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SpeckOnEarth
Doo-Gooder


Registered: 11/21/19
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Loc: Alta California
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Bacteria galore, forge on or start over?
#26379102 - 12/13/19 11:51 AM (4 years, 1 month ago) |
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New guy, so please bear with me. I obtained spore syringes from a Shroomery supporter and commenced with what I learned in Bod’s agar TEK. Used an inoculation loop to start 8 of 10 plates and just dribbled directly from the syringe on the last two plates. After a couple of days there was definite growth of something. Very exciting for me because I’m new to this. Lots of microbiologists where I work, one of which is a trusted friend and I brought a plate for her to check out. Felt a bit deflated when she looked at the plate and said, “looks like bacteria.” A sample under the microscope revealed what looked to me like little beans wiggling around. She did a stain test (I think that’s what she said) and confirmed that the growth is in fact bacteria. I felt that I must have fouled up sterile technique-wise but she said that the fact that there is no other signs of any other little buggers and that the plates that were dribbled in directly from the syringe look exactly the same means that it’s most likely from the spore syringe... almost definite. Apparently my technique with the inoculating loop is laughable, she sent me some papers that I can learn from, but other than that, she advised that I wait to see if there’s any growth that I had hoped for since they should be slower to develop. Then try to isolate that, but she admitted that her expertise is not exactly this hobby and I should pose the question to you all. Sorry it took so long to get to my question, but for those of you familiar and who may have encountered this issue; should I toss these plates and try a syringe from a different source or would you advise waiting and trying to isolate any colonies that might develop? I swear the TEKs I read made it seem so much more cut and dry. Pictured is one of the plates I inoculated with the loop. Thank you for taking the time to read this.
 My question
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The Mycologist
Explorer

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Re: Bacteria galore, forge on or start over? [Re: SpeckOnEarth]
#26379110 - 12/13/19 11:59 AM (4 years, 1 month ago) |
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I trash the bacterial ones.
-------------------- "That you are here—that life exists, and identity; That the powerful play goes on, and you will contribute a verse.” ― Walt Whitman, Leaves of Grass

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Growtech
Stranger
Registered: 11/09/19
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Re: Bacteria galore, forge on or start over? [Re: SpeckOnEarth]
#26379116 - 12/13/19 12:02 PM (4 years, 1 month ago) |
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That’s bacteria most likely from the syringe. Spore syringes are inherently not sterile. I get that same sort of bacteria in probably 40% of syringes. The good news is that the bacteria will stop growing and the (eventual) mycelium will over take it. It’s probably even okay to go syringe straight to grain because the mycelium will most likely overpower it. However, I like to clean it up first personally and since you are already doing agar, I’m guessing you do too. I’d let it germinate and transfer some good mycelium away from the contamination. Might take 2 transfers since you streaked up the plate pretty good, but you might be able to do it in a single transfer.
One of the last syringe to agar plates I did was similar... but also ended up with some of the best rhizomorphic growth I’d ever gotten from a ms start, so I was glad I kept the plate and was worth doing the clean up work.
One thing you might want to do is make a new, “less streaky” plate with that syringe so when mycelium starts growing, you have more clean areas you could transfer away from.
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AyePlus
Stony Danza



Registered: 12/18/14
Posts: 3,393
Loc: Fairfield, Connecticut
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Re: Bacteria galore, forge on or start over? [Re: Growtech]
#26379125 - 12/13/19 12:06 PM (4 years, 1 month ago) |
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You need a new spore syringe, you can try germinating on brf cakes and transferring a bit of the cake back to agar, but you’d be better off with new cleaner spores.
You could also wait and see if any myc pokes out through the bacteria but thats pretty bad.
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The Mycologist
Explorer

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Re: Bacteria galore, forge on or start over? [Re: Growtech]
#26379127 - 12/13/19 12:08 PM (4 years, 1 month ago) |
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Quote:
Growtech said: That’s bacteria most likely from the syringe. Spore syringes are inherently not sterile. I get that same sort of bacteria in probably 40% of syringes. The good news is that the bacteria will stop growing and the (eventual) mycelium will over take it. It’s probably even okay to go syringe straight to grain because the mycelium will most likely overpower it. However, I like to clean it up first personally and since you are already doing agar, I’m guessing you do too. I’d let it germinate and transfer some good mycelium away from the contamination. Might take 2 transfers since you streaked up the plate pretty good, but you might be able to do it in a single transfer.
One of the last syringe to agar plates I did was similar... but also ended up with some of the best rhizomorphic growth I’d ever gotten from a ms start, so I was glad I kept the plate and was worth doing the clean up work.
One thing you might want to do is make a new, “less streaky” plate with that syringe so when mycelium starts growing, you have more clean areas you could transfer away from.
You have to watch out for this tho. Bacteria can ride along with mycelium and can effect yields and flushes.
-------------------- "That you are here—that life exists, and identity; That the powerful play goes on, and you will contribute a verse.” ― Walt Whitman, Leaves of Grass

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StygianKnight
A Mushroom

Registered: 03/12/12
Posts: 2,717
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Re: Bacteria galore, forge on or start over? [Re: Growtech]
#26379131 - 12/13/19 12:11 PM (4 years, 1 month ago) |
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This is the whole point of agar, let the spores germinate and then transfer the mycelium away onto a new plate. With luck you can grab some clean mycelium but transferring bacteria is fine, the goal is to get a plate that’s clean except for the mycelium and possibly bacteria transfer in the center, then allow the mycelium to outrun the bacteria and take another transfer from the clean agar, and put it to a new plate. With luck in only a few transfers you can get a clean sample from a dirty one.
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Growtech
Stranger
Registered: 11/09/19
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Re: Bacteria galore, forge on or start over? [Re: AyePlus]
#26379183 - 12/13/19 12:38 PM (4 years, 1 month ago) |
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Quote:
AyePlus said: You need a new spore syringe, you can try germinating on brf cakes and transferring a bit of the cake back to agar, but you’d be better off with new cleaner spores.
You could also wait and see if any myc pokes out through the bacteria but thats pretty bad.
Well really you are better off not using a spore syringe. But you have to start somewhere. The fact that he's going syringe to agar is the right way to do it. It's not practical to replace a contaminated syringe because a truly clean syringe is pretty rare even from the best sources. You're "replacement" syringe will most likely be contaminated as well. Spend the time and clean it up (a couple transfers and you should hopefully have a clean plate), then never use syringes again. Use clones, LC, master plates/slants, etc. Also some spore prints as backup once you can.
But straight up throwing away a syringe because it has a little bacteria in it is silly.
To give you an example, these are 2 different syringes (from an "official" sponsor here), with what looks like similar bacteria:

A few weeks later, they look like this (obviously 1 didn't germinate):
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TeaforTwo
Flight Attendant




Registered: 09/22/19
Posts: 1,005
Loc: Psychonaut City, Mile Hi!
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Re: Bacteria galore, forge on or start over? [Re: StygianKnight]
#26379202 - 12/13/19 12:47 PM (4 years, 1 month ago) |
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we got a badly bacterