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OfflineChowder
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[many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations
    #22989671 - 03/09/16 04:42 PM (8 years, 21 days ago)

Thanks a ton for your help in advance!!!

So I've had a lot of good success with BRF cakes in the past and some okay results with rye+verm+coir. But the last 6-7 times I'm getting only failures on all fronts. Last three times I've tried to germinate spores on agar with no results, and now I'm tryig to go back to innoc BRF - just so I can get some shroomz.

Here are the results of my last try. I've also done some extra experimentation on agar - these are the results 14 days after innoc(22-24C).:

(1) - Amazonian spores from a friend (I kinda doubt his cleanliness) - no growth
(2) - Same spores - contam. (dirt from the print??)
(3) - Same spores - no growth
(4) - PE6 spores (1 year old, own prints, take as clean as I could) - no growth
- PE6 spores - no growth
- PE6 spores - no growth
(7) - own B+/GT spores (that I've working with since beggining / 1-2 year old print) - slimey contam all over (what contam is this??)
(8) - idem as above
(9) - idem as above
(10) - touched with clean sterile loop - no growth => clean loop:thumbup:
(11) - unopened dish => good agar sterlilization :thumbup:
(12) - exposed 10sec in SAB - no growth => clean air :thumbup:
(13) - exposed 60 sec in SAB - no growth => clean air :thumbup:
(14) - exposed 10 sec outside SAB - contam => indeed SAB works!:thumbup:
(15) - exposed 60 sec outside SAB - contam => yep.:thumbup:



Next,  I've made the good ol' PF cakes - 48 of them! Syringes were made from my own prints. After 10 days I only got growth on 2 of them (this and this), and only at one of the 4 inoculation points. Whats going on?! I normally get growth after 5-6 days and my contam rate is below 10%.
Also,all jars have a slightly blackened area at the point of inoculation. What is this?

=Questions=
This is my third batch of agar dishes, with no growth or contamination. I've been following PastyWhite's agar tek to the T.
A.Is my agar not nutritious enough? (Maybe the instant potato flakes are not good? I'm using Smash brand witch is 98% potato + E numbers)
B.Are my spores not viable? (I can't see how- they been kept in several layers of protection)
C.How come my PF jars didn't germinate?
D. Why did they germinate at only one of the four points? Also, the myc looks different- is this because of the multispore?

I'm innoculating tonight another batch of agar dishes (that were kept in the fridge all this time, should be fine, right?)
This time with some B+ spores that I got from a Shroomey sponsor vendor. This is the only thing I can think of.... If this doesnt work, that I have no idea what do try next...

Thanks for your time, any feedback welcomed!


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OfflineSloppyJoseph
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Chowder]
    #22989690 - 03/09/16 04:55 PM (8 years, 21 days ago)

How much agar are you using? What is your recipe? It looks stiff but that's kind of impossible to tell. IME spore tend to germinate better on softer agar. So if you are using a tsp of agar for 2 tsp of potato flakes and 1/2 cup of water, try making it with half as much agar. So a half teaspoon of agar.

I used a normal recipe and got 1/4 plates to germinate. Then I cut it down and got 8/12. So it definitely helps to make your culture plate a little softer.


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OfflineChowder
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: SloppyJoseph]
    #22989714 - 03/09/16 05:01 PM (8 years, 21 days ago)

My recipe is: 210ml water + 4.3g agar + few drops of honey + 3.5 tsp potato flakes.
Okay, I'll make a softer agar, thanks!

But this doesnt account for the PF cakes....where germination shouldn't be a problem (spores were left to be properly hydrated beforehand)


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OfflineSloppyJoseph
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Chowder]
    #22989726 - 03/09/16 05:06 PM (8 years, 21 days ago)

Yeah that's definitely a stiff ratio.

Maybe your syringes aren't good :shrug: you'll get it dude, just keep questioning your process and make sure you dot your ts and cross your is


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OfflineChowder
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Chowder]
    #22989868 - 03/09/16 05:45 PM (8 years, 21 days ago)

Thats the strange thing - if the syringes weren't good, I wouldn't have gotten two growths. Can it be that simply 99% spores were not viable? How could they get unviable? I thought those fuckers can resist outer space radiation... Especially since it took utmost care to do the prints and store them.

I hope more experienced mycologist can help me out. I have so many questions, and I'm at the end of the line with this.


