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OfflineRotnpins
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Let's talk about Water Agar!! šŸ’§ * 31
    #27900393 - 08/12/22 10:03 AM (1 year, 5 months ago)

When I first started working with agar, I had an abundance of plates that I thought were too contaminated to clean up.. I thought about using antibiotics, but i had read that it could also hinder the mycelium growth and I didn't want to get stuck using it as a crutch.. So I decided to try making some water agar..

What is "water agar"?
Water agar is exactly what it sounds like, water and agar without any added nutrients.. the mycelium grows out very quickly (compared to the contams) looking for food.. this gives it a chance to out grow any contamination that may be riding along.

How to make water agar:
The recipe I use is 7g Agar powder per 500ml H2O

I leave the plates clear and add a different food coloring to each of my nutritional plates so I can easily distinguish between the type of plates.. (for example: H2O Agar=clear, MEA= blue, YMEA= yellow, etc,.)



You would prepare the water agar the same way as your nutritional agar plates.. the method I use is this:

1: add 100ml cold/room temp H20 to media bottle.
2: measure out remaining water (400ml for a 500ml batch) and heat water using preferred method. I personally bring it to a light boil on the stove.
3: while the water is heating, measure out your agar powder (7g per 500ml) and add it to your media bottles. Then give the bottle a little bit of a shake/stir.
4: once it is hot, add the remaining water to the media bottle. Put the lid on and shake it up.
5: prepare your PC with enough water for a 20 minute cycle.. put the water agar media bottle in the pc. If you have any nutritional agar, instruments that need sterilizing, no pour plates, etc,. you can throw them in the PC at the same time.
6: vent steam from PC for 10-15 minutes, add your weight, bring PC up to 15-17psi, reduce heat to low,, set a timer ā² and let cook for 20 minutes.
7: when PC cycle is done, turn the heat off and wait for pressure to drop to 0 on its own.
8: take your bottle(s) to your work space and wait for them to cool to pouring temp (I usually pour between 140-120 degrees)

How to use water agar:
Once your plates have cooled, it's time to do some transfers..

I make my transfers the same way I would when transferring to a nutrient plate.. I find an area that seems to have the most organized growth and I take a small transfer to the new plate.. smaller transfer wedges may be a little more difficult to work with at first, but they are less likely to carry contamination to the new plate. So try practicing taking smaller transfers each time until you're able to easily transfer a piece that is about the size of a grain of rice.

Wait for the mycelium to grow out.. it will be very thin and whispy, and it will grow out toward the edges much quicker than any contamination that tags along.

When the mycelium has grown out enough that I feel confident it has outgrown the contamination (its a good idea to let it at least half way to the edge, or all the way to the edge for more piece of mind), I take a few transfers from the water agar plate and move them to nutritional plates.

Next step is easy: watch your new plate as the mycelium grows out clean!

Edit:
Here's a video I found that discusses a couple applications for water agar that I didn't mention



Pictures added 8/28:

Here are some pictures to show you guys the growth on a few plates.. they aren't the best pictures,  but you should be able to see the leading edge to get an idea of the speed of growth.

Plate stared 8/10 (18 days ago):



8/11 (17 days ago):



8/12 (16 days ago):




Agar to grain comparison
Water Agar vs Nutrient Agar

10/28:
I'm on a mission to start using up some of my ridiculously large plate collection.. So, I decided to do a side-by-side, plate to grain, comparison using 2 fully grown out Phobos plates.. One plate is water agar, the other is MYA. I decided to do 3 jars from each plate.

Quick picture before getting to work:


Here are the plates I'll be using


Plate 1 (MYA- cut into 3 wedges, leaving the center and outer ring behind:


Used plate 1 to inoculate 3 jars of oats:


Here is plate 2 (WA- cut in a similar pattern to plate 1)


Used plate 2 to inoculate the other 3 oat jars:


I plan to post updates comparing any differences in the length of time it takes for the mycelium to jump off, how long it takes the jars to colonize, etc...

11/3

The WA plates are finally starting to jump off to the grain a little bit.. they are definitely running a few days behind the jars that were inoculated using nutrient agar.. like I said in a previous post, this could be due to the age of the plates.. it is a possibility that the mycelium was in a dormant state, due to the fact that they're around 3 months old.. I still plan to run another test with fresh plates to see if the results differ from this batch.

Enigma (WA)


Phobos (WA)


Phobos (MYA)



Edited by Rotnpins (11/03/22 10:56 AM)


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Invisiblenosf3r4tu


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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 5
    #27902961 - 08/14/22 09:48 AM (1 year, 5 months ago)

I'm a lazy fuck and I just flip the bacterial plates and take a small sample from the other side. Myc grows through the agar, bacteria doesn't.


