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InvisibleMushenstein
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When to give up on Agar Plates???
    #24021097 - 01/18/17 07:19 PM (7 years, 12 days ago)

Ok, so I have made dozens of Pasty Plates with fairly high success rate. I just started "Pour Agar" with Petri and substituting Pasty's PDA for Dog Food Agar and LMEA. The only two that have shown growth are the two transfers from PDA "Pasty Plates", shown below. Which really seem to like the DFA.


The other petri's, which were inoculated with spore prints,


and a spore syringe,


Have shown no growth after almost 2 weeks. The petri I knocked up with the syringe actually has dark spots where the drops of spores were laid.


When do I give up on these and throw them out and start again? Old threads say to leave them for two weeks. Well they have had 12-13 days. I don't think they are going to spring to life tomorrow. Any ideas what could be wrong with the agar? Transfers liked it. Why don't spores???


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OfflineDactylium
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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24021123 - 01/18/17 07:29 PM (7 years, 12 days ago)

Spores take better to softer agar. Try using a little less agar in your recipe. Munchauzen has a good recipe for soft agar if you don't trust your own judgment. Spores can also just be plain stubborn. I waited weeks for the ones in my journal.


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Re: When to give up on Agar Plates??? [Re: Dactylium]
    #24021154 - 01/18/17 07:39 PM (7 years, 12 days ago)

That's what I like about PDA. If going for long term storage use a thick mix with more potato less karo. Off indoor prints go softer. For wild prints have patience, they are tough, even if clean as can be.

When I'm germinating dry spores I use 6g agar, 1.5 cups water drop of karo pinch of potato. Transfers 7g agar 1.5 cups water, drop of karo and a bit more potato. Just don't make too sugary agar when doing wild prints. Also when moving from syringe to plate, get the needle red hot, eject, saturate a swab and streak the plate. It's less messy and more predictable. You'll know where growth is and isn't supposed to be, and long swabs keep your hands away from your work.


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Re: When to give up on Agar Plates??? [Re: Adden]
    #24021179 - 01/18/17 07:48 PM (7 years, 12 days ago)

I would remove the swab from the equation as it's just one more vector for contam...you can streak the spores by rolling the drop around on the plate.


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24021210 - 01/18/17 07:56 PM (7 years, 12 days ago)

I'm pretty new to agar, but I had some AA+ spores take more than 3 weeks to germinate in my first round. They were scraped from a print onto a plate that I'm sure was mixed too stiff. I noticed the growth when I was about to toss them, so there's definitely no harm in keeping them around unless you absolutely need the room.


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InvisibleMushenstein
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Re: When to give up on Agar Plates??? [Re: Adden]
    #24021228 - 01/18/17 08:02 PM (7 years, 12 days ago)

So your saying its definitely not the type of agar, save for the sugar, but its just too damn stiff?
I hadn't factored that into the equation. I guess the softer it is the more access to water the spores have. If that is even a consideration.

How much longer should I give these ones before chucking them for another try??:rocket:


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Re: When to give up on Agar Plates??? [Re: pheasant327]
    #24021243 - 01/18/17 08:07 PM (7 years, 12 days ago)

Why toss them? Just distract yourself with another project to keep the anxiety down. Maybe they will surprise you.  :goodluck:


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InvisibleMushenstein
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Re: When to give up on Agar Plates??? [Re: Voltageloss]
    #24021288 - 01/18/17 08:19 PM (7 years, 12 days ago)

Quote:

Adden said:
When I'm germinating dry spores I use 6g agar, 1.5 cups water drop of karo pinch of potato. Transfers 7g agar 1.5 cups water, drop of karo and a bit more potato. Just don't make too sugary agar when doing wild prints.




It doesn't sound like you put a lot of nutes in your agar? Drop of karo and a pinch of potato in 1.5 cups of water is probably the least nutes I've seen in a recipe. Do you get fast rhizo growth?


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Re: When to give up on Agar Plates??? [Re: Voltageloss]
    #24021294 - 01/18/17 08:21 PM (7 years, 12 days ago)

When my agar is too thick I'll add 7 or 8 drops of solution, let it sit for 24 hours, then turn it upside down and wait.  Incubating upside down prevents condensation.  Either that or germinate in turbinado lc, inoculate from the lc, and be ready to transfer if there is any sign of contamination.


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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24021315 - 01/18/17 08:28 PM (7 years, 12 days ago)

Turbinado???


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OfflineDactylium
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Re: When to give up on Agar Plates??? [Re: Voltageloss]
    #24021326 - 01/18/17 08:31 PM (7 years, 12 days ago)


Same spore syringe, softer recipe on the right. The one on the left is a week older too.


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24021350 - 01/18/17 08:39 PM (7 years, 12 days ago)

Sugar in the raw.  5 grams per cup of water and pc with an injection point... spores germinate pretty quickly.  The only thing is how often syringes have some contamination so a couple of transfers might be necessary. 

I aspirate off the extra uncolonized lc, then pull out the mycelium and mix it with about 50 ml of distilled water.


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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24021388 - 01/18/17 08:57 PM (7 years, 12 days ago)

This isc why i like lme. Retarded simple. 50ml water to 1g agar / 1g lme. For spores I do .08g agar.


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InvisibleMushenstein
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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24021406 - 01/18/17 09:05 PM (7 years, 12 days ago)

Quote:

RealityCzech said:
I aspirate off the extra uncolonized lc, then pull out the mycelium and mix it with about 50 ml of distilled water.




2 things, Why use turbinado? is it better then honey??

Second thing...How do you aspirate off the excess LC. I need to know this!


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InvisibleAdden
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Re: When to give up on Agar Plates??? [Re: Mycolorado]
    #24021452 - 01/18/17 09:27 PM (7 years, 12 days ago)

Quote:

Adden said:
That's what I like about PDA. If going for long term storage use a thick mix with more potato less karo. Off indoor prints go softer. For wild prints have patience, they are tough, even if clean as can be.

When I'm germinating dry spores I use 6g agar, 1.5 cups water drop of karo pinch of potato. Transfers 7g agar 1.5 cups water, drop of karo and a bit more potato. Just don't make too sugary agar when doing wild prints. Also when moving from syringe to plate, get the needle red hot, eject, saturate a swab and streak the plate. It's less messy and more predictable. You'll know where growth is and isn't supposed to be, and long swabs keep your hands away from your work.




Quote:

Mycolorado said:
I would remove the swab from the equation as it's just one more vector for contam...you can streak the spores by rolling the drop around on the plate.




Risk vs benefit. A clean Z on a plate and it keeps your hands away. Swabs should be used for prints too for this reason. You're not using a Qtip, you're using sterile swabs with 6 or 8 inch handles that you open in a SAB. Regardless, if it contams you have a lot more areas to pick myc from.


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Re: When to give up on Agar Plates??? [Re: Adden]
    #24021615 - 01/18/17 10:40 PM (7 years, 12 days ago)

Rolling the drop around is tooless. Or you could use a loop. This one is huge but works fine.



This plate was inoculated with this loop and dragged in a z pattern like you suggest.  Id rather torch a tool then use a Pre sterilized swab...just me.



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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24021641 - 01/18/17 10:55 PM (7 years, 12 days ago)

Quote:

Mushenstein said:
So your saying its definitely not the type of agar, save for the sugar, but its just too damn stiff?
I hadn't factored that into the equation. I guess the softer it is the more access to water the spores




MEA and DFA are great.  Typically, media are made with a 2% agar content, which is pretty stiff. The plate pictured on the other post was 1.5% agar and the difference was noticeable. Those spore germinated in 3 days.  They were also very fresh. Either way, if you want to adjust your agar content, use weight to weigh not weight to volume as suggested.  A ml of water weighs 1g so that makes it pretty easy on ya.  Your transfers look solid.  Hope this helps!


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Re: When to give up on Agar Plates??? [Re: Mycolorado]
    #24021739 - 01/19/17 12:02 AM (7 years, 12 days ago)

Loops are annoying but just as easy. I only got comfortable with them when I built my own rig with different gauge copper wires. I found the others clunky. (SAB could be bigger). If I had plates and a flow hood I'd use them all day every day. I did use my Noc loop last time I did transfers tho. Stabbed into filament mycelium on the outer edges of a plate. Didn't even look like I was transferring anything. They look sexy it's a week later I should check on them.. I'm trying to get a fast KSS.


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InvisibleMushenstein
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Re: When to give up on Agar Plates??? [Re: Mycolorado]
    #24022197 - 01/19/17 07:59 AM (7 years, 11 days ago)

3 days! Wow that sounds pretty quick for a spores on agar. My pasty plates usually take no less than 5 days to germinate and spring to life.

I understand the weight factor of water per ml, but agar is not as dense. My scale doesn't read tenths, or past whole numbers, so it might be 1.5 grams and shows only 1 gram. It could be 2.9 grams but it still reads 2.0. So I have to try to adjust for volume. i.e. 1 teaspoon = 2.5 grams of powdered agar or at least over 2 grams.

Under this method it would be impossible to measure 2% solution of agar and then reduce to 1.5%. My scale will not allow me to do that. Do you have any suggestions other then cutting back by eyeing up the teaspoon, which is what I will be doing?


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24022383 - 01/19/17 09:19 AM (7 years, 11 days ago)

My scale was 20$ at the pipe store. They're even cheaper on Amazon


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Re: When to give up on Agar Plates??? [Re: bodhisatta]
    #24022625 - 01/19/17 10:48 AM (7 years, 11 days ago)

Yeah you can get a pocket scale for small stuff like this for $6.50 on amazon


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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24022721 - 01/19/17 11:32 AM (7 years, 11 days ago)

Thanks for the directions. I had no idea they were so cheap.
Back to my last question, if anyone knows.
Quote:

RealityCzech said:I aspirate off the extra uncolonized lc, then pull out the mycelium and mix it with about 50 ml of distilled water.




How do you aspirate excessive LC off of the Myc in an LC container? Is this even possible or is this guy full of it???


