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Offlinehedgeclipper
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Registered: 08/02/12
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Last seen: 1 year, 9 months
Agar success! What's next?
    #23804317 - 11/05/16 06:40 PM (7 years, 2 months ago)

I'm trying to do agar strain isolation, rye berrie jars, and monotub fruiting. I've got fresh syzygy spores, 3 year old albino A+spores from my last grow, and some spore syringes I made with Indian Oriza. So for my A+ spores haven't worked with either direct inoculation or hydration in a syringe, and my syzygy haven't worked direct, but I'm trying a syringe now. The Oriza, though, have been going quite well. I got 4 or 5 nice, but very tomentose plates, transfered the most rhizomorphic versions, and I have fully colonzed second transfer ("t2") plates now. Here are some of my second generation of transfers. They're looking much nicer than the first set. Also, I got a 0% contamination rate on this transfer! :cool:





http://imgur.com/a/R0XJw

I have a few questions about strain isolation:

What is the real importance of rhizomorphic vs. tomentose mycellium, and is there any problem with throwing my old plates on grain? I don't see the point of wasting good mycelium.

For this second transfer batch, I used more dilute agar solution, but I also used the most rhizomorphic sectors from my previous set of plates. Not a very good control, I know, but am I getting more rhizomorphic growth due to reduced nutrient content on my medium, or is it because I am isolating something good? Also, my plan was to put some of these plates into jars, and save the nicest one (the top image) for transfer to a next batch of plates. Does this plan make sense? Besides jars, where should I go from here? Should I just keep transferring the best sectors? And once I end up with a minifridge stuffed with colonized plates, what do I do with 'em all?

Are there any caveats to watch out for in agar -> jars?

I'll be using 1 pint jars with plastic lids/polypropylene filters for gas exchange, autoclaved at 15-17 psi. Do I just drop a little piece of colonized agar in there and shake it about? What's the most likely way I'd fuck this up?

Any tips for improving my success with agar colonization for my other two strains?

Also, I am trying to start two other strains from spore prints (one is fresh, the other is 3 years old), but I'm having minimal success with my inoculation loop. I decided to make syringes with them to hydrate them. I haven't tested the fresh ones yet, but the syringes with the old spores aren't colonizing. Anyone have suggestions on spore print -> agar technique?

any help/suggestions would be appreciated. I'll try to get some higher res pics of my plates soon.


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Looking for Albino A+ spores


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Offlinemrmazdarx9
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Re: Agar success! What's next? [Re: hedgeclipper]
    #23804344 - 11/05/16 06:46 PM (7 years, 2 months ago)

Less nutrition usually makes more rizo growth


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InvisiblemushboyMDiscord
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Re: Agar success! What's next? [Re: mrmazdarx9]
    #23804414 - 11/05/16 07:02 PM (7 years, 2 months ago)



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Offlinetump
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Re: Agar success! What's next? [Re: mrmazdarx9]
    #23804516 - 11/05/16 07:28 PM (7 years, 2 months ago)

Tiger drop works well for spore print to agar. Just swab the print and put it in the middle of the agar plate.


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OfflineAlCapone2k
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Re: Agar success! What's next? [Re: mrmazdarx9]
    #23804708 - 11/05/16 08:41 PM (7 years, 2 months ago)

I also face one problem you have: Really good plates I don't want to inoculate with, cause I think I can make them even better with more transfers and maybe even isolating some good genetics.

But at the end it's a stupid decision, cause good Agar Plates are easy to reproduce. I started with about 5 plates, 2 of them were good. Now I have about 10 plates, that are really good and every few days I add 3 or 4 of them by making more transfers.

So at the end you will always have clean and good plates so use them for inoculation.

It would be stupid to store really good plates and just use the "not sooo good"-plates for inoculation. Because the jar colonization is the "risky point" in this hobby, so it's better to take the best plates you have for that.

Just my opintion as a noob :smile:


What I do, if I really like a plate is both: First make a small transfer to another plate to "save" the culture and after that I inoculate a jar with it.


