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InvisibleMushenstein
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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21442364 - 03/22/15 09:29 AM (9 years, 1 month ago)

I started agar again yesterday. Everybody seems pretty positive this is the best way to go. My question is if you use agar and say you see a green spot is the entire agar dish bad or can you cut out a slice or myc from a different part of the myc specimen, say on the opposite side of the dish, and use it in your jar, or is the whole dish supposedly contaminated?


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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21443670 - 03/22/15 04:23 PM (9 years, 1 month ago)

Quote:

Jimmy Slump said:
I started agar again yesterday. Everybody seems pretty positive this is the best way to go. My question is if you use agar and say you see a green spot is the entire agar dish bad or can you cut out a slice or myc from a different part of the myc specimen, say on the opposite side of the dish, and use it in your jar, or is the whole dish supposedly contaminated?





If you see a green spot on a dish you don't want to transfer from that dish into a jar, you want to transfer from that dish into another dish until you don't have any weird shit growing alongside it, THEN use that in a jar.


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21443759 - 03/22/15 04:46 PM (9 years, 1 month ago)

You probably wont see a green spot though, I mostly see bacteria on my first plates - looks like spots of slime.

As soon as you see healthy germination transfer a tiny piece of the best looking, leading edge thats futherest away from any contams.

Repeat 2 or 3 more times (or as many as nesscary) until you are confident your culture is clean to put to grains


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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21443769 - 03/22/15 04:49 PM (9 years, 1 month ago)

True that, if you have mold on a plate it's going to also be white, and chances are you'll have a hard time telling the difference between tomentose cube mycelium and mold during the beginning stages.  Watch for anything not white, and also for white growing in places that cube mycelium never touched.  May as well aim for rhizomorphs since other shit doesn't do that and you'll at least know you have cube myc.


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21444163 - 03/22/15 06:31 PM (9 years, 1 month ago)

You say that nothing else rizomorphs? So if it is string like and white its good?

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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21444278 - 03/22/15 07:05 PM (9 years, 1 month ago)

OK, here is my problem(s)! I inoculated these with Shitake and Lion's Mane. They grew this shit. And fast. It looks like cob web but it is rhizomorphic. How is that? Please looks at these pics and tell me what this shit is. Do wood loving myc look like this?

I included a quart jar of Shiitake inoculated spawn and a Jar of colonized grain spawn of PE. The PE has like small traces of black something in the bottom. But the PE came from my own homemade LC. So contam is probable.

At the end I put my last agar attempt. I can see Myc in it, but it turned the green agar back to its original color??? does this look ok? I put green food coloring in it like Pasty says to do but I didn't think the myc would eat it. Do these two agar's look ok to use?


Shiitake and Lions mane PF Tek jars. Is this COB Web, it looks Rhizomorphic? Really, it does!


These next two are PE, from my own LC. Do they look ok?


This is a Shiitake inoculate grain jar. Is this going down hill?


This is agar I put green food coloring in and the myc turned it back to agar color. Does this look ok? Can I use this?


This is a another agar. You can see the green food coloring spot. It's not a contam spot. Does this look ok to use???


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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21444818 - 03/22/15 09:15 PM (9 years, 1 month ago)

The PE, hard to tell but *could* have bacteria it has that kind of creamy look. Give them a shake and smell through the filter

The agar, I cant see the culture but thats weird about it filtering out the food coloring, myc is amazing! With agar, you want to transfer a healthy piece as soon as you see germination, dont want to let the first plate to grow out too far.

Is that the intial plate?


As far as the edibles, Im not familiar with those mycs youd have to have someone else chime in on that


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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21444988 - 03/22/15 09:52 PM (9 years, 1 month ago)

All I'm going to address is that the cob-webby looking jars do not look nearly as rhizomorphic as you suggest.  That's not at all what I think of when I think of rhizomorphs.





Otherwise, that pretty much all looks funky to me.


