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 Diggler
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Registered: 10/03/17
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 Please help me understand what's going on with these APE plates 
#25025174 - 02/26/18 11:18 PM (6 years, 1 month ago) |
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Below are 2 sets of plates. The first are 2 that were inoculated via MS syringe about 3 weeks ago (maybe a little more).
Plate one (which I did all my transfers from)
Plate 2: If you look carefully you can see little shades of brown more on the right side of the dish
Plate 2 side view: Is the brown discoloration contamination or something else?
The 2nd set of plates were inoculated 10 days ago. All transferred from plate 1.
Plate 3:
Plate 4:
Plate 5:
Plate 6:
Plate 7
If you notice, in all but plate 3, the agar is much more translucent and spotty. I think what happened is that I let the jar cool too much after PCing, and when I went to shake/swirl it before pouring, some of the BRF part of the solution may have coagulated towards the bottom of the pour jar (I didn't actually use all of the jar, so this seems right.) Can someone confirm that's what happens when you let an agar pour jar cool too much? I'm almost certain I didn't mess up the recipe.
Anyway, one of the plates in the 2nd set (plate 3) was actually an uncolonized plate that was poured the same time as the 1st set. You can see the diffference in the agar. The mycelium that is growing on that seems to be more robust than the other plates, but I have to think it's because of the better agar, since they were all taken from the same spot on the same dish (dish 1) .
So, can someone with more experience than me look at these plates and let me know what they think and how I should proceed? I did end up inoculating five jars off of the 1st dish. It's going very very slow. Like after 10 days, this is what i have:
I understand the need for patience, but I have the sense that these jars are kind of stalled.
When I inoculated these jars, they were with a tiny little wedge from the dish using a scalpel. Now I'm thinking that because APE is such a notoriously slow colonizer, I should try again with much a much larger piece of mycelium? Can I use any and/or all of the plates in the 2nd group? Does anybody see any contamination that I need to work around?
The first set of dishes that are almost fully colonized: should I wait and see if they pin and then clone? Or does that mycelium look healthy enough to use for jars, even though it's first generation from a MS syringe?
Thanks for any and all replies!
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 elasticaltiger
Like Tigers in Coitus
Registered: 06/24/13
Posts: 8,438
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25025186 - 02/26/18 11:25 PM (6 years, 1 month ago) |
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How long since you inoculated the jars?
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First time growing cakes? DON'T make a Shotgun Fruiting Chamber
The Shmuvbox. - The Old TC's Like it  Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger! No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit. "The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT
Pastywhite's Easy Agar Tek (PastyPlates)
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EZEKIEL 23:20
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: elasticaltiger]
#25025192 - 02/26/18 11:30 PM (6 years, 1 month ago) |
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Quote:
elasticaltiger said:
How long since you inoculated the jars?
At the same time as I did the transfers to the 2nd plates. About 10 days ago. I was advised to do a transfer first "for safety" which is what I did, but I figured it couldn't hurt to take some wedges and inoculate a few jars to see if they would colonize as the same time as I did the transfer to the new plates.
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elasticaltiger
Like Tigers in Coitus
Registered: 06/24/13
Posts: 8,438
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25025197 - 02/26/18 11:34 PM (6 years, 1 month ago) |
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What temperature are the jars at? That's pretty tame colonization for 10 days unless temps are low.
Since you already opened the jars it probably wouldn't hurt to drop a bigger wedge in. But you should also start new jars too.
Might have something to do with your grain prep. I don't see any obvious signs of contamination but if the grain was soaking wet when you loaded the jars that might affect colonization times.
--------------------
First time growing cakes? DON'T make a Shotgun Fruiting Chamber
The Shmuvbox. - The Old TC's Like it Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger! No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit. "The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT
Pastywhite's Easy Agar Tek (PastyPlates)
Tiger Drop Video Demos By munchauzen
Van Gogh would’ve sold more than one painting if he’d put tigers in them.―Bill Watterson
EZEKIEL 23:20
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: elasticaltiger]
#25025227 - 02/26/18 11:59 PM (6 years, 1 month ago) |
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Quote:
elasticaltiger said:
What temperature are the jars at? That's pretty tame colonization for 10 days unless temps are low.
Since you already opened the jars it probably wouldn't hurt to drop a bigger wedge in. But you should also start new jars too.
Might have something to do with your grain prep. I don't see any obvious signs of contamination but if the grain was soaking wet when you loaded the jars that might affect colonization times.
The jars are being stored around 75 degrees.
I'm cool letting those keep colonizing there's a possiblility that they will keep going. But they feel kinda stalled.
I'm going to try some new jars. Which plates should I use and how much mycelium should I transfer per jar? Should I dunk the whole piece of colonized agar from each plate into a jar? Or maybe half the colonized plate?