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OfflineRotnpins
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 5
    #28018148 - 10/26/22 06:33 PM (1 year, 3 months ago)

One last photo drop from today's agar session...

The original Enigma plate I was sent was bacterial and had visible metabolites.. I ended up cleaning it up with WA and sent a transfer back to nutrient agar.. that plate produced the 2 successful bags of Enigma that I ran (they were a little bacterial, but it was from a grain prep failure. The nutrient plate was clean).. so I'm fairly confident that all but the center of this plate is clean..

I decided to send some of my old WA plates to grain to see how things went..

Here is the Enigma plate:


This is how I cut the plate.. the goal is to leave the center (and hopefully the contamination) behind and use the rest of the plate:


Here is a shot of the jar I inoculated:


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OfflineSmellyhobbit
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 5
    #28020116 - 10/27/22 07:28 PM (1 year, 2 months ago)

You guys want to debate a bro science topic for 8 pages? Really fuck this thread up?


--------------------
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OfflineSirPsycho
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Smellyhobbit] * 5
    #28020120 - 10/27/22 07:30 PM (1 year, 2 months ago)

Quote:

Smellyhobbit said:
You guys want to debate a bro science topic for 8 pages? Really fuck this thread up?



Listen here you little shit


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Offlinehazyhorse
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 4
    #28020279 - 10/27/22 08:32 PM (1 year, 2 months ago)

for real, you’re doing some amazing stuff for the community with this thread & it’s all great info to have. the more we know the more we can apply our knowledge & streamline shit.

but the way we treat plants & fungus is just so hilarious on a meta level. cutting it down to a few cells with a hot knife & forcing it to do all this stress response shit, shaking it up as it’s trying to colonize & consume nutrients, breaking it up & forcing it to recolonize & we’re out here like ā€œoh yeah, the myc LOVES itā€ lol


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InvisibleBaba Yaga
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Re: Let's talk about Water Agar!! šŸ’§ [Re: hazyhorse] * 4
    #28020539 - 10/27/22 11:25 PM (1 year, 2 months ago)

Honestly using water agar is like cheating, where is the craft and art in this? No need for taking great care to cut the tiniest sample over and over again just to find out there is still a contam riding along. I was throwing this pin onto a water agar plate and taking transfers when it had barely grown out 10mm just to get 4 pristine clean plates on the first try and I could probably have gone straight to LC with this making this whole thing soooooooo easy boooooooring. You young folks are missing out on a painful but character building experience.




What challenge has life got ready next? Painting by numbers maybe?

This is utterly ridiculous. :oldman::lol:


Edited by Baba Yaga (10/27/22 11:39 PM)


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OfflineRotnpins
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 4
    #28021784 - 10/28/22 05:01 PM (1 year, 2 months ago)

I'm on a mission to start using up some of my ridiculously large plate collection.. So, I decided to do a side-by-side, plate to grain, comparison using 2 fully grown out Phobos plates.. One plate is water agar, the other is MYA. I decided to do 3 jars from each plate.

Quick picture before getting to work:


Here are the plates I'll be using


Plate 1 (MYA- cut into 3 wedges, leaving the center and outer ring behind:


Used plate 1 to inoculate 3 jars of oats:


Here is plate 2 (WA- cut in a similar pattern to plate 1)


Used plate 2 to inoculate the other 3 oat jars:


I plan to post updates comparing any differences in the length of time it takes for the mycelium to jump off, how long it takes the jars to colonize, etc...

I may continue to update through the first flush, but I'm not really sure if it's relevant to this thread or not continuing to watch the grow until completion, or if it should stay limited to agar work and jar colonization :shrug:

Edit: updated OP to include this comparison :mushroom2:


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OfflineRotnpins
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 4
    #28030951 - 11/03/22 10:38 AM (1 year, 2 months ago)

WA vs MYA- side by side inoculation:

The WA plates are finally starting to jump off to the grain a little bit.. they are definitely running a few days behind the jars that were inoculated using nutrient agar.. like I said in a previous post, this could be due to the age of the plates.. it is a possibility that the mycelium was in a dormant state, due to the fact that they're around 3 months old.. I still plan to run another test with fresh plates to see if the results differ from this batch.

Enigma (WA)


Phobos (WA)


Phobos (MYA)


I'll update with some pre-shake pictures in a few days.