--------------------
Since I am from Germany, my english is not perfect but I try my best :smile:




My small Agar to Oyster Open Air Grow Trek


Edited by AlCapone2k (11/05/16 08:43 PM)


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Offlinehedgeclipper
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Re: Agar success! What's next? [Re: AlCapone2k]
    #23804891 - 11/05/16 09:35 PM (7 years, 2 months ago)

Quote:

mushboy said:
really good tek/write up for agar clean up and such




I understand that, but then why am I trying to isolate rhizo sectors if it's just a function of how nutrient-dense my agar is? I don't get it.

Quote:

tump said:
Tiger drop works well for spore print to agar. Just swab the print and put it in the middle of the agar plate.




I just swabbed my sporeprint with a sterilized nickel inoculation loop and swabbed the agar with it. I could even see bits of black spore aggregates on the surface of the agar, but I got no growth or contamination.

Quote:

AlCapone2k said:
I also face one problem you have: Really good plates I don't want to inoculate with, cause I think I can make them even better with more transfers and maybe even isolating some good genetics.





maybe this is nooby of me too, but I don't see why I shouldn't just keep working on my one good plate, but also make a bunch of jars with it. Otherwise, I'd just be wasting valuable time. I should be able to do 2 or 3 plates and 10 pint jars with one fully-colonized plate, right?

How much agar mycellium do you need to put in a jar for it to colonize?


--------------------
Looking for Albino A+ spores


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OfflineAlCapone2k
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Re: Agar success! What's next? [Re: hedgeclipper]
    #23804940 - 11/05/16 09:54 PM (7 years, 2 months ago)

Quote:



I just swabbed my sporeprint with a sterilized nickel inoculation loop and swabbed the agar with it. I could even see bits of black spore aggregates on the surface of the agar, but I got no growth or contamination.


maybe this is nooby of me too, but I don't see why I shouldn't just keep working on my one good plate, but also make a bunch of jars with it. Otherwise, I'd just be wasting valuable time. I should be able to do 2 or 3 plates and 10 pint jars with one fully-colonized plate, right?

How much agar mycellium do you need to put in a jar for it to colonize?





1. Maybe your sporeprints are too old? Also give them some time, I had germination of the spores on agar after 2 weeks, sometimes they need time :smile: You cool your inoc loop before going into the spore print, right? Because if you flame sterilize it and then directy touch the spore print, you "kill" the spores.

Therefore first cool the inoc loop on the edge of your agar, this will also make the loop sticky so you can grab a bunch of spores with it.

If it doesn't work at all, get some sterile swabs from the pharmacy and try with them, for me they are great and I don't use my inoc loop anymore. Just go over some agar to make the cotton swab "wet" and then grab some spores. For me works well.


2. You could colonize 2 new plates and 10 jars with one fully colonized plate but that's not so smart. First reason is that you will only have a very, very small wedge per jar, which will increase colonization time and therefore increase the risk of contamination. So better go for 4-6 jars per fully colonized plate.

And I also suggest not to let the plate fully colonize, because the edges of a agar plate are a place for hidden contaminations. So better let it just colonize until 7/8 oder even 3/4.

Then just transfer to two new plates small wedges and divide the rest of the plate into 4-6 wedges and colonize your jars. Just cut out the mycelium in the middle and let the edges of the agar stay in the petri dish :smile:



The next step will be Grain 2 Grain anyway, with this method you could make 10 jars out of one fully collonized jar and then 100 and then 1000 :wink: starting with ONE Matser jar. You could read spitball jedis tek.

But first of all I recommend to go on with agar to grain colonization :smile:



Sorry for my english, I'm from germany and it's 5 am here rightnow :laugh:


--------------------
Since I am from Germany, my english is not perfect but I try my best :smile:




My small Agar to Oyster Open Air Grow Trek


Edited by AlCapone2k (11/05/16 09:57 PM)


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Offlinehedgeclipper
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Re: Agar success! What's next? [Re: AlCapone2k]
    #23804998 - 11/05/16 10:19 PM (7 years, 2 months ago)

Thanks for your organized response AlCapone2k

1. I'm pretty sure I cooled my inoculation loop well enough, but maybe I didn't. It's a very very thin piece of Ni though, so the heat dissipates very quickly.

