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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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When to give up on Agar Plates???
#24021097 - 01/18/17 07:19 PM (7 years, 12 days ago) |
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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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Dactylium
Don't touch me I'm sterile


Registered: 08/12/16
Posts: 696
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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) |
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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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Adden

Registered: 06/04/03
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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) |
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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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Mycolorado
Hobbyist


Registered: 07/23/16
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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) |
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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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pheasant327

Registered: 10/04/16
Posts: 133
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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) |
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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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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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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) |
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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??
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Voltageloss
Stranger everyday

Registered: 11/23/16
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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) |
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Why toss them? Just distract yourself with another project to keep the anxiety down. Maybe they will surprise you.
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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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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) |
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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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RealityCzech
Entropy enthusiast


Registered: 12/29/16
Posts: 90
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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) |
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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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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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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) |
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Turbinado???
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Dactylium
Don't touch me I'm sterile


Registered: 08/12/16
Posts: 696
Last seen: 10 months, 13 days
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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) |
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 Same spore syringe, softer recipe on the right. The one on the left is a week older too.
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RealityCzech
Entropy enthusiast


Registered: 12/29/16
Posts: 90
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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) |
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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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Intelligentxfruit
Earth Hippy


Registered: 01/06/13
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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) |
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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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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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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) |
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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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Adden

Registered: 06/04/03
Posts: 39,201
Loc:
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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) |
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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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Mycolorado
Hobbyist


Registered: 07/23/16
Posts: 8,529
Loc: Interdimensional Bootcamp
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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) |
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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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Mycolorado
Hobbyist


Registered: 07/23/16
Posts: 8,529
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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) |
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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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Adden

Registered: 06/04/03
Posts: 39,201
Loc:
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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) |
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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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Mushenstein
Brain Damage



Registered: 02/19/15
Posts: 680
Loc: At The Gates
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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) |
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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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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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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) |
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My scale was 20$ at the pipe store. They're even cheaper on Amazon
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