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OfflineTakeatrip
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Registered: 09/10/23
Posts: 195
Last seen: 3 months, 14 days
How to get spore syringe to germinate on agar
    #28676870 - 02/26/24 12:16 PM (10 months, 15 days ago)

I am unsure what happens when I try to get spores to germinate on agar or when transferring mycelium from a BRF cake to agar. I have only had one successful plate with some B+.
I get what I can only describe as white snot on my plates regardless of vendor. I have tried using an inoculation loop with zero success even when I blow torch it beforehand. All I see weeks later is just a line where I went with the loop.
I am currently using BRF cakes to get them to germinate which does actually work and then transferring that mycelium to agar without success also.
I get results that are not covered in mould, just surrounded by a jelly like substance.


When you all order spore syringes what is your method to get your spores to germinate successfully as often as possible and what agar specifically are you using?? I would be grateful for any help

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InvisibleBaba Yaga
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Registered: 09/13/20
Posts: 4,496
Loc: Try 'n' Error Boot Camp
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Re: How to get spore syringe to germinate on agar [Re: Takeatrip]
    #28676877 - 02/26/24 12:28 PM (10 months, 15 days ago)

Yep this is bacteria. Germinating a bacterial MSS on BRF pucks is the way to go. Looks like you are dragging things over to the plate.

How much are you letting the mycelium grow out on the BRF puck before taking the transfer? I would place some spore solution on one side of the plate and wait till the mycelium has grown all the way to the middle or even the other side.


Just to cover the very basics: you are flaming the scalpel and you are not drenching your hands in ISO so it does run down your scalpel?

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OfflineTakeatrip
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Registered: 09/10/23
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Re: How to get spore syringe to germinate on agar [Re: Baba Yaga]
    #28676892 - 02/26/24 12:49 PM (10 months, 15 days ago)

Quote:

Baba Yaga said:
Yep this is bacteria. Germinating a bacterial MSS on BRF pucks is the way to go. Looks like you are dragging things over to the plate.

How much are you letting the mycelium grow out on the BRF puck before taking the transfer? I would place some spore solution on one side of the plate and wait till the mycelium has grown all the way to the middle or even the other side.


Just to cover the very basics: you are flaming the scalpel and you are not drenching your hands in ISO so it does run down your scalpel?



Quote:

Baba Yaga said:
Yep this is bacteria. Germinating a bacterial MSS on BRF pucks is the way to go. Looks like you are dragging things over to the plate.

To answer your question regarding how long I am letting the mycelium grow out on the BRF puck, see these pictures



How much are you letting the mycelium grow out on the BRF puck before taking the transfer? I would place some spore solution on one side of the plate and wait till the mycelium has grown all the way to the middle or even the other side.


Just to cover the very basics: you are flaming the scalpel and you are not drenching your hands in ISO so it does run down your scalpel?


As for dropping a drop or even two onto some agar I just get what looks like gel or whitish gel like substance no mycelium, its super frustrating and has cost me enough in agar and plates etc. I would like a little success now.

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InvisibleBaba Yaga
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Registered: 09/13/20
Posts: 4,496
Loc: Try 'n' Error Boot Camp
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Re: How to get spore syringe to germinate on agar [Re: Takeatrip]
    #28676907 - 02/26/24 12:57 PM (10 months, 15 days ago)

Ah OK, I thought you were using pucks, so you are not having any success with PF-Tek?

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OfflineYahra
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Registered: 03/06/23
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Re: How to get spore syringe to germinate on agar [Re: Takeatrip]
    #28676915 - 02/26/24 12:59 PM (10 months, 15 days ago)

Have you tried water agar? That's agar without nutrients.

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OfflineTakeatrip
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Registered: 09/10/23
Posts: 195
Last seen: 3 months, 14 days
Re: How to get spore syringe to germinate on agar [Re: Yahra]
    #28677572 - 02/26/24 09:56 PM (10 months, 15 days ago)

Quote:

Yahra said:
Have you tried water agar? That's agar without nutrients.



I have watched a video where a guy has around 300ml of water in a jar, the lid has a small breather hole and an injection port hole.
He then adds about 12ml of light corn syrup and stirs it into the water. He then sterilises it for 15 mins at 15 psi in a pressure cooker. Once cool he then adds some spores from a syringe and within a week or so mycelium starts growing in the water.
It looked simple enough but I can’t see why I have to do this when others can put a few drops of spores onto agar and the job is done. This hobby isn’t straight forward and doesn’t always go down the path you expect it to sometimes. I guess some strains just won’t grow on agar? I have tried different agar aswell and still am as I don’t get why B+ was so simple to do and yet others have taken months with zero results, it is interesting but frustrating at the same time.

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InvisibleWyoMX
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Registered: 07/06/15
Posts: 2,351
Loc: PNW
Re: How to get spore syringe to germinate on agar [Re: Takeatrip]
    #28677636 - 02/26/24 11:21 PM (10 months, 14 days ago)

Whoever you watched do that please do not follow what they do. That is called liquid culture not so much agar. And putting spores in that is a terrible thing to do with a snaller success rate. If there's one speck of anything in your syringe which there apparently is since all you get is bacteria on your agar plates, it would ruin the entire liquid culture. I'm not sure the other challenges you're facing but taking spores to a nutritional broth is asking for bad news.

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OfflineTakeatrip
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Registered: 09/10/23
Posts: 195
Last seen: 3 months, 14 days
Re: How to get spore syringe to germinate on agar [Re: WyoMX]
    #28678071 - 02/27/24 11:08 AM (10 months, 14 days ago)

Quote:

WyoMX said:
Whoever you watched do that please do not follow what they do. That is called liquid culture not so much agar. And putting spores in that is a terrible thing to do with a snaller success rate. If there's one speck of anything in your syringe which there apparently is since all you get is bacteria on your agar plates, it would ruin the entire liquid culture. I'm not sure the other challenges you're facing but taking spores to a nutritional broth is asking for bad news.



Ok, well perhaps I will give that a miss then. But surely even from different vendors the results would be different but they are not.
I am experimenting with some different agar this week.
What I find the most frustrating is I have successfully isolated two clean cultures and they are very impressive so I can do it, it’s just these last three are very problematic.

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Invisiblejoze
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Registered: 11/10/20
Posts: 1,056
Loc: PNW
Re: How to get spore syringe to germinate on agar [Re: Takeatrip]
    #28678090 - 02/27/24 11:19 AM (10 months, 14 days ago)

As others have stated, you're getting serious bacterial contamination.

What is your procedure for sterilizing your inoculation loop? You should be flaming until red hot, then allowing it to cool until it stops glowing red. Then, before you use your inoculation loop to pick up spores, you need to open up your agar dish and gently press your inoculation loop against a clean, uncolonized portion of the agar. You will hear a brief sizzle. Now you are ready to pick up some spores.

If you do not properly cool down your inoculation loop, it will be too hot and will burn any cubensis spores in your solution. All you'll be left with is contaminants, because some bacterial endospores can survive these extreme temperatures.

Personally, I love using sterile cotton swabs for spreading spores. I use this TEK to sterilize some cheap cotton swabs. When I need to use one, I just unwrap it from the tinfoil, put a drop of spore solution onto the swab, and then zig-zag it across some agar. Very convenient.


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