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OfflineNonagon Infinity
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First time working with agar! * 1
    #27063859 - 11/30/20 12:42 AM (3 years, 1 month ago)

Greetings, friends.

Been a little while since I've been active on the forums. I've been doing a lot more reading rather than posting, since I've been dealing with some contamination during my last couple of grows (I was doing spore to grain - never again). Ultimately, I decided to level up my technique: I built a SAB and started working with agar, so I think those two things should help reduce my contamination.

Anywho, I decided to go with pasty plates, since they're cheap and super easy. I made a batch of them on Nov. 26, 2020, PC'd them, let them cool in the PC overnight, and then moved them into my prepped SAB the next evening. On the evening of Nov. 27, 2020, I inoculated each plate with some spores (different varieties - each labeled for convenience).

I have two different questions:

1. From what I've read, most people prefer to just put a single drop of liquid spores in the middle of the agar so that it's obvious where the myc are growing from when they germinate. I had a *really* hard time getting just a single droplet out of the syringes. It seemed like I was either not pressing hard enough and absolutely nothing was coming out, or it sprayed out like a fire hydrant, with no in-between. I was able to get spores on my agar, but it was always way more than just a drop. I'm guessing this increased my probability of contamination, since multispore syringes are never completely sterile. Any suggestions on spore syringe technique so that I can really just get one drop on there?

2. I started seeing something in one of the plates tonight (Nov. 30, 2020) - pic attached. It looks white and fluffy, suggesting it's mycelium, but it seems way too good to be true. It's only been about 48 hours since I inoculated these bad boys, and this plate in particular was inoculated with APE spores, which have a reputation for being slow colonizers. None of the other plates are showing any signs of anything yet, which seems normal (I've read 1-3 weeks for germination on agar is about average, so that's what I expected). The growth is showing near the edge of the agar, which is consistent with where the liquid wound up spraying (see my first question). Is 2 days for germination too good to be true, or am I just incredibly lucky here?


I know there's a lot of condensation in there. I was a bit worried about that at first, but after reading a bit on other posts, I've found that condensation on pasty plates usually goes away within a few days. Indeed, this is better than it was at the time of inoculation.

Feel free to ask any questions if you need more detail and feel free to give me more advice. I'm here to learn!


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OfflineBlueMushies
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Re: First time working with agar! [Re: Nonagon Infinity]
    #27064073 - 11/30/20 07:23 AM (3 years, 1 month ago)

I usually flame the needle and presquirt in a empty container then inoculate, it helps loosen the plunger and also cools down the needle when I inoculate grains through the ship. Also ms can take a week or so to see growth so you most likely have contams or mold growing if it’s showing that early. Just my opinion


Edited by BlueMushies (11/30/20 07:23 AM)


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OfflineSold Out Online
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Re: First time working with agar! [Re: BlueMushies]
    #27064088 - 11/30/20 07:34 AM (3 years, 1 month ago)

About a month ago I started going from spores to agar for the first time. Everything that grew within 3 days ended up being mold. Took closer to a week for mycelium, but that was from spore prints, and these guys might've done better from being hydrated in the syringe.

Anyway, the lesson I learned is to be skeptical of early growth. I'm on T5 now trying to get away from the mean green.


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OfflineBrian Jones
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Re: First time working with agar! [Re: Nonagon Infinity]
    #27064114 - 11/30/20 08:02 AM (3 years, 1 month ago)

I don't have good luck with spore syringes to agar. Hopefully you will, but just in case here's another way to proceed. Grow some shrooms with Bodhi's "Easy AF Cheapest way to get started..."  It's just PF tek cakes that you fruit in the jars with no fruiting chamber. Then clones these to agar. Also take spore prints from some of them.

It's just about foolproof and the easiest way IMO to get from point A to point B.


