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Offlineavocajoe
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Registered: 01/26/23
Posts: 5
Last seen: 1 year, 6 months
Agar bacterial contam?
    #28185432 - 02/13/23 12:37 PM (1 year, 10 months ago)

Hey y'all, looong time lurker, first time poster. Trying to get some insight on my agar cups.

This is my first run at agar. I followed PGT easy 1,2,3 recipe. 4g corn syrup, 8g agar, 12g potato flakes, 400ml water.  PC'd in my instant pot (I know it's not 15psi) for 60 minutes. Let the cups sit at room temperature for close to a week to allow for any contam to show. I did one drop of MSS in each cup in my SAB. Syringes are from a trusted vendor here. After a few days, where the water solution had been turned a yellow color, which from my research, is usually bacterial. I also did a cotton swab zig zag on 6 leftover cups and those are starting to show itty bitty yellow dots (really hard to see in the picture though, sorry). I also knocked up 8 PF jars with GT and those seem to be growing quickly and healthy.

My questions are these:

1. Does this look bacterial? I have 5 cups of GT and 5 cups of B+ (same vendor) and each cup looks the exact same.

2. If bacterial, is it tied to potentially the syringes? Or maybe my sterile technique? I'm leaning more towards the latter being that 2 different strains/syringes are showing the same thing but same batch of agar.

3. If not bacterial, is it tied to the agar recipe? Should I adjust that in the future?

Thanks in advance.






Edited by avocajoe (02/13/23 12:50 PM)

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OfflineTheMagicConch
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Registered: 09/11/20
Posts: 841
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Last seen: 1 year, 2 months
Re: Agar bacterial contam? [Re: avocajoe]
    #28185577 - 02/13/23 02:07 PM (1 year, 10 months ago)

1. Yes

2. Likely from the syringe, make sure you're flaming the needle and squirting a little bit out to cool it down before you drop it into the dish. Though if bacteria is present in the syringe it'll still grow. After doing that, try squeezing the sides of the syringe to get just a little drop out. Make sure it's shaken well before hand too.

3. You could try a different recipe if you want, but the agar recipe doesn't necessarily mean you'll get bacterial plates more often. Try a recipe with LME.


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"You're not losing your mind, your mind is losing you"

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OfflineSeaSalt
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Registered: 12/08/21
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Re: Agar bacterial contam? [Re: TheMagicConch] * 1
    #28185603 - 02/13/23 02:23 PM (1 year, 10 months ago)

I'd advise making brf pucks. A couple drops of spore solution and you'll have mycellium. Grab some mycellium from the top of the puck and put it on agar. Let the puck colonize and it will pin. Grab the pins and put those on agar.
Take the first mushroom from your cakes and make a spore print.


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Offlineavocajoe
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Registered: 01/26/23
Posts: 5
Last seen: 1 year, 6 months
Re: Agar bacterial contam? [Re: SeaSalt]
    #28185678 - 02/13/23 03:11 PM (1 year, 10 months ago)

Quote:

SeaSalt said:
I'd advise making brf pucks. A couple drops of spore solution and you'll have mycellium. Grab some mycellium from the top of the puck and put it on agar. Let the puck colonize and it will pin. Grab the pins and put those on agar.
Take the first mushroom from your cakes and make a spore print.




I've never thought of pucks till you just said it but it makes perfect sense. I was actually planning on doing exactly that with the cakes once they started pinning but to do it on a smaller scale with a puck didn't cross my mind. I'll def do that and bypass the baccy syringe. I appreciate it!

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Offlineavocajoe
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Registered: 01/26/23
Posts: 5
Last seen: 1 year, 6 months
Re: Agar bacterial contam? [Re: TheMagicConch]
    #28185680 - 02/13/23 03:13 PM (1 year, 10 months ago)

Quote:

TheMagicConch said:
1. Yes

2. Likely from the syringe, make sure you're flaming the needle and squirting a little bit out to cool it down before you drop it into the dish. Though if bacteria is present in the syringe it'll still grow. After doing that, try squeezing the sides of the syringe to get just a little drop out. Make sure it's shaken well before hand too.

3. You could try a different recipe if you want, but the agar recipe doesn't necessarily mean you'll get bacterial plates more often. Try a recipe with LME.




I flamed the needle between each cup and changed needles when switching between the strains. I'll give a LME recipe a shot too. Got plenty of it laying around from my home brewing. Guess I'll just toss these pups. Oh the joys of mycology!

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OfflineHappinessStan
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Registered: 10/10/12
Posts: 1,860
Loc: Worcester, UK
Last seen: 2 days, 4 hours
Re: Agar bacterial contam? [Re: avocajoe]
    #28185719 - 02/13/23 03:48 PM (1 year, 10 months ago)

Transfer some clean mycelium from one your pf jars to agar to clean up.
This is basically how I start any syringe or swab these days. Straight to brf puck, then pull clean myc to agar, then clean up and inoculate.
Like Seasalt said, you can let the puck grow out and clone a pin from the puck.
Try brf pucks, they work.
This is why your pf jars are looking OK but your plates are doing nothing.


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Offlineavocajoe
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Registered: 01/26/23
Posts: 5
Last seen: 1 year, 6 months
Re: Agar bacterial contam? [Re: HappinessStan]
    #28185808 - 02/13/23 04:53 PM (1 year, 10 months ago)

Quote:

HappinessStan said:
Transfer some clean mycelium from one your pf jars to agar to clean up.
This is basically how I start any syringe or swab these days. Straight to brf puck, then pull clean myc to agar, then clean up and inoculate.
Like Seasalt said, you can let the puck grow out and clone a pin from the puck.
Try brf pucks, they work.
This is why your pf jars are looking OK but your plates are doing nothing.




Much appreciated! Got some baby food jars with BRF/verm cooking as we speak. Seems like a more efficient way to get spore to agar with less contam risk.

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