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InvisibleInocuole
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Chowder]
    #22990323 - 03/09/16 07:55 PM (8 years, 21 days ago)

Could be the stiff agar recipe just making it hard for the spores to germinate possibly.  I doubt the prints are the issue.  I always get weird growth for about 2 transfers until it looks good, but I do a cup of water to 5g agar.


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Offlinemushpunx
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Inocuole]
    #22990707 - 03/09/16 09:59 PM (8 years, 21 days ago)

Yea try a softer agar, or make a spore syringe and inoculate a small (half pint or less) jar of grains. Then fork a colonized grain with your scalpel and drop on agar.

Some spores are just reluctant to germinate on agar. I've been driving myself nuts with a Pan Cyan spores recently.. soft , stiff... different amounts of nutrients, dosent wanna grow :lol:

As for the contams, unless your print is filthy you should be able to pull something clean off it


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Offlinethemadextractor
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: mushpunx]
    #22991136 - 03/10/16 01:16 AM (8 years, 21 days ago)

I've been having this issue as well. Fi ally got some to germ on agar with the softer recipe but still only got one to germinate. I did however get great germination on whole oats with brown rice flower in mini rounds.

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Invisibleblindingleaf
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: themadextractor]
    #22991355 - 03/10/16 06:09 AM (8 years, 20 days ago)

when u swipe ur spores or drop ur spores from a syringe, try making scratches on the agar surface to create little hills and valleys for the spores to land in.  that will surround them with a moist nutritious media.



i think thats why BRF/corn meal plates work so well with spore germination.  its not a flat surface, and the texture kind of "cradles" the spores while they try and germintate.


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OfflineKizzle
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: blindingleaf]
    #22991510 - 03/10/16 07:52 AM (8 years, 20 days ago)

Be sure you make a streak with the spores so that more spores make contact with the agar. Don't just dab them in one place.


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OfflineChowder
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Kizzle]
    #22992618 - 03/10/16 01:32 PM (8 years, 20 days ago)

Thanks all for the good answers.
I will make softer agar and get those spores inside the media by steaking.

But this still doesn't explain the failed germination on PF cakes...


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Offlinemushpunx
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: blindingleaf]
    #22992939 - 03/10/16 03:02 PM (8 years, 20 days ago)

Quote:

blindingleaf said:
when u swipe ur spores or drop ur spores from a syringe, try making scratches on the agar surface to create little hills and valleys for the spores to land in.  that will surround them with a moist nutritious media.



i think thats why BRF/corn meal plates work so well with spore germination.  its not a flat surface, and the texture kind of "cradles" the spores while they try and germintate.






This is great advice man! Its also why I prefer using a wire loop


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OfflineChowder
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: mushpunx]
    #22995640 - 03/11/16 11:08 AM (8 years, 19 days ago)

Any ideas about the PF cakes? Cause this is what makes my germination problem more serious, it's not just on agar.
I'm not sure whether to wait and see if anything will grow (it's been 12 days with barely any signs of growth), or just scrap my current jars and make a new batch with the newly bought spores. But I wonder what would be different? Just the spores?

Thanks guys!!


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Offlinemushpunx
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: Chowder]
    #22996237 - 03/11/16 02:43 PM (8 years, 19 days ago)

Quote:

Chowder said:
Any ideas about the PF cakes? Cause this is what makes my germination problem more serious, it's not just on agar.
I'm not sure whether to wait and see if anything will grow (it's been 12 days with barely any signs of growth), or just scrap my current jars and make a new batch with the newly bought spores. But I wonder what would be different? Just the spores?

Thanks guys!!





Well 12 days isn't terribly long they can take up to 3 weeks. Id give them some more time.

But go ahead and do a round with the new syringe too!


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OfflineWellEndowedPenguin
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Re: [many pics] Countless failed germinations on agar + BRF. Need some pro help. -detailed explanations [Re: blindingleaf]
    #22996336 - 03/11/16 03:07 PM (8 years, 19 days ago)

Quote:

blindingleaf said:
i think thats why BRF/corn meal plates work so well with spore germination.  its not a flat surface, and the texture kind of "cradles" the spores while they try and germintate.




Do what blindingleaf said, here's the link https://www.shroomery.org/forums/showflat.php/Number/22252599
If the spores germinate in the brf plates you can then transfer mycelium to agar plates.

If that fails, I don't think there's going to be much hope. Maybe you could try making a syringe to hydrate the spores and inoculate agar with the syringe (make a little indentation in the agar, so that the spore solution can deposit there), and there's also the sandwich agar tek and forced hydration tek. But you said your prints aren't that old, right?


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