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: BlueMushies]
    #27064439 - 11/30/20 11:41 AM (3 years, 1 month ago)

Quote:

BlueMushies said:
I usually flame the needle and presquirt in a empty container then inoculate, it helps loosen the plunger and also cools down the needle when I inoculate grains through the ship. Also ms can take a week or so to see growth so you most likely have contams or mold growing if it’s showing that early. Just my opinion



That sounds like a good technique to loosen up the plunger. Don't know how I didn't think of doing that myself.

Yeah, I figured that MS would take up to a week, so it definitely does seem too good to be true. I woke up this morning and it turns out that, out of all the plates I inoculated, the two I inoculated with APE are the only two showing signs of growth. Maybe that's just a dirty syringe. I know the syringe at least contains APE spores, since I've been able to grow some APE fruits using other methods from this syringe, but it might also contain a lot of mold spores. Shoot.

Follow-up question: Is there any way to distinguish mold mycelium from mushroom mycelium on agar (other than the rate of germination)? I thought the whole point of using agar was so that you could see contamination before taking the next step.


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: Sold Out Online]
    #27064444 - 11/30/20 11:43 AM (3 years, 1 month ago)

Quote:

cdnpsychonaught said:
About a month ago I started going from spores to agar for the first time. Everything that grew within 3 days ended up being mold. Took closer to a week for mycelium, but that was from spore prints, and these guys might've done better from being hydrated in the syringe.

Anyway, the lesson I learned is to be skeptical of early growth. I'm on T5 now trying to get away from the mean green.




Thanks for sharing your experience. How did you determine that those growths were mold? Were you able to tell from looking at the agar, or did you have to wait until you actually spawned to find out the bad news?

The mean green is also what I'm trying to deal with. Trich is a nasty motherfucker.


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: Brian Jones]
    #27064448 - 11/30/20 11:45 AM (3 years, 1 month ago)

Quote:

Brian Jones said:
I don't have good luck with spore syringes to agar. Hopefully you will, but just in case here's another way to proceed. Grow some shrooms with Bodhi's "Easy AF Cheapest way to get started..."  It's just PF tek cakes that you fruit in the jars with no fruiting chamber. Then clones these to agar. Also take spore prints from some of them.

It's just about foolproof and the easiest way IMO to get from point A to point B.



Thanks for the advice, man. I actually started out with that tek when I first started cultivating, so I won't even have to re-learn it. I will definitely go with this route if I end up having a really rough ride with spore -> agar.

I'll also have to learn how to clone... but I was going to learn that anyway, eventually!


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Offlineadhoc
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Re: First time working with agar! [Re: Nonagon Infinity]
    #27064988 - 11/30/20 04:29 PM (3 years, 1 month ago)

Also just started working with agar and I have definitely been having the same syringe issues.

I had some luck with making sterilized swabs, squirting from the syringe onto the swab, and then streaking the swab onto plates.  No idea if it's a good idea or if I just got lucky, but I did manage to get something that at least looked like it might be healthy myc.

One thing I have been wondering - if you're only going to agar is there any reason why the spore solution needs to stay in the syringe?  Could I PC an empty jar and lid next time I run it, and then just dump an entire syringe into it?  From there I'd just dab a sterile swab into the solution and streak it on plates.  Would keep it in the fridge when not using. 

I feel like if this were a good idea I'd have probably read about it by now, but is there any obviously stupid reason why this wouldn't work? 

Quote:

Nonagon Infinity said:
Follow-up question: Is there any way to distinguish mold mycelium from mushroom mycelium on agar (other than the rate of germination)? I thought the whole point of using agar was so that you could see contamination before taking the next step.




In the main agar thread someone mentioned recently that a lower-nutrient agar mix will promote rhizomorphic growth, which seems easier to pick out contams on to my newbie eye.  I transferred something from one of my swab plates to a lower-nutrient plate and the growth looks a lot more like the pictures of healthy plates than I was getting before.


