting the gene pool with a fresh spore print will give me a better chance of finding a really good culture for future use.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26849231 - 07/28/20 03:53 AM (3 years, 5 months ago)

Quote:

krock said:
I agree it makes sense to do both a spore print and clone it! I will do both if I actually get some fruits :laugh:

When I wrote about restarting would be better, I was thinking more about what makes sense in terms of what is best for the future. The collection of strains that I have right now in the culture, even if they are able to fruit, are probably kind of shitty because of their origin. These strains just happened to be lucky enough to survive among the contamination. I also don't know how many strains are in there, it could be that there are very few strains and thus very little diversity. My reasoning is that restarting the gene pool with a fresh spore print will give me a better chance of finding a really good culture for future use.




Exactly bruv, you can do both. Or just the print. You are right on both counts. As far as cloning goes, most growers here would say go for the largest fruit in a cluster.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26858360 - 08/02/20 08:13 AM (3 years, 5 months ago)

It's time for a small update!


Day 7 shoeboxes



B2T3X2; I am surprised this tote is colonizing the fastest. It was from the jar that I shook and then it didn't grow much. But look at it now colonizing the coir.

B2T3Y2; From the jar that was 100% colonized and had grain cake consistency. It is a bit behind B2T3X2 for some reason.

A4T4; Don't know what to say... It has grey mycelium so it is really hard to see if it is colonizing the coir or not...


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26858419 - 08/02/20 08:49 AM (3 years, 5 months ago)

Im still not sure about these. The fact that one of the jar didn't recover. And one looked like it never colonized to start with right? But only time will tell.

Hang in there.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat] * 1
    #26858628 - 08/02/20 11:07 AM (3 years, 5 months ago)

Those all look doomed to me.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26859989 - 08/03/20 02:52 AM (3 years, 5 months ago)

I agree it doesn't look good. I'll let it ride out, maybe I am lucky and get something.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26872763 - 08/10/20 06:18 AM (3 years, 5 months ago)

Update time! Day 15 totes

The top two totes seem to have some kind of black metabolites?







All the totes have begun breaching the casing. When opened all of the totes smelled like coir.

B2T3X2: The casing got a bit wet because there was condensation on the lid that dropped down when I opened up to take the picture.

B2T3Y2: Surface growth actually looks a bit promising.

A4T4: Seems to have come along the most. Surface growth looks whiter than side growth. But growth is kinda loose, mold like?


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock] * 1
    #26872881 - 08/10/20 08:31 AM (3 years, 5 months ago)

All those totes are full of mold. Get them out of the house ASAP. I highly doubt there is any living mushroom mycelium in there at all.


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte]
    #26872890 - 08/10/20 08:39 AM (3 years, 5 months ago)

Yeah dude--
:hugitout:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal]
    #26873504 - 08/10/20 02:43 PM (3 years, 5 months ago)

FUCK!!

Well at least I tried my best. I feel like I've learned a lot from this experience, I've become better at pouring plates, doing small transfers and also I was able to try out new techniques like proper streaking and hot pour.

With this post I will officially end my efforts in cleaning up that liquid and I will discard the last 1 ml. This has been a wild ride that started out with my first shroomery post 7 months, 18 days ago.
However I am not going to give up on growing cubes. I will acquire a legit print and grow it good, like I should.

I want to thank all of you who have stayed by my side along the way and I will reward you all with 5 shrooms! Thank you! :cheers:


Edited by krock (08/10/20 03:05 PM)


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: krock]
    #26873516 - 08/10/20 02:52 PM (3 years, 5 months ago)

Quote:

krock said:
However I am not going to give up on growing cubes. I will acquire a legit print and grow it good, like I should.

I want to thank all of you who have stayed by my side along the way and I will reward you all with 5 shrooms! Thank you! :cheers:




:yess:


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Re: 4th attempt at cleaning up my yeasty spore solution [Re: coAsTal] * 1
    #26873519 - 08/10/20 02:55 PM (3 years, 5 months ago)

Kudos to the OP for sticking with things and learning from the experience. Rated 5 :mushroom2:


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Offlineredhandmat
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Pastywhyte] * 1
    #26873528 - 08/10/20 03:02 PM (3 years, 5 months ago)

I too rated you OP. Btw, I think you should head to the trade forum and see if there are giveaways. Im sure you will find someone to send you prints within the week (may take longer to receive them). You're a nice guys, and gave it your best: sure someone will help!


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OfflineKyngwhatt
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: redhandmat]
    #26873547 - 08/10/20 03:13 PM (3 years, 5 months ago)

Ok i Have a few contaminated syringes and thought if i made a liquid culture there would be a mass of myc in the jar i could suck into a syringe and put on agar. Thoughts??

and what was the verdict of the experiment that was cobweb the whole time?


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Whatt


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InvisiblecoAsTal
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Re: 4th attempt at cleaning up my yeasty spore solution [Re: Kyngwhatt]
    #26873552 - 08/10/20 03:15 PM (3 years, 5 months ago)

Quote:

Kyngwhatt said:
Ok i Have a few contaminated syringes and thought if i made a liquid culture there would be a mass of myc in the jar i could suck into a syringe and put on agar. Thoughts??




If you have contaminated syringes, you put the syringe liquid to plate agar to isolate good mycelium away from the contamination via transfers-- THEN and only then do you use LC made with the good, clean mycelium.


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