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TripBalls
Rocketfart



Registered: 01/14/14
Posts: 170
Loc: harambe
Last seen: 5 days, 10 hours
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Why doesn't agar like me? I mean really, what the hell..
#23862412 - 11/24/16 01:30 AM (7 years, 3 months ago) |
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Lately I've been having a tough one with my pasty plates. I literally am getting NO visible growth whatsoever.. I have done a few transfers successfully but lately, if I get any recovery at all, the growth is VERY light and almost undetectable around the wedge.. Sometimes I'll get what looks to be a few drops amount of water that run around the agar, which I believe is from condensation.. I've been taking action to reducing condensation, but after dropping some spores on a few plates that seemed relatively dry, I still have no growth after over a week.
At this point, I'm convinced I have a clear bacterial infestation among me, Or I'm killing the spores/wedges by not allowing the needle/blade to cool enough (which I doubt) Maybe the incubation/germination environment isn't warm enough? (74'f)
I have no idea why the transfers didn't recover.. I PC my PDA pastywhyte plates for 30 mins, flame sterilizing the blade/needle in between plates.. Hell, my syringes are from a very reputable sponsor I've done business with for years!
What could be going wrong here? These were supposed to be easier!
I tried taking some pictures but they really don't do justice..
Any advice is greatly appreciated. I really need some guidance!
-------------------- I Followed Frank's Footsteps This guy is a hero. Bod’s system saved my life as well.. if these guys can’t get you where you need to be, you’re in the wrong hobby. Rate me cause I'm a nice guy? Not all who wander are lost, but I sure as fuck am
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Tira



Registered: 11/20/10
Posts: 1,202
Loc: Turkey
Last seen: 6 years, 4 months
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: TripBalls]
#23862464 - 11/24/16 02:28 AM (7 years, 3 months ago) |
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If you're using spore syringe as inoculant, that happens with syringe to agar, sometimes they don't germinate. Spore print to agar using an inoculation loop is more convenient. But since you're also having problems with your transfers of already germinated mycelium, it seems like that is not the problem. Maybe it's your recipe? Qhat agar recipe do you use?
To get rid of condensation you can just wait for a few days before inoculating, agar absorbs the water after a while.
-------------------- Useful Links for Beginners The Basics AMU Teks Frank''s Teks Agar Noob Forum Reccomended Teks Agar for guaranteed spawn, Proper pasteurization for guaranteed substrate.
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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
Last seen: 9 months, 22 days
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: Tira]
#23862606 - 11/24/16 05:31 AM (7 years, 3 months ago) |
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Quote:
Or I'm killing the spores/wedges by not allowing the needle/blade to cool enough (which I doubt)
Flame the needle, shake the syringe, flame it again. Push some drops of spore solution out of the syringe to cool the needle. Then use one drop of spore solution to inoculate the pasty plates.
Quote:
Maybe the incubation/germination environment isn't warm enough? (74'f)
It is a good temperature.
Cool the scalpel by cutting the agar of the dish you want transfer to.
Quote:
If you're using spore syringe as inoculant, that happens with syringe to agar, sometimes they don't germinate. Spore print to agar using an inoculation loop is more convenient.
I experienced the opposite. Especially with old spore prints. I had to rehydrate them in spore syringes to get germination on agar.
Can you post your agar recipe?
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ComebackKid
Multispore Enthusiast



Registered: 05/27/16
Posts: 3,951
Loc: ked in the trunk of a car
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: enlightenment]
#23862634 - 11/24/16 06:04 AM (7 years, 3 months ago) |
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I'm going to assume he's using pasty recipe
1 tsp / 2.5g agar agar powder 2 tsp potato flakes (~8g) 1/2 cup water (125 ml) 1 drop honey
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Substrate surface conditions / Monotub prep and care
Look around you... Everything you see exists inside the mind. Consciousness, the awareness that is experiencing this mind, is peering in from outside the universe. Our individual experiences are all part of the universe's experience of itself
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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
Last seen: 9 months, 22 days
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: ComebackKid]
#23862651 - 11/24/16 06:16 AM (7 years, 3 months ago) |
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//edit
wrong thread
Edited by enlightenment (11/24/16 06:17 AM)
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WeavieWonder
Time Travel Sucks



