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Liminal_God
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Registered: 06/05/14
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Last seen: 4 years, 2 months
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Isolating Away from Contamination on Agar
#20643942 - 10/01/14 11:18 AM (9 years, 5 months ago) |
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So here's my situation. About a month ago I inoculated a bunch of brf cakes only to find that the syringe was infected. I was worried that my next syringe may be infected as well, so I decided to start working with agar for my next attempt. A few days ago I knocked up 20 petri dishes with the other syringe I had. Within a day I saw promising white growth on all the dishes. Fast forward to today, and my initial suspicions were confirmed, another contaminated syringe as all the growth started to turn greenish. Now, the reason I switched to agar was because I was under the impression that I would be able to isolate healthy mycelium growth away from the infection. Just a glance at them and it seems the infection may be to aggressive to do that. I'm hoping someone will chime in and tell me I can recover from this.
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Sagescruffy
CH
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Re: Isolating Away from Contamination on Agar [Re: Liminal_God]
#20644023 - 10/01/14 11:45 AM (9 years, 5 months ago) |
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Interesting that whatever spores were put to agar germinated within a day. The last pic (far right) on the top row might have had cubensis mycelium on the outer perimeter and that would have been what you'd have cut out and transfered to another plate.
-------------------- Love.
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Callingmehandsome
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Re: Isolating Away from Contamination on Agar [Re: Sagescruffy]
#20644089 - 10/01/14 12:10 PM (9 years, 5 months ago) |
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I have transferred at least a hundred times and all your agar p.dishes look contaminated only one I see with possible potential is pic #7 ( I can see clear enough on some of them ) but it will need to be grown out more then transfer a healthy wedge and grow that out to see how it holds up and if it's needed transfer again until no contams are visible. All it takes is a small piece of healthy myc to transfer
Also if started from spores and you seen growth after 1 day that's not normal you was probably seeing some type bacteria grow
-------------------- F*CK YOU ........ PAY ME! I read this members post and laughed so hard I started tearing up he's scared shitless and you can see it in his post repteur said: Well i don't think you guys are doing the same acid we did in the 70's. I only did it once. IT was called windowpaine. I was on the ride of my life for 16 hours seeing colors and everything else. I would never do it again. It scared the shit out of me. However today i see guys talking about doing 2 hoffmans. You would never do 2 hits of the acid in the 70's you would flip seriously out. Probably fry yourself for life. LSD25 Patients is the key to cultivation success .... Ego can be your downfall.... And shortcuts get you short yields ....... Words of wisdom
Edited by Callingmehandsome (10/01/14 12:16 PM)
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Fivel
Crazy Smurf
Registered: 07/09/14
Posts: 521
Loc: Midwest
Last seen: 2 years, 10 days
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How are you inoculating? Have you tried a swab or inoculation loop? Also, sometimes a fucked syringe will do better on grain than on agar. I have a PE syringe I have been dealing with for a couple weeks now. I put it to agar. Twice. Both were no germination and contam city. I put it to grain jars. Then I put it to anti-bacterial agar using a swab.(Squirted some spore solution on a pressure cooked cotton swab) So far I have growth in one of the grain jars and I have what I believe is germination on the agar plate and it has no visible contams after 5 days which is a win in my book considering the other times I put this syringe to agar is slimed over in less than 2 days.
Looks like there are multiple types of contamination in there. If you ask me I would look at either A) your sterile technique. or B) Where you got the syringe from. Also where did you get your agar? Are you confident in your pour technique if you pour? etc etc. It could be a number of things.
-------------------- "The only way to deal with an unfree world is to become so absolutely free that your very EXISTENCE is an act of rebellion." - Albert Camus Have Orissa Prints for trade! In search of PF albino, PF redspore, PF classic "... the associations between sadism and GAIT (Global Assessment of Internet Trolling) scores were so strong that it might be said that online trolls are prototypical everyday sadists."
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Edmunter
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Re: Isolating Away from Contamination on Agar [Re: Fivel]
#20644159 - 10/01/14 12:36 PM (9 years, 5 months ago) |
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So really transferring the first growth is the best as it has a decent trait of being fast and at this stage it will still have loads of other genetics with it.
RR when growing from pins said to transfer the first growth as quick as poss and then do it again from this transfer. This way you re cleaning and getting the best genetic with it.