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Re: First time working with agar! [Re: adhoc] * 1
    #27065108 - 11/30/20 05:41 PM (3 years, 1 month ago)

I was PCing a shot glass wrapped in foil and injecting about a CC in it to use for inoculation loop and streak plate.  Now I just use a really small mason jar.  I'll PC it with Agar for like 20-30 mins. 



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OfflineSold Out Online
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Re: First time working with agar! [Re: Nonagon Infinity]
    #27065117 - 11/30/20 05:45 PM (3 years, 1 month ago)

Quote:

Nonagon Infinity said:
Quote:

cdnpsychonaught said:
About a month ago I started going from spores to agar for the first time. Everything that grew within 3 days ended up being mold. Took closer to a week for mycelium, but that was from spore prints, and these guys might've done better from being hydrated in the syringe.

Anyway, the lesson I learned is to be skeptical of early growth. I'm on T5 now trying to get away from the mean green.




Thanks for sharing your experience. How did you determine that those growths were mold? Were you able to tell from looking at the agar, or did you have to wait until you actually spawned to find out the bad news?

The mean green is also what I'm trying to deal with. Trich is a nasty motherfucker.




It took me a couple days to realize what was going on. My first clue was that the little white fuzzy guys that showed up didn't show up on the streak. My inoculation loop left a little faint trail that I was able to see if I looked closely. The second tip-off was that they turned green. I've since learned that you want to catch things before the green shows up, because that's the mold sporulating. If you have mycelial growth, and some un-connected white bumps show up, transfer your mycelium quick.


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OfflineSold Out Online
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Re: First time working with agar! [Re: Sold Out Online]
    #27065135 - 11/30/20 05:59 PM (3 years, 1 month ago)

This is a good example of the bad kind of little white dot.

Had to transfer away from it today.



As you can see, hard to tell it's a bad guy if it's the first thing to show up on a plate. It's not the end of the world though. If you get some mycelium germinating you can just grab a little transfer and get it away from the mold. Seeing the mold doesn't automatically mean you need to toss immediately.


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: adhoc]
    #27065530 - 11/30/20 09:55 PM (3 years, 1 month ago)

Quote:

adhoc said:
I feel like if this were a good idea I'd have probably read about it by now, but is there any obviously stupid reason why this wouldn't work? 




Sounds like you're just wasting a bunch of perfectly good spores if you dump an entire syringe onto one agar plate. It only takes one drop from a syringe to germinate on agar. Why use a whole syringe on one plate when you could make hundreds of plates using a single syringe?


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: Sold Out Online]
    #27065535 - 11/30/20 09:58 PM (3 years, 1 month ago)

Quote:

cdnpsychonaught said:
The second tip-off was that they turned green.



That was what I was mostly curious about. The main problem I've been dealing with during my grows is trichoderma. I was curious about whether or not trich still sporulates on agar, or if it waits until there's more FAE. I've read a few posts from trusted cultivators that suggest light is what causes trich to sporulate, so for that reason, it's recommended to keep your agar plates in the dark until they're mostly colonized with cubensis.


Quote:

cdnpsychonaught said:
As you can see, hard to tell it's a bad guy if it's the first thing to show up on a plate. It's not the end of the world though. If you get some mycelium germinating you can just grab a little transfer and get it away from the mold. Seeing the mold doesn't automatically mean you need to toss immediately.



Absolutely. Thanks for all those tips. As I mentioned, this is my first time, so I don't even have any mycelium to transfer to another plate yet. I'm sure I'll get the hang of it with enough persistence and a willingness to learn!


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Offlinetiptrippy
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Re: First time working with agar! [Re: Nonagon Infinity]
    #27065600 - 11/30/20 10:52 PM (3 years, 1 month ago)

Quote:

Nonagon Infinity said:
I was curious about whether or not trich still sporulates on agar





Here's some Trich on agar lol



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Re: First time working with agar! [Re: Nonagon Infinity]
    #27065614 - 11/30/20 11:11 PM (3 years, 1 month ago)

Quote:

Nonagon Infinity said:


Follow-up question: Is there any way to distinguish mold mycelium from mushroom mycelium on agar (other than the rate of germination)? I thought the whole point of using agar was so that you could see contamination before taking the next step.




