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OfflineTranscendent Other
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Registered: 04/11/20
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First Agar attempt fail: What is the likely cause?
    #26831973 - 07/19/20 05:40 AM (3 years, 6 months ago)

So two weeks ago I made my first agar petri's, and obviously they failed! In the sense that not even bacteria are interested. So where did I go wrong?


STERILISATION
Given that there is no growth at all, my thinking is it should be safe to rule this out? MEA was mixed and sterilised in my PC for 45 mins, then poured in an SAB.

AGAR MEDIA (MEA)
Perhaps the agar media? However, It doesn't seems to me to be a non-nutritious mix, so why do mycelium and bacteria not think it is incredibly delicious?

INOCULUM
GT
4x GT spore print (must be about 6 months old)
4 x GT spore syringe (3 months old, stored in the fridge)
2 x not inoculated (also showing no sign of contam)


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OfflineAtmozFear
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Re: First Agar attempt fail: What is the likely cause? [Re: Transcendent Other]
    #26831996 - 07/19/20 06:03 AM (3 years, 6 months ago)

I wouldn't call this a 'fail'... inject some more shit into it if there is not even any bacteria  :2cents:


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Offlineredhandmat
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Re: First Agar attempt fail: What is the likely cause? [Re: AtmozFear]
    #26832004 - 07/19/20 06:16 AM (3 years, 6 months ago)

Try again? There are no failures my friends, only experience to be gained.

So you used an inoculation loop or qtips?


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: redhandmat]
    #26856593 - 08/01/20 02:53 AM (3 years, 5 months ago)

So a per the suggestion I reinoculated the agar a week ago...

Nada! Not a microscopic sausage!

My aim now is to try to establish whether the cause is the agar or the innoculums.

I've seen some posts suggesting testing the nutrition of the agar by putting some dirt on it or something. I'm thinking of using a small drop of yogurt as its already cultured, what do you think?


Edited by Transcendent Other (08/01/20 02:55 AM)


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: redhandmat]
    #26856595 - 08/01/20 02:57 AM (3 years, 5 months ago)

Hey, no I used a spore syringe on 4 plates and a spore print and scalpel on another 4


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Invisiblealaskappalachian
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Registered: 10/22/19
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Re: First Agar attempt fail: What is the likely cause? [Re: Transcendent Other]
    #26856603 - 08/01/20 03:24 AM (3 years, 5 months ago)

How much agar did you use?  Softer agar helps.  I find that with really fucked over spores that won't germ on MEA, GSWA, etc., going back in time and making some thick brf pucks/prepours (no agar)  will work a miracle.  I've never met spores I can't germ with brf paste.  Ever.  Then grab your myc and slap it on your agar plate.  Voila.  Equally valuable: syringe to a few brf cakes then grab myc off one in a SAB.  Another gem.  But softer agar often helps quite a bit.  I've had spores take over 2 weeks.  Let those plates ride and try a., b., or c. as suggested if not.  You'll get some germ action,  bud.  Best of luck.


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: alaskappalachian]
    #26856631 - 08/01/20 04:14 AM (3 years, 5 months ago)

I didn't use a lot of agar, probably a few millimetres at the base of each plate.

Hmm, i'm not sure how thick it is! I'll leave it a while as you suggested and if it still fails i'll give it a prod. I followed this recipe: FreshCapMushrooms MEA but divided by 4 as I calculated that it would be plenty for my 10 plates.

Okay great, thanks for the advice! I ordered some new plates so i'll probably leave these ones to it and then start some new ones with your suggestions.

Thanks for the support! Fingers crossed.


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Invisiblealaskappalachian
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Re: First Agar attempt fail: What is the likely cause? [Re: Transcendent Other]
    #26856634 - 08/01/20 04:22 AM (3 years, 5 months ago)

No problem.  Wasn't referencing the depth though,  just for reference.  Took me a second to figure you were using premixed MEA.  Was referring to amount of agar in the amount of H20.  A lot of the time those premixed MEA powders are (per the manufacturer's directions) like 20g per  500ml.  You can cut the amount of premix down to almost half and it'll still set up.  You just want it softer,  despite the pour depth.  Hell... hit Wally world up for a bag of brown rice and a two dollar pack of 4oz. Ziploc twist and lock containers and make a few of the pucks  I mentioned and you'll be gold.  Let us know how it goes!
Edit: yup- checked their mix and just as I suspected: the amount they use in their recipe contains 20g agar to 500ml water lol.  Cut the amount of powder by almost half for the same amount of water.  Way too firm for hard to germ spores. 


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"First we build the tools, then they build us."

THE 49th MYCOJOURNAL: Exotics, Auroras, and Entities


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: alaskappalachian]
    #26856837 - 08/01/20 08:25 AM (3 years, 5 months ago)

Nah it wasn't premixed, I mixed it myself.

My ingredients:


I only remembered earlier that when I mixed my ingredients they clumped up a little bit! So there's also the chance that the mix isn't nutritious enough...

Okay cool, Thanks for checking in on that! As I have to wait for the new plates, and considering the above, i'll test a couple to see if they're nutritious at all.

I'm intrigued by the situation of the media firmness - I'm a novice I know, but i thought mushroom mycelium preferred denser media? Or is it just that its too dense?


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Invisiblesandman420
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Re: First Agar attempt fail: What is the likely cause? [Re: Transcendent Other]
    #26856873 - 08/01/20 08:53 AM (3 years, 5 months ago)

Maybe dead syringes from hot storage or UV light exposure.

Leave out yeast flakes IMO. Plain old MEA is great for cubes.


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Edited by sandman420 (08/01/20 08:54 AM)


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: sandman420]
    #26868102 - 08/07/20 10:40 AM (3 years, 5 months ago)

UPDATE

I'm going to have another crack tomorrow, but here's what one of my plates looks like after I infected it:



Not sure why its gone red and yellow, but looks pretty funky!

I'll keep you posted with the results of the next stage.


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Offlineredhandmat
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Registered: 05/09/19
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Re: First Agar attempt fail: What is the likely cause? [Re: Transcendent Other]
    #26868274 - 08/07/20 12:34 PM (3 years, 5 months ago)

Quote:

Transcendent Other said:
UPDATE

I'm going to have another crack tomorrow, but here's what one of my plates looks like after I infected it:



Not sure why its gone red and yellow, but looks pretty funky!

I'll keep you posted with the results of the next stage.




Oh god that is sooo nasty :prayer:


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OfflineTranscendent Other
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Re: First Agar attempt fail: What is the likely cause? [Re: redhandmat]
    #26869541 - 08/08/20 06:08 AM (3 years, 5 months ago)

Hahaha!

It does look it it could be the star of a horror movie.


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