Im useless at agar so dont listen to a word I wrtte........
Edited by Edmunter (10/01/14 12:37 PM)
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Liminal_God
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Re: Isolating Away from Contamination on Agar [Re: Fivel]
#20644261 - 10/01/14 01:06 PM (9 years, 5 months ago) |
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Quote:
Fivel said: How are you inoculating? Have you tried a swab or inoculation loop? Also, sometimes a fucked syringe will do better on grain than on agar. I have a PE syringe I have been dealing with for a couple weeks now. I put it to agar. Twice. Both were no germination and contam city. I put it to grain jars. Then I put it to anti-bacterial agar using a swab.(Squirted some spore solution on a pressure cooked cotton swab) So far I have growth in one of the grain jars and I have what I believe is germination on the agar plate and it has no visible contams after 5 days which is a win in my book considering the other times I put this syringe to agar is slimed over in less than 2 days.
Looks like there are multiple types of contamination in there. If you ask me I would look at either A) your sterile technique. or B) Where you got the syringe from. Also where did you get your agar? Are you confident in your pour technique if you pour? etc etc. It could be a number of things.
I worked in a clean empty room, no ventilation, and I did all the work in a SAB. I made the agar(500ml-h2o,9g agar agar, 10g LME) and it was PCed. Agar was poured and left to cool for a day inside the SAB. All my syringes have been from a forum sponsor. I inoculated by flaming the syringe (outside SAB), bringing it inside, cracking petri dish open, poking syringe a little bit into agar and squirting a bit. As you can see on the one dish, my first squirt was a BIG squirt haha. Overall I'm confident in my sterile techniques. What makes me think it was the syringes is that in the syringe I used for the brf jars, I noticed a small white speck floating around inside. And for the syringe I used for the dishes, I noticed a clearish white film surrounding the clumps of spores.
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Liminal_God
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Re: Isolating Away from Contamination on Agar [Re: Edmunter]
#20644720 - 10/01/14 03:25 PM (9 years, 5 months ago) |
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Quote:
Edmunter said: So really transferring the first growth is the best as it has a decent trait of being fast and at this stage it will still have loads of other genetics with it.
RR when growing from pins said to transfer the first growth as quick as poss and then do it again from this transfer. This way you re cleaning and getting the best genetic with it.
Im useless at agar so dont listen to a word I wrtte........
This was my initial impressions of agar work for shrooms. However, researching it further, all I could find was people letting the mycelium inhabit almost the whole plate before selecting specific areas to wedge and transfer.
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Edmunter
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Re: Isolating Away from Contamination on Agar [Re: Liminal_God]
#20644728 - 10/01/14 03:28 PM (9 years, 5 months ago) |
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Try both
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Edmunter
Mr
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Re: Isolating Away from Contamination on Agar [Re: Edmunter]
#20644732 - 10/01/14 03:29 PM (9 years, 5 months ago) |
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Pasty gave me good advice. When making pda to grow. Use low nute to grow quick and high nutes to store.......
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Edmunter
Mr
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Re: Isolating Away from Contamination on Agar [Re: Edmunter]
#20644738 - 10/01/14 03:30 PM (9 years, 5 months ago) |
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Liminal_God
Stranger
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Re: Isolating Away from Contamination on Agar [Re: Edmunter]
#20644812 - 10/01/14 03:58 PM (9 years, 5 months ago) |
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Yeah I had seen and read through the beginning of that thread the other day. I'll have to read some more of it when I get a chance.
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Fivel
Crazy Smurf
Registered: 07/09/14
Posts: 521
Loc: Midwest
Last seen: 2 years, 10 days
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Re: Isolating Away from Contamination on Agar [Re: Liminal_God]
#20644898 - 10/01/14 04:18 PM (9 years, 5 months ago) |
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Try a swab or a loop. Also, You could try violets culturing tek and use a "whole brown rice petri" If it's from a former sponsor I would have to wonder why they are a "former" sponsor. All in all if they syringe is bad I would request another one.
-------------------- "The only way to deal with an unfree world is to become so absolutely free that your very EXISTENCE is an act of rebellion." - Albert Camus Have Orissa Prints for trade! In search of PF albino, PF redspore, PF classic "... the associations between sadism and GAIT (Global Assessment of Internet Trolling) scores were so strong that it might be said that online trolls are prototypical everyday sadists."