I did two direct inoculations of my fresh (as in recently printed) syzygy spores. One with just a little swipe, and one where I actually put a shitload of spores onto the agar. The one with many spores has actually started to get some fuzz in and around the place where the spores are, but it doesn't really look right, and it took over a week to show up. There was no initial contamination though so I don't know what else it could be.

The old spore print (3 years old) is actually more important to me. I really really liked the mushrooms it came from and I really want to find a way to resurrect them. I've only tried syringes with those spores though. Not direct wipe. Maybe I should try direct wipe.

and yeah, inoc. loop is really meant for liquid cultures. It's not really the best tool for sporeprints.

2. I haven't let my plates fully colonize. I put them in the fridge right after I took the above photographs.

You're saying I should only use the middle of the mycellium agar, not the outer edges, for my jars?

Do I just toss the agar with mycellium on it into the grain and shake, or do I have to slice the mycellium off the top?

No worries about the English. I'm sorry I don't speak German. I love Germany/Austria/Switzerland.


--------------------
Looking for Albino A+ spores


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OfflineAlCapone2k
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Re: Agar success! What's next? [Re: hedgeclipper]
    #23805074 - 11/05/16 10:52 PM (7 years, 2 months ago)

1. If you just inoculated the agar plate one week ago, then just wait for a week or two. It could take 3 weeks until mycelium show after inoculation with spores :smile:

Hard to say, but I think your three years old spore print is lost. You could try direct inoculation with the print but don't expect too much.

2. I'm a noob, so I don't want to tell you what to do, but I have read a lot and I read a lot of times that the edges of a agar plate could hide some contamns, so I do it this way. If i have a mycelium culture colonized frome the middle to the edges to about 3/4 of the plate, then I cut a circle around the mycelium cultre, then cut it into 4 wedges (like a pizza) and then I inoculate the jars. What stays in the plate is just the edge of the plate.

I don't know if this is necessary but I make it this way and it sound logic :P

If you understood if this way, that I only use the middle part of the mycelium, then no, I use the whole mycelium, but the unoclonized edge stays in the petri dish. I hope you understand what I mean :smile:


And no, you don't have neither to shake nor to slice the myc off the top. Just flame sterilize your scalpel, cool it by stabbing into the mycelium-less agar edge to cool down and cut a wedge of agar which the mycelium has fully colonized, stab it with the scalpel like with a spear and carry it to your jar and let it fall into the jar through a small crack by opening the lid a bit.

Then don't shake, just "roll" the jar a bit so that the wedge is at the glass edge of the jar (so that you can see it) and that it's totally covered by grains. Don'T shake. Just shake after 30% of the visible grains are colonized :smile:

This way is how you want it: https://www.shroomery.org/forums/showflat.php/Number/20380256



P.S. I hope you like germany the most and not austria and switzerland :laugh:


--------------------
Since I am from Germany, my english is not perfect but I try my best :smile:




My small Agar to Oyster Open Air Grow Trek


Edited by AlCapone2k (11/05/16 10:53 PM)


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Offlinehedgeclipper
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Re: Agar success! What's next? [Re: AlCapone2k]
    #23849781 - 11/19/16 08:02 PM (7 years, 2 months ago)

heya. My jars are looking pretty nice. Is it time to shake them yet??



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Looking for Albino A+ spores


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Offlinespore-ty
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Re: Agar success! What's next? [Re: hedgeclipper]
    #23850582 - 11/20/16 06:30 AM (7 years, 2 months ago)

Ya shake those bad girls and spank em


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Offlinehedgeclipper
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Re: Agar success! What's next? [Re: spore-ty]
    #23853455 - 11/21/16 07:36 AM (7 years, 2 months ago)

Quote:

spore-ty said:
Ya shake those bad girls and spank em




I slapped em around, put 'em in their place.


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Looking for Albino A+ spores


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