You think I could use any from those initial plates or should I let them try and pin so maybe I could clone?
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99.99
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25025240 - 02/27/18 12:11 AM (6 years, 1 month ago) |
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That's very very slow what kind of agar are you using
I used premade mea agar from fungi Perfecti...
You might try putting dry powderized horse poo in with yours 50/50
It really helps
But what's not right is the mycelium in your grain it should be taking off regardless of what the agar does
I would try to redo the APE from scrach
This is my recent try with it
https://www.shroomery.org/forums/showflat.php/Number/24810964/page/1/fpart/all
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: 99.99]
#25025249 - 02/27/18 12:16 AM (6 years, 1 month ago) |
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Quote:
99.99 said:
That's very very slow what kind of agar are you using
I used premade mea agar from fungi Perfecti...
You might try putting dry powderized horse poo in with yours 50/50
It really helps
But what's not right is the mycelium in your grain it should be taking off regardless of what the agar does
I would try to redo the APE from scrach
This is my recent try with it
https://www.shroomery.org/forums/showflat.php/Number/24810964/page/1/fpart/all
I'm using 200ml water
3g BRF
4g Agar
My initial plates seemed to do well with this recipe. I think I screwed up the 2nd time around by letting the jar cool too much before pouring. (I think).
If you look at plates 1,2, and then the 3 (the newest), they seem to be doing well with the Agar.
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: elasticaltiger]
#25025259 - 02/27/18 12:21 AM (6 years, 1 month ago) |
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Quote:
elasticaltiger said:
Might have something to do with your grain prep. I don't see any obvious signs of contamination but if the grain was soaking wet when you loaded the jars that might affect colonization times.
I actually think that maybe the WBS is a bit too dry. This time I used a 20 min soak in water that was brought to a rolling boil. I did add a small layer of verm at the bottom, which wasn't in the original tek. But I didn't shake it enough where the verm mixed in with rest of the grain in the jar. I noticed that the WBS didn't expand in the way that I'm used to seeing when I soak it in hot water for 90 min, which is what I usually do.
So maybe the grain is too dry?
I tried the shorter soak time this go around, because in my prior jars, the colonized grain is usually very "stuck together" and hard to scoop out of the jar.
I think next time around I'm going to try a 60 min soak. Kind of "split the difference."
Edited by Diggler (02/27/18 12:31 AM)
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Diggler
Stranger
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Re: Please help me understand what's going on with these APE plates [Re: 99.99]
#25025984 - 02/27/18 10:20 AM (6 years, 1 month ago) |
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This is my recent try with it
https://www.shroomery.org/forums/showflat.php/Number/24810964/page/1/fpart/all
Now you're just showing off. lol Dude that's awesome. I was under the impression that APE was a slow colonizer. Did I get that wrong and that it's only slow to pin?
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Kizzle
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25026098 - 02/27/18 11:19 AM (6 years, 1 month ago) |
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BRF is not completely soluble. You need to filter out the solid bits if you want a nice homogeneous and transparent agar.
As for the plates, #7 is only one that is clearly contaminated. Using a culture that was inoculated directly with spores or a plate that has fully colonized to inoculate spawn is not a good idea as spotting contaminants is difficult. They should only be used for starting a fresh culture on a clean plate.
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: Kizzle]
#25028324 - 02/28/18 09:19 AM (6 years, 1 month ago) |
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Quote:
Kizzle said:
BRF is not completely soluble. You need to filter out the solid bits if you want a nice homogeneous and transparent agar.
As for the plates, #7 is only one that is clearly contaminated. Using a culture that was inoculated directly with spores or a plate that has fully colonized to inoculate spawn is not a good idea as spotting contaminants is difficult. They should only be used for starting a fresh culture on a clean plate.
Thanks for the reply. Where do you see the contamination on Plate 7? BTW the little black spot is actually a weevil that I accidentally missed that was in my container of BRF.
Also, assuming that I can use the rest of the 2nd generation plates to inoculate jars, how much from each plate should I use? Can I use all the mycelium from a single plate for one jar?
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JHOVA
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25028449 - 02/28/18 10:10 AM (6 years, 1 month ago) |
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Quote:
Diggler said:
Quote:
Kizzle said:
BRF is not completely soluble. You need to filter out the solid bits if you want a nice homogeneous and transparent agar.
As for the plates, #7 is only one that is clearly contaminated. Using a culture that was inoculated directly with spores or a plate that has fully colonized to inoculate spawn is not a good idea as spotting contaminants is difficult. They should only be used for starting a fresh culture on a clean plate.
Thanks for the reply. Where do you see the contamination on Plate 7? BTW the little black spot is actually a weevil that I accidentally missed that was in my container of BRF.