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Liminal_God
Stranger
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Re: Isolating Away from Contamination on Agar [Re: Fivel]
#20645029 - 10/01/14 04:44 PM (9 years, 5 months ago) |
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Fivel, what would be the proper procedure for using a swab or loop? I've done a lot of agar work with yeast, and I always use a loop for that, and understand it's use and benefit. But, for spores I'm still not familiar with all the methodology. And they are actually a current sponsor. I contacted them, and still have to wait to hear what they're going to do for me.
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supernewfie
some fella
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Re: Isolating Away from Contamination on Agar [Re: Liminal_God]
#20645065 - 10/01/14 04:52 PM (9 years, 5 months ago) |
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Damn man looks like it was a trich MS syringe
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bodhisatta
Smurf real estate agent
Registered: 04/30/13
Posts: 61,891
Loc: Milky way
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Re: Isolating Away from Contamination on Agar [Re: Liminal_God]
#20645071 - 10/01/14 04:54 PM (9 years, 5 months ago) |
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Quote:
Liminal_God said: Fivel, what would be the proper procedure for using a swab or loop? I've done a lot of agar work with yeast, and I always use a loop for that, and understand it's use and benefit. But, for spores I'm still not familiar with all the methodology. And they are actually a current sponsor. I contacted them, and still have to wait to hear what they're going to do for me.
squirt spore solution onto a cooled loop, flame loop outside of SAB bring into sab cool on agar then squirt spores onto the cooled loop not over the dish, open the dish back up put spores on the dish. all the pictures in your OP are fucked there's nothing to isolate away from.
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Liminal_God
Stranger
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Re: Isolating Away from Contamination on Agar [Re: bodhisatta]
#20645182 - 10/01/14 05:16 PM (9 years, 5 months ago) |
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Gotcha, thanks. There's one thing that I'm going to try and do to isolate. I'm going to try it tomorrow morning and see how it goes. In the mean time I'm just going to get some new dishes ready.
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Fivel
Crazy Smurf
Registered: 07/09/14
Posts: 521
Loc: Midwest
Last seen: 2 years, 10 days
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Re: Isolating Away from Contamination on Agar [Re: bodhisatta]
#20645184 - 10/01/14 05:17 PM (9 years, 5 months ago) |
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same procedure with a swab except I wrap my swabs in a makeshift aluminum foil envelope and pressure cook them. I open the aluminum foil envelope in my sab, flame needle, squirt a drop or two on swab, swipe on agar.
-------------------- "The only way to deal with an unfree world is to become so absolutely free that your very EXISTENCE is an act of rebellion." - Albert Camus Have Orissa Prints for trade! In search of PF albino, PF redspore, PF classic "... the associations between sadism and GAIT (Global Assessment of Internet Trolling) scores were so strong that it might be said that online trolls are prototypical everyday sadists."
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Callingmehandsome
KingDingaLing
Registered: 07/25/14
Posts: 184
Loc: EastSide
Last seen: 8 years, 8 months
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Re: Isolating Away from Contamination on Agar [Re: Edmunter]
#20645579 - 10/01/14 06:38 PM (9 years, 5 months ago) |
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Quote:
Edmunter said: Pasty gave me good advice. When making pda to grow. Use low nute to grow quick and high nutes to store.......
That is good advice for growing and storing. A lot of ppl gave you good advice try a few different ways to get the best result for you. I let mines grow out so I can locate the strongest mycelium. Some ppl transfer right after growth starts some ppl use loops ask a question and you will get a bunch of good proven ways to do things
-------------------- F*CK YOU ........ PAY ME! I read this members post and laughed so hard I started tearing up he's scared shitless and you can see it in his post repteur said: Well i don't think you guys are doing the same acid we did in the 70's. I only did it once. IT was called windowpaine. I was on the ride of my life for 16 hours seeing colors and everything else. I would never do it again. It scared the shit out of me. However today i see guys talking about doing 2 hoffmans. You would never do 2 hits of the acid in the 70's you would flip seriously out. Probably fry yourself for life. LSD25 Patients is the key to cultivation success .... Ego can be your downfall.... And shortcuts get you short yields ....... Words of wisdom
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