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21445042 - 03/22/15 10:04 PM (9 years, 1 month ago)

That plate looks like a snowflake, gorgeous


Heres one from me of a plate, pretty rhizo sector and a very tometose sectoring



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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21445050 - 03/22/15 10:05 PM (9 years, 1 month ago)

Quote:

mushpunx said:
That plate looks like a snowflake, gorgeous


Heres one from me of a plate, pretty rhizo sector and a very tometose sectoring






I have some really rhizo plate right now but I haven't taken any pictures of any of that in forever.  This was a better example than mine.


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21446841 - 03/23/15 11:32 AM (9 years, 1 month ago)

On the agar plate, once I inoculate do I need to cover up the needle hole with another piece of micro-pore tape or do I just leave is how it is. Wasn't sure if contams could get in the inoculation hole.


I tried a grain to grain transfer last night. Just as I took the lids off my wife let the damn dog in a and he went running all around me. Is there any chance these would be ok? I didn't have the holes on the glove box covered with the garbage bag. I took it off per your direction.

I like G2G but I it just feels like a lost cause unless you have a flow hood. That's why liquid cultures were so attractive to me. I stoped making the liquid cultures all to together and made another batch of agar last night.

My third question is I want to test the remaining liquid cultures for contamination by putting them on agar. If they are contaminated is the contamination throughout the syringe. Meaning that if I squirt a little on agar and it doesn't appear contaminated is the whole syringe clean. Put another way is it possible that if it is contaminated is it possible that it would not show on 1 agar sample.

Thanks


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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21446953 - 03/23/15 11:56 AM (9 years, 1 month ago)

If a syringe shows contamination on one plate the yea the whole syringe is bad.

With Agar, youre going to have to take the lid off eventually :lol: so no real point in making a hole or injection point. After germination, you are going to need to flame sterilize a scalpel and take a transfer from plate A to plate B.


The dog running around probably didnt help but as long as you used proper sterile technique and controlled, desicive hand motions you will probably be OK.



A still air box is just as usefull a tool as a flow hood. In some ways it is actually preferable to use a SAB. The vast majority of home cultivators dont own or use a flow hood, there are countless growers with much more experience and skill than I have that do all thier work in a SAB with very high sucess rate!


Have you watched RR's Lets grow mushrooms?


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Edited by mushpunx (03/23/15 11:57 AM)

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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21447058 - 03/23/15 12:20 PM (9 years, 1 month ago)

Ill write a few tips on using a still air box.

To prep your SAB, optionally you can give it a quick wipe down with ISO alchohol, but then mix a little dish soap and water in a spray bottle and spritz the walls and floor of the SAB, give the air inside a quick mist.
The idea is that contaminants in the air will settle and stick to the water.

Leave the SAB to settle for a few minutes, in the mean time wash up. I shower before I work, but at least wear fresh clean clothes out of the wash. Scrub your hands and arms like a nurse, put on gloves, and tyvek sleeves (if you dont have them yet get some).

Anything amd everything going inside needs to be wiped down woth a fresh paper towel and ISO. Jars, get the lid and crevice underneath as best you can. Wipe your plates and tools.

Slather your gloves with ISO, flame sterilize any tools between any contact with media.


Inside the SAB, once a jar or plate is open only leave it open as long as you need it. Work fast but steadily, dont rush and be clumbsy.

Dont move your hands over any open lid or jar/plate, try not to swirl up the air use controlled, descicive hand motions.

Dont move anything over an open vessle!


This is all basic sterile procedure, its pretty much the same regardless of working in a SAB or in front of FH. Hope it helps!


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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21447641 - 03/23/15 03:08 PM (9 years, 1 month ago)

Quote:

mushpunx said:
If a syringe shows contamination on one plate the yea the whole syringe is bad.





This is true and not true depending on how hard you're willing to work.  APE and PE syringe are notorious for having more bacteria than a regular cube.  I was never able to get a clean culture out of my APE syringe but I was able to get a small amount of mycelium to spawn on grain, alongside a shit ton of bacteria.  2-3 transfers later, with one of the transfers being a single grain to agar, I had a good clean culture.