Edited by Rotnpins (11/03/22 10:56 AM)


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Offlinezilker
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Re: Let's talk about Water Agar!! šŸ’§ [Re: baz] * 3
    #27907494 - 08/17/22 11:25 PM (1 year, 5 months ago)

You beat me to it. 

I found an uncommon shroom in my back yard last weekend, Reddening Lepiota (red lip sounds better :cool:).  I left the spore print in open air until the ID was confirmed, then scraped into foil with a razor so it's contaminated to hell and back. Figure this is a pretty good study on the difference between the two agars?

Made 40 plates last night, half LMEY & half WA. Last task of the night was adding the spores to the plate. LMEY plate was cooled enough but had to wait on the WA until this morning...9 hour time difference.  Although I'm not sure what the myc is supposed to look like for this genus/species, I assume it doesn't resemble bacteria, as suspected in the LMEY pic.

Time difference...even after ~equal time, WA still looks less contaminated 9 hours later than the LMEY did.  I'm going on memory, didn't take pic, but I'll have time-equal pics soon and will edit post. Not sure either of these will make it, but hopefully it gives somewhat of a controlled study on how contam reacts to the WA??

Haven't USFSE, but wonder if anyone has tried spraying ISO on a spore plate with extreme bacteria contam, prior to myc growth?





8/18 - About 24hrs from last post, 48hrs from LMEY noc, and 36hrs from WA noc. LMEY is contaminated AF! WA is holding the contams back for the most part. WA has myc growth in two spots. Not visible in the pic and I barely caught it with a ray of sun today, but it's there.

The dark spot in the 1st microscope pic appears to be a mushroom crumb from the MS sample I saved.  2nd appears to be spore germination. 3rd is closeup of 2nd pic. Probably transfer WA to WA tomorrow.

Mind you, this mushroom was growing in the yard, finger fucked to death w/o gloves, and the spore print was left in open air to dry for well over 12hrs.  Pretty impressive IMO.

Again, nice job Rotn.

LMEY:


WA:


Microscope 1:


Microscope 2:


Microscope 3:


8/25 - Updated pics on the two plates.  Never attempted to transfer Plate #1 which is the nutrient plate b/c it never showed signs of growth.  Going in the trash today.  Made 3 transfers from Plate #2 on 8/19.  As you can see, although it did a great job keeping the bacteria at bay, it's now showing signs of the same mold (stachybotrys chartarum?) that #1 has.  I originally had plans to transfer to nutrient, but I think I'm going to transfer to water one more time to be safe.





I'll end the progress reports here and say that WA did the job it was supposed to do.  Took something dirty AF, cleaned it up nicely in 7 days, whereas it would have been trash with a standard nutrient plate.  Any future transfers are SOP and any future reporting would be redundant.  Cheers!


Edited by zilker (08/25/22 10:41 AM)


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Re: Let's talk about Water Agar!! šŸ’§ [Re: rumfor69] * 3
    #27913660 - 08/22/22 01:08 PM (1 year, 5 months ago)

Rotnpins- water tek master? :sailboat:


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InvisibleStipe-n CapMDiscord
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #27921291 - 08/27/22 01:49 PM (1 year, 4 months ago)

Yup that's basically it.

When there are ample environmental resources the mycelium will be in an exploitation phase of growth, minimal requirement for exploratory growth;

Water agar is void of nutritional resourse which will trigger the mycelium to enter exploratory phase;

By adding a discrete resource packet we may be able to direct the exploratory growth in one direction, while bacteria, etc, remain mostly in place.

Might even be able to correlate visual growth patterns like tomentose/rhizomorphs to phases of growth in real time which would be cool as well. Rhizomorphs being aggregate structures formed for exploratory hyphal elongation...this is the Hypothesis at least.

I don't mean to hijack, but seems like an awesome way to expand on the water agar topic.

I'll stop interfering and let you do your thing, I'll be following along with interest.


Edited by Stipe-n Cap (08/27/22 02:01 PM)


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InvisibleBaba Yaga
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #27928387 - 08/31/22 11:17 PM (1 year, 4 months ago)

I second that. For a while I'm using agar with 1% LME for everything including germination with not problems. Thinking about lowering it to 0.5% and see how that performs.


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OfflineRotnpins
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #27935562 - 09/05/22 03:17 PM (1 year, 4 months ago)

I started the experiment p9 and I were talking about



I may do a couple versions once I have some different things to work with. Such as putting bacteria in one corner and nutrients in the other, but for now I just did a very basic starting version to see what happens..