You can if you pour agar in petris and have a microscope. Otherwise you have to be able recognize subtleties, and change over time as well since our eyes can't make out an indistinct white spot very well.

And as for germination time, it's taken several (6+) weeks for some spores to germinate for me. I used to hate getting spores to germinate more than most things in this hobby. Recently my success has been better, taking far less time and probably more than 50% of the plates I inoculate with a fresh syringe germinate. I had one very recently show signs of growth probably 2-3 days in. I was skeptical that it was cubensis until a few days after that though.

I wouldn't bother with a plate that wasn't showing growth by 7 days in now, just make up more. A good strategy in this hobby is to keep yourself busy, which I haven't been doing for a while but am starting to recently.

Glad to see another KGLW fan. I'll be seeing them at Red Rocks next year


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Edited by Lemgrub (11/30/20 11:11 PM)


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: Lemgrub]
    #27065644 - 12/01/20 12:02 AM (3 years, 1 month ago)

Quote:

Lemgrub said:
I wouldn't bother with a plate that wasn't showing growth by 7 days in now, just make up more. A good strategy in this hobby is to keep yourself busy, which I haven't been doing for a while but am starting to recently.




Yeah, making pasty plates is so easy there's basically no reason not to do it all the time haha. I'm sure I'll get something going soon enough. Appreciate all the advice! I guess I'll just keep an eye on these plates and see what happens.

Quote:

Lemgrub said:
Glad to see another KGLW fan. I'll be seeing them at Red Rocks next year



Fuck yeah, dude. They're the best :smile:


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Re: First time working with agar! [Re: Nonagon Infinity]
    #27067130 - 12/01/20 08:16 PM (3 years, 1 month ago)

Quote:

Nonagon Infinity said:
Quote:

adhoc said:
I feel like if this were a good idea I'd have probably read about it by now, but is there any obviously stupid reason why this wouldn't work? 




Sounds like you're just wasting a bunch of perfectly good spores if you dump an entire syringe onto one agar plate. It only takes one drop from a syringe to germinate on agar. Why use a whole syringe on one plate when you could make hundreds of plates using a single syringe?




Oh no, I was talking about dumping the syringe into a sterilized jar and then use a loop to streak plates as I need them.  I did some searching earlier today and found a few threads where people have done it and it seems to work.  Sorry if my earlier post wasn't clear, I definitely wasn't suggesting to dump an entire syringe onto an agar plate.


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OfflineNonagon Infinity
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Re: First time working with agar! [Re: adhoc]
    #27067447 - 12/02/20 12:26 AM (3 years, 1 month ago)

Update: I am still seeing some pretty stable growth on that plate I photographed. For my first batch, I used APE spores on two plates, both of which are showing the same growth. Either these APEs are uncharacteristically fast colonizers, or this syringe is filled with something nasty.

I made another batch of pasty plates tonight. They're cooling off overnight. I've read advice from some trusted cultivators who recommend letting the plates cool in the pressure cooker, and I've read others who recommend letting the plates cool outside the pressure cooker. I tried inside for my first batch and outside for this batch. I'll see what works better for me.

As I mentioned earlier, I've been having trouble with trich during my last few grows. However, during my most recent grow, I was able to produce one healthy-looking APE fruit before the trich really started taking over. I figured that if this one fruit was strong enough to rise up in the face of a parasitic mold, then it probably had decently trich-resistant genetics. So, I'm going to take a stab at cloning it onto a couple of these plates. I understand that the outside of the fruit is probably contaminated with mold spores at this point, so I'm going to split the mushroom open inside my SAB and take a tissue sample from the inside of the stipe and transfer to a couple of my new pasty plates :smile: Wish me luck!


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