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21447677 - 03/23/15 03:18 PM (9 years, 1 month ago)

You answered the question in reverse. I'm trying to detect contam that has not shown itself. If I have a syringe and it has given me a clean agar plate sample is there still a chance the syringe is contaminated? More specifically, if a LC has contam in it, is the contam throughout it or could you get a clean agar plate and think it is ok, but then wind up with contam in your substrate.

Should I do more then one plate with a single syringe. If I only do one plate can I be guaranteed there is no contam in the LC if the plate is clean. I hope I'm clear. I can try to be more specific.

I did 10 plates of agar and inoculated with 10 different syringes. One for each plate. Or one plate for each syringe. I wanted to see which syringes were bad and which one weren't. Is one plate a good enough test for one syringe, or should I do several plates for each syringe?


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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21447889 - 03/23/15 04:13 PM (9 years, 1 month ago)

If an LC has a contam, the whole LC is fucked.  If a syringe has a little bit of bacteria, you can keep trying and trying again.  Ideally you'd want to put the syringe to agar and transfer away from the bacteria but you may have better luck doing what I described in my last post.  Mycelium and bacteria seem to coexist better on grain than on agar.  From there you can transfer a colonized grain to a batch of agar with antibiotic added, or you can use mad transfer skillz to get it without that.  I haven't had antibiotic agar handy til recently and I only use it when I'm at death's doorstep with a culture and I can't afford to fuck around.  (Like when I realize I failed to slant something and the last agar dish I have of that culture has a bacterial contamination off to the side, so I HAVE to save that one)


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Re: And on the 8th day god made Trich!!! [Re: Inocuole]
    #21448097 - 03/23/15 05:07 PM (9 years, 1 month ago)

Oh yea when he said syringe I thought he meant syringe full of LC


A dirty spore syringe can usually be cleaned up on agar. An LC, if you test it and its contam toss it

I sugguest you start a brand new agar culture from spore


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InvisibleMushenstein
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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21448159 - 03/23/15 05:22 PM (9 years, 1 month ago)

OK, I think I got it. I tested 10 syringes of LC, each on 1 pasty agar plate. If it any came out clean I was going to use it on grain. But I guess my best bet would be to go to the agar and try to clean it up with transfers or if it is clean in the agar just use the agar slice.

I was afraid if I tested it on agar and it came up clean I was going to use it on the jar. But I guess it is possible for it to come up clean in the agar and still fuk my grain. I see why agar is the medium of choice


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Re: And on the 8th day god made Trich!!! [Re: Mushenstein]
    #21448316 - 03/23/15 06:14 PM (9 years, 1 month ago)

Cleaning up a liquid culture on agar kind of defeats the purpose of LC unlesd you dont have spores


Do you not have any spores, a couple drops left in a syringe or a print?

I really sugguest you save your agar plates, take a drop of spore solution or a swipe from a print amd inoculate your first plate with that man


10 syringes of LC? You dont store your LC in jars? I really think LC is where all your problemd are coming from man.


Put spores to agar, make 3 or 4 transfers, put agar to grain, G2G

Thats the best way to get clean spawn IMHO


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Re: And on the 8th day god made Trich!!! [Re: mushpunx]
    #21448415 - 03/23/15 06:40 PM (9 years, 1 month ago)

I have a syringe and a half of spores. I made a shit tone of liquid cultures so I would have excess. I went through about 5 or 6 syringes and two liquid cultures from SW. As I ran low I made LC to keep it going. When the jar got full of LC I transferred to syringe so I could store in the frige. My wife would not go for 6 quart jars of white floating shit in piss yellow liquid being stored with the food. So I wrapped up my syringes in foil and bagged them. I wanted to test them for contams, but the process is a bitch to get the LC into a syringe without getting contams. I doubt I was successful on any of them.

But I digress, this is about the best I have going except my PF Tek cakes which after 1 week only have 1 pin.

These are two monotubs I started about a week ago, well exactly a week ago. I used the 50/50 coir method. One has a thick sheet plastic glued and sealed with silicone one the lid. This is of course to see through.

The other is more difficult to see through. It has two holes for sight that were covered with plastic bags and taped. I was using it as a hydroponic Deep Water Culture, then switched to mycology, so he holes were convenient. Let me know what you guys think.



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