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24022728 - 01/19/17 11:37 AM (7 years, 11 days ago)

Quote:

Mushenstein said:
Thanks for the directions. I had no idea they were so cheap.
Back to my last question, if anyone knows.
Quote:

RealityCzech said:I aspirate off the extra uncolonized lc, then pull out the mycelium and mix it with about 50 ml of distilled water.




How do you aspirate excessive LC off of the Myc in an LC container? Is this even possible or is this guy full of it???




Yes, this dude is clueless.

Just eyeball it until you get a proper scale.  I'd also just let those plates ride a while as suggested...they might pop eventually....


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24022934 - 01/19/17 01:26 PM (7 years, 11 days ago)

Quote:

Mushenstein said:
How do you aspirate excessive LC off of the Myc in an LC container? Is this even possible or is this guy full of it???




The myc tends to stay in a little cloud in the LC near the bottom.  There is a lot of "clear" media that remains uncolonized.  Use a large syringe through your port to suck the clear media off the top.  Once you get as much as you can without getting the myc cloud, use a second syringe to draw out the mycelium and stop when you've got most of it leaving some of the "clear" media that was in the bottom behind.

I'll then add the myc to distilled water in a small bottle with a SHIP.  Right now I'm using some cobalt blue dropper bottles that I flipped the dropper bulb over and filled the bulb with silicon.  I don't know if it is necessary, but I PC the distilled water in another bottle and PC the bottle with 10 ml in it.  Once cooled, I close them up so there isn't a vacuum and add the myc and distilled water that was PCed separately.

It doesn't get ALL the liquid media out, but with the distilled water it dilutes it quite a bit.  Once well shaken it will break up the mycelium and allow you to easily draw and inject it.  It isn't super thick like some of the LC syringes that you can buy, but there is plenty of myc and it keeps for a long time in the fridge.

Use the bottle like you would a med draw... if you're adding liquid to it, draw out 1 ml of air from the top for every ml you injected after injection.  When you go to draw liquid out, fill your syringe with air and inject it before you draw out the liquid (again, equal volume of air going in and liquid coming out).  This will keep the pressure in the bottle relatively stable over time... At least long enough to use up the LC.

I am not home at the moment, but I've got a jar of golden oyster LC that's just about ready for this at home.  I'll snap a pic of that and of one of the cobalt bottles that's running low tonight and post them so you can see what I mean.


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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24023162 - 01/19/17 02:58 PM (7 years, 11 days ago)

As promised...  Note how the mycelium is sitting on the bottom of the jar?  Use that to your advantage and use a syringe to aspirate off the extra liquid before you pull the myc out.



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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24023163 - 01/19/17 02:59 PM (7 years, 11 days ago)

I was under the impression that an LC isn't done until the water is 90% filled with the myc. Your saying that this is having too high of an expectation??? When do you consider the LC done and ready to go to syringe?


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24023208 - 01/19/17 03:14 PM (7 years, 11 days ago)

I can't speak to other media recipes, but with the turbinado spores seem to germinate in 2-3 days and it slows to a crawl about 5 days later with 33-50% of it colonized.  Maybe I'm impatient and could get more, but when I don't see noticeable growth for a couple days I'll go ahead and pull it,  run it on agar for contaminates,  and call it a day.

There are a lot of little variables and your results may vary... I'm just giving you the easiest thing I've found.  If it helps,  great.  If not,  that's fine too.


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Re: When to give up on Agar Plates??? [Re: RealityCzech]
    #24023811 - 01/19/17 07:00 PM (7 years, 11 days ago)

When should I transfer from these plates? Do I wait until they are 100% colonized or does it matter. They will be totally done in a few days, but if it doesn't matter I will cut um up now!


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24024161 - 01/19/17 08:51 PM (7 years, 11 days ago)

New to agar but it's my understanding you want to transfer from a leading edge, so full colonization is not the goal.


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Re: When to give up on Agar Plates??? [Re: Voltageloss]
    #24024581 - 01/20/17 12:37 AM (7 years, 11 days ago)

^this is my understanding as well, Ive also heard this is because it touching the sides can be a contam vector... not sure how esp in a pasty plate that should be 100% sterile, but as far as ive seen people tiger drop at about 80% colonization


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Re: When to give up on Agar Plates??? [Re: Voltageloss]
    #24024981 - 01/20/17 08:28 AM (7 years, 10 days ago)

Quote:

Voltageloss said:
New to agar but it's my understanding you want to transfer from a leading edge, so full colonization is not the goal.




This is sound logic. Unless someone argues differently I will cut from the leading edge. Since these are transfers, the plates that colonize from these cuts will be going to grain, so avoiding contam factors is paramount. I'd rather not have to clean it up again.

I have become very impressed with the performance of Dog Food Agar as opposed to Agar recipes with more elegant ingredients. I made some more last night and had a slight accident with the addition of food coloring, in which much more was added then should have been. like 20 drops as opposed to 3. I wonder if it will have any impact on sustaining the growth or germination of fungal strain.


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Re: When to give up on Agar Plates??? [Re: Mushenstein]
    #24025737 - 01/20/17 02:01 PM (7 years, 10 days ago)

Definitely transfer now. That looks nice and clean. You do want the leading edge. It's the freshest mycelium.


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