Registered: 08/18/16
Posts: 1,455
Loc: USA
Last seen: 2 months, 4 days
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: enlightenment]
#23862962 - 11/24/16 08:41 AM (7 years, 3 months ago) |
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From research on agar work so far, I've found that germinating spores from syringe on agar can be tricky. I've read people will inoculate a small amount of grain first, wait for it colonize a bit, then transfer the grain to agar to clean it up. Anti bacterial agar might be useful too. I haven't tried using a syringe on agar yet, but will in the future, as PE from vendors are only available in a syringe. Btw, syringes are usually contaminated, I don't care what vendor you get them from. There has to be a better solution than inoculating a shit load of plates in hopes that one will give you something to transfer over. Thoughts anyone?
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Ferather
Mycological



Registered: 03/19/15
Posts: 6,325
Loc: United Kingdom
Last seen: 1 year, 3 months
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: WeavieWonder]
#23863099 - 11/24/16 09:50 AM (7 years, 3 months ago) |
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I don't want you to waste money, however as WeavieWonder mentioned, you could germinate on grain then clean it.
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Mycolorado
Hobbyist


Registered: 07/23/16
Posts: 8,557
Loc: Interdimensional Bootcamp
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: WeavieWonder]
#23863119 - 11/24/16 09:57 AM (7 years, 3 months ago) |
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I noc a few plates at a time (3-5). IME, some have no germination, some are loaded with bacteria and mycelium and some appear very clean. I don't think you need to noc a shitload of plates, but you'll have more options if you at least do a few at a time.
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krypto2000
Unknown


Registered: 12/05/06
Posts: 11,579
Last seen: 4 years, 4 months
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: Mycolorado]
#23863213 - 11/24/16 10:32 AM (7 years, 3 months ago) |
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A week isn't that long. Ime spores germinate slower on agar than grain, I'd wait at least a month before calling it bunk. Myclium transfers should recover and show growth within less than a week though, if not you probably have some bacteria going on.
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TripBalls
Rocketfart



Registered: 01/14/14
Posts: 170
Loc: harambe
Last seen: 5 days, 10 hours
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: Ferather]
#23876711 - 11/28/16 10:26 PM (7 years, 3 months ago) |
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Quote:
WeavieWonder said: From research on agar work so far, I've found that germinating spores from syringe on agar can be tricky. I've read people will inoculate a small amount of grain first, wait for it colonize a bit, then transfer the grain to agar to clean it up. Anti bacterial agar might be useful too. I haven't tried using a syringe on agar yet, but will in the future, as PE from vendors are only available in a syringe. Btw, syringes are usually contaminated, I don't care what vendor you get them from. There has to be a better solution than inoculating a shit load of plates in hopes that one will give you something to transfer over. Thoughts anyone?
I have 2 jars fully colonized and consolidating.. I can't believe i never thought to do a G2P isolate. Will definitely drop a few plates.. Let's just hope I can isolate from there with no sweat! Surely I could transfer what grains I don't use on the plates to other jars...
I hate contam vectors.. I'm very squeamish to new stuff for this reason.
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krypto2000 said: A week isn't that long. Ime spores germinate slower on agar than grain, I'd wait at least a month before calling it bunk. Myclium transfers should recover and show growth within less than a week though, if not you probably have some bacteria going on.
Jesus! Really? I'm going for speed, this time around.. I heard about slurries and got into LC forums a bit more.. I can't/won't waste the time like I used to with multispore cultivation. I thought agar was supposed to be the faster route! I'll keep a few around, and hope for the best!
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Ferather said: I don't want you to waste money, however as WeavieWonder mentioned, you could germinate on grain then clean it.
I'm almost not as worried about starting a culture now as I am isolating it! The grain to plate transfer was an idea I hadn't even thought of yet, and I'm sure it will help tons.. But then once I get a culture, how do I know I'm not going to end up with a bunch of stalled-out wedges?
I'm still clueless as to why my transfers didn't recover? Seriously, it's like I have a completely invisible contam. Never had that problem before!
Thanks a bunch Shroomery! You guys are great!
-------------------- I Followed Frank's Footsteps This guy is a hero. Bod’s system saved my life as well.. if these guys can’t get you where you need to be, you’re in the wrong hobby. Rate me cause I'm a nice guy? Not all who wander are lost, but I sure as fuck am
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Moabfighter
Tam Fighter