One sample is a transfer of Semperviva from a MYA plate and the other is a clean sample from the same plate. The hypothesis is that the mycelium will seek out the nutrient sample instead of just branching out evenly in all directions


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OfflineRotnpins
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #27941574 - 09/09/22 07:22 AM (1 year, 4 months ago)

Here's an update on the first nutrient sample test plate day 4:



So far, the growth is stretching out in all directions.. however I'm taking note of a few things that could be influencing the results...

1) the size of the mycelium transfer I took from the nutrient plate... I was working pretty quickly and since the donor plate was clean, i just took a normal sized transfer without giving it a second thought..

It looks like the mycelium is still feeding off the nutrients from the original plate.. the growth is still rather strong/aggressive.. It should be interesting to see what happens once it's grown out a little further, and its starting to run out of food.

2) The proximity of the nutrient sample.. Theoretically, the nutrient sample wouldn't need to be placed very far away from the transfer on the WA plate...

If our goal is to direct the mycelium in one direction, away from bacterial contamination, and then grab a clean transfer to a new plate; it would then make sense that we want the nutrient sample to be far enough that it wouldn't provide food for the bacteria, but close enough that we could use this strategy to grab a quick sample of clean growth.

3) As Baba Yaga pointed out.. the amount of time that the nutrient sample is on the WA plate before the test could play a role.. if the nutrients are allowed to start to leaching into the WA plate, it could be easier for the mycelium to detect trace amounts, while still remaining far enough to not effect the growth of any contamination that is present.

These are just a few of the thoughts/observations I have so far.. it's still pretty early to make any judgements about the outcome.

I plan to test out a few different distances for the nutrient sample in future tests. I'll also do some tests with smaller sized transfers, and try comparing plates that are prepared ahead of time vs ones that have the nutrient sample added at the time of the test..

Even if the mycelium continues to go out in every direction, the nutrient sample could make it easier to identify different types of mycelium growth when choosing where to take a sample when transferring back to your nutrient plate.


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Re: Let's talk about Water Agar!! šŸ’§ [Re: Baba Yaga] * 3
    #27998756 - 10/14/22 02:12 PM (1 year, 3 months ago)



Aaayyyee! Rusty Whyte back on nutes for about 3 days. Not a lot to look at, but does look pretty clean so far.

:disco:

And in current events completely unrelated to water agar, my Choco-Tat has visibly germinated. Too early to get a decent pic, but I’m excited to share the news anyway.


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OfflineSirPsycho
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #28020089 - 10/27/22 07:20 PM (1 year, 2 months ago)

Ok hol up. I think y'all are talking a bit past eachother here.

I think the main point Rusty is making is about cloning a pin from a grow as opposed to an actual mature fruit since you have no real idea about characteristics with a pin.

While Rotn is mostly focusing on the cleaning aspect of WA which I don't think Rusty is really questioning.



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OfflineSmellyhobbit
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Rotnpins] * 3
    #28020094 - 10/27/22 07:21 PM (1 year, 2 months ago)

Water agar thread poppin off tonight


--------------------
A Love Letter to New Growers
A Guide for New Growers
Growth 2023 - A Year In Review

Grow more shrooms. Eat more ass. :mushroom:



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Offlinehazyhorse
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Smellyhobbit] * 3
    #28020141 - 10/27/22 07:37 PM (1 year, 2 months ago)

Quote:

Smellyhobbit said:
You guys want to debate a bro science topic for 8 pages? Really fuck this thread up?




hobbit i laughed out loud when i read this


--------------------
you're not the first to set foot here, just another
===================================
i love glass petris & you can too!!
posts i constantly refer back to
new to mushroom cultivation?? read this!!
===================================

šŸ…ƒ šŸ„“ šŸ„° šŸ„¼    šŸ„² šŸ„» šŸ„ø šŸ„½ šŸ„¶ šŸ…† šŸ… šŸ„° šŸ„æ


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Offlinehazyhorse
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Re: Let's talk about Water Agar!! šŸ’§ [Re: Smellyhobbit] * 3
    #28020232 - 10/27/22 08:16 PM (1 year, 2 months ago)

abusing the fuck out of this organism for
:yields:

very based


--------------------
you're not the first to set foot here, just another
===================================
i love glass petris & you can too!!
posts i constantly refer back to
new to mushroom cultivation?? read this!!
===================================

šŸ…ƒ šŸ„“ šŸ„° šŸ„¼    šŸ„² šŸ„» šŸ„ø šŸ„½ šŸ„¶ šŸ…† šŸ… šŸ„° šŸ„æ


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