Also, assuming that I can use the rest of the 2nd generation plates to inoculate jars, how much from each plate should I use? Can I use all the mycelium from a single plate for one jar?
Agar2grain is slow. Use a whole plate whole, or chopped into 4 pieces.
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: JHOVA]
#25028628 - 02/28/18 12:10 PM (6 years, 1 month ago) |
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Agar2grain is slow. Use a whole plate whole, or chopped into 4 pieces.
What would be the most sterile way to transfer the whole thing (or in quarters) to grain?
Should I loosen it with a scalpel and then hold the whole plate upside down and let it drop? It seems like that may be inviting contamination if I'm hovering over the mouth of the jar with the whole plate, since the outside of the plate isn't sterile.
I've never moved slices that big before. It seems like maybe it would be a challenge to balance pieces that big on a scalpel to move to the jar.
Just trying to be clear before I do it so I don't have any mis-steps that might increase my chances of contamination, so any advice is appreciated.
Thanks!
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JHOVA
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25028668 - 02/28/18 12:33 PM (6 years, 1 month ago) |
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Take the receiving jar and tilt it to one side. Te pocket that you created drop ithe agar in that part
Stab it gently. Drop it in a grain jar. Dont put your hand over it at any time and font flail your arms its easy as that..
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: JHOVA]
#25028691 - 02/28/18 12:45 PM (6 years, 1 month ago) |
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Quote:
JHOVA said:
Take the receiving jar and tilt it to one side. Te pocket that you created drop ithe agar in that part
Stab it gently. Drop it in a grain jar. Dont put your hand over it at any time and font flail your arms its easy as that..
Sorry I'm confused by your description. Not sure what you mean by "the pocket."
I think your saying.... with one hand tilt the grain jar, then in the other hand slice out a quarter, then stab it and move it over to the grain jar and drop it?
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JHOVA
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Re: Please help me understand what's going on with these APE plates [Re: Diggler]
#25029299 - 02/28/18 02:58 PM (6 years, 1 month ago) |
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If you dont tilt your grain jar its dofficult to get an agar wedge anywhere other than on the very top of the jar. Titlting the jar creates a valley where the agar can rest when dropped into the grain jar.
Iso wipe hands, jar
Tilt jar so your grains lean to one side
Unscrew lid
Leave lid loose
Flame scalpel
Gently open your plate
Spear the agar or cut it into pieces
Transfer the agar to the valley you created
Repeat if you cut your agar up
Close the jar
Tilt the jar to bury your agar wedge
Leave it alone until 30% colonization then shake
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RomeoPapa
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Re: Please help me understand what's going on with these APE plates [Re: JHOVA]
#25029321 - 02/28/18 03:06 PM (6 years, 1 month ago) |
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I usually shake and beat the jar like it owes me money after I inoculate with agar wedges. I'm going to try it this way next go round.
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It's better to have it and not need it
Than it is to need it and not have it.
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elasticaltiger
Like Tigers in Coitus
Registered: 06/24/13
Posts: 8,438
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Re: Please help me understand what's going on with these APE plates [Re: RomeoPapa]
#25029342 - 02/28/18 03:15 PM (6 years, 1 month ago) |
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Quote:
RomeoPapa said:
I usually shake and beat the jar like it owes me money
--------------------
First time growing cakes? DON'T make a Shotgun Fruiting Chamber
The Shmuvbox. - The Old TC's Like it Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger! No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit. "The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT
Pastywhite's Easy Agar Tek (PastyPlates)
Tiger Drop Video Demos By munchauzen
Van Gogh would’ve sold more than one painting if he’d put tigers in them.―Bill Watterson
EZEKIEL 23:20
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Diggler
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Last seen: 5 years, 7 months
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Re: Please help me understand what's going on with these APE plates [Re: elasticaltiger]
#25057216 - 03/11/18 09:02 PM (6 years, 1 month ago) |
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Just an update. Here's 2 of the best jars out of the 5.
One thing I learned: If you poor your plates thin, it makes going agar to grain much harder as the agar doesn't really want to stay on the scalpel. Lesson learned.
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Diggler
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Re: Please help me understand what's going on with these APE plates [Re: JHOVA]
#25057248 - 03/11/18 09:19 PM (6 years, 1 month ago) |
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Quote:
JHOVA said:
If you dont tilt your grain jar its dofficult to get an agar wedge anywhere other than on the very top of the jar. Titlting the jar creates a valley where the agar can rest when dropped into the grain jar.
Wouldn't that kind of ensure that the agar is up against the side of the jar? Does it even matter if it is?
Also, it would seem tricky (to me) to tip the jar and then lift the lid with one hand and then manage the agar slice with the other.....if that is what your describing.
In any event, I got it done. Did learn a few things this time around. I don't appear to have any contams but I guess we'll see when I open them to spawn.
Thanks for your input.
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