Registered: 12/13/15
Posts: 2,710
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: TripBalls]
#23876739 - 11/28/16 10:39 PM (7 years, 3 months ago) |
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I use 2.0 g agar, half cup water, one teaspoon of potato flakes and one drop of Karo corn syrup.
Lest nutes give me nice rhizomorphic growth
-------------------- KSSS And PE WBS.
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afflictis
ModestMycologist
Registered: 03/09/12
Posts: 334
Last seen: 5 years, 10 months
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: Moabfighter]
#23876903 - 11/28/16 11:57 PM (7 years, 3 months ago) |
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Quote:
Moabfighter said: I use 2.0 g agar, half cup water, one teaspoon of potato flakes and one drop of Karo corn syrup.
Lest nutes give me nice rhizomorphic growth
how long do you pc for
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enlightenment
alchemist


Registered: 08/09/09
Posts: 1,647
Loc: Europe
Last seen: 9 months, 22 days
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: afflictis]
#23877071 - 11/29/16 02:06 AM (7 years, 3 months ago) |
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I usually use MEA and PC it for ~20 minutes and let it cool slowly. 20 minutes is also a good time for PDA IMO. Don't PC it too long to prevent Maillard reaction.
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Moabfighter
Tam Fighter


Registered: 12/13/15
Posts: 2,710
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: enlightenment]
#23877740 - 11/29/16 09:58 AM (7 years, 3 months ago) |
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I PCd last night 11:20 to 12am.
So 40 mins.
My recipe really makes nice vigorous growth. Try it if you're having issues.
-------------------- KSSS And PE WBS.
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Spore Ninja
PsychoMycoPhile



Registered: 10/25/16
Posts: 161
Last seen: 5 years, 7 months
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Re: Why doesn't agar like me? I mean really, what the hell.. [Re: TripBalls]
#23878201 - 11/29/16 12:48 PM (7 years, 3 months ago) |
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Quote:
TripBalls said:
I'm still clueless as to why my transfers didn't recover? Seriously, it's like I have a completely invisible contam. Never had that problem before!
Thanks a bunch Shroomery! You guys are great!
Is your PDA homemade?
Do you always use the same agar recipe?
I did some PDA recently and accidentally made it double strength. I should have brought the volume back up to 1000ML after boiling the potatoes, but since I was only making 500ML, I fubar'd it and only brought it back to 500ML. (Lesson learned, now i only use 100 G of potatoes and bring it back to 500ML) I cloned onto the plates and have had almost zero growth. It was puzzling for a few days before I realized I screwed up the agar.
If the agar is the same in all your failed attempts- I'd look at changing up the agar...do something completely different - dog food agar is cheap and easy and has always worked well for me.
-------------------- I'm interested in Dr. Pollock's work and his (especially older) strains. Anyone have pix of his Shroommobile RV?? PSA: Protonmail offers free, encrypted email hosted in privacy loving Switzerland. Red Phone / Signal offer free encrypted phone calls and texting between users. All of these programs are available free for PC and Android or apple and are available in the respective stores or online by googling the names. Stay safe out there! The Electronic Freedom Foundation's review of text and messaging program's security is here>>> https://www.eff.org/node/82654
Edited by Spore Ninja (12/20/16 01:06 PM)
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