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Sits
Shady Vagrant


Registered: 09/09/13
Posts: 157
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First try agar fail *DELETED*
#26742323 - 06/13/20 05:50 PM (3 years, 7 months ago) |
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Post deleted by Sits
Reason for deletion: Don't think it'll be helpful to others.
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Sits
Shady Vagrant


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Re: First try agar fail [Re: Sits]
#26784425 - 06/26/20 11:28 AM (3 years, 6 months ago) |
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Update, second try:
So I've tried the GSA method a few times now. I use the water from boiling the oats from Bod's Oat Prep Tek along with 8g of Agar Agar mixed and boiled, then PC'd for about 25 min at 15 PSI. I poured 20-30 plates each time at the right temp. everything seems according to plan at this point.
I noc'd these plates up with MS spore solutions from about 5 different syringes from 2 different sponsor sites (Reliable Spores and Sporeworks) using different methods like dropping straight on the agar, dropping on agar then smearing with a sterilized loop, dropping liquid into a dish first and then using a loop to smear it, etc.
The most recent batch is 10 days in and one syringe was clearly bacterial as I can see bacteria forming directly where the liquid was. The ones from the other site are pretty clean except for one case of satellite mold from my technique presumably. There is absolutely nothing growing on some of these plates after 10 days. What am I doing wrong?
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juvec
Stranger
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s [Re: Sits]
#26784442 - 06/26/20 11:32 AM (3 years, 6 months ago) |
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s
Edited by juvec (06/17/21 09:48 AM)
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Roger Clemency
Smile


Registered: 03/23/20
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Re: First try agar fail [Re: Sits]
#26784451 - 06/26/20 11:35 AM (3 years, 6 months ago) |
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Others disagree with me often but I still shout my disdain for going spore syringe to agar. It’s the worst in my experience. I almost always end up with a bacterial mess wherever spore solution touches and the spores never get a chance to germinate.
Try sterilizing a half pint (or whatever small jar) of grain and nocc that up with a syringe and if it germinates then pull a grain out and take it to a plate to clean from there. I’ve seen that classic character RR recommending this for agar unfriendly syringes.
Or just order some beautifully dry spore prints
-------------------- Sour grapes, sweet revenge Heaven starts right where hell ends
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Sits
Shady Vagrant


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I have some grain jars going that I'll be able to clone from soon.
One set of plates is totally clean though which is what is confusing me. No mycelium, no bacteria or mold, nothing.
It feels like I messed up the agar recipe somehow? Not sure how as the recipe is so simple.
I made some MEA plates the other day to test that theory out, hopefully will see results soon.
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juvec
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s [Re: Sits]
#26784493 - 06/26/20 11:52 AM (3 years, 6 months ago) |
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s
Edited by juvec (06/17/21 09:49 AM)
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juvec
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s [Re: Sits]
#26784512 - 06/26/20 11:58 AM (3 years, 6 months ago) |
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s
Edited by juvec (06/17/21 09:49 AM)
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Sits
Shady Vagrant


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Re: First try agar fail [Re: juvec]
#26784582 - 06/26/20 12:19 PM (3 years, 6 months ago) |
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Quote:
juvec said: Same experience i had pretty much. Things i think it might be:
-Time spent pouring agar -Re-using petri dishes -Lots of transfers/inoc using same scalpel/loop, re-entering SAB a lot.
Other than that i'm completely lost. Factory new petri dishes came out clean on my second agar batch though, the ones i washed and wiped with alcohol (thoroughly) were the ones that showed bacteria the second time around. I did get some bacteria on my first batch too though, which was 100% factory new petri dishes.
I haven't ever reused a petri. I don't think my particular situation has anything to do with my inoculation technique as I've tried everything I can think of.
It's the amount of bacteria in the innoculant and possibly my agar recipe as far as I can tell. I should have SOME mycelium on some of these plates after 10 days.
Anyway, should know more after my MEA plates have a few days to grow.
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juvec
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s [Re: Sits]
#26784643 - 06/26/20 12:38 PM (3 years, 6 months ago) |
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s
Edited by juvec (06/17/21 09:50 AM)
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Sits
Shady Vagrant


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Re: First try agar fail [Re: juvec]
#26786669 - 06/27/20 08:22 AM (3 years, 6 months ago) |
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The only way I'm not getting viable spores onto the agar is if maybe I'm not squirting ENOUGH out after flaming the needle? This seems extremely unlikely to have happened 50+ times though. Like I said, I have grain jars going that are performing just fine (although probably have a good amount of bacteria too) from some of the same syringes. I'm pretty stumped if the MEA plates have the same results.
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Sits
Shady Vagrant


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Re: First try agar fail [Re: Sits]
#26786683 - 06/27/20 08:31 AM (3 years, 6 months ago) |
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UPDATE: Is this myc or cobweb?
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AtmozFear
just a shade of myself


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Re: First try agar fail [Re: Sits]
#26786718 - 06/27/20 08:44 AM (3 years, 6 months ago) |
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I've been through all this with the pour method. It's either your homemade agar or, since it's bacteria, not sealing up the plate properly with parafilm, saran wrap, or a plastic baggie.
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LadysKnight
Hello Ladies


Registered: 10/09/15
Posts: 1,670
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Re: First try agar fail [Re: Sits]
#26786913 - 06/27/20 10:02 AM (3 years, 6 months ago) |
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Quote:
Sits said: Like I said, I have grain jars going that are performing just fine (although probably have a good amount of bacteria too) from some of the same syringes.
If it's working in the jar, do what RC said, sacrafice one in a SAB, transfer some good growth to a few plates. If it's actual myc, you will have some cultures to keep you going.
That pic is mold.
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Sits
Shady Vagrant


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I have a couple jar that are almost ready for g2g, so will probably just take a few grains out of those when I transfer them.
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Sits
Shady Vagrant


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Re: First try agar fail [Re: Sits]
#26789238 - 06/28/20 08:54 AM (3 years, 6 months ago) |
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Just for reference, here's what all the plates that were inoculated by dropping 1 drop of spore solution directly onto the center of the plate look like:
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Tight Lunchbox
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Re: First try agar fail [Re: Sits]
#26789349 - 06/28/20 09:40 AM (3 years, 6 months ago) |
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Quote:
Sits said: I nocc'd up about 30 plates from 3 different syringes to get some practice. Some, I did a regular dilution swipe, some I tried a serial swipe. I got what looks like bacteria here on all of them and somehow zero visible mycelium growth.
I used a SAB, alcoholed/flamed everything before every step, I squirted about 2 mL of spore solution into a new blank petri to swipe with the loop, I shook the bejesus out of the syringes before starting too.
Also, in the serial dilutions, the bacteria only shows up where there was actual liquid from the spore solution and no contams or anything on the 2nd and 3rd streaks.
Going to try squirting spore solution directly on the loop and also directly onto the agar next time to test it out, kinda confused why I'm getting zero healthy growth when the grain jars I noc'd up the same day are starting to colonize nicely.



I have a PE syringe and a galindoi syringe that give me nothing but bacteria. I try them about twice a year and it's always the same story. Syringes fucking suck imo.
Swabbing a print gives me far less trouble. Try that if you can.
-------------------- "it's all a joke between mom contractions and coffin fittings" The most useful tool for noobs
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alaskappalachian
Entitiologist


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If you can't seem to get shit for growth any way you inoculate your plates, you still have a couple tricks up your sleeve. Let me save you some trouble. First method I use for this is to make up a handful of brf cakes which get inoculated with said syringe(s). Once you get some good growth going, take the cake into your SAB to open and grab a tiny piece of myc. Works splendidly. Also making a thick brf paste (search for brf pucks or the like) and swab/dab a little solution on it like you were inoculating any other dish. BRF for the win. I'd try the cake method first, but the pucks/brf paste pre-pours are a gem as well. Much more forgiving than agar with sketchy spore solution. Best of luck, bud.
-------------------- "First we build the tools, then they build us." THE 49th MYCOJOURNAL: Exotics, Auroras, and Entities
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Dorfnob
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Re: First try agar fail [Re: Sits]
#26789473 - 06/28/20 10:28 AM (3 years, 6 months ago) |
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Quote:
Sits said:
Quote:
juvec said:
It's the amount of bacteria in the innoculant and possibly my agar recipe as far as I can tell. I should have SOME mycelium on some of these plates after 10 days.
My experience it took at least 2 weeks for spores to germinate I would be patient. The bacteria thing sucks I feel yah I’m still battling contam rates.
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Sits
Shady Vagrant


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Re: First try agar fail [Re: Dorfnob]
#26789913 - 06/28/20 01:58 PM (3 years, 6 months ago) |
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Ah, that is comforting that germination can take over 2 weeks! I had no idea. Ok, another round of cobweb or myc? Obviously, the blob in the middle is bacteria.



Note: these are 3 separate plates and were all noc'd up by one drop to the middle.
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Sits
Shady Vagrant


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Re: First try agar fail [Re: Sits]
#26791470 - 06/29/20 06:47 AM (3 years, 6 months ago) |
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I'm thinking it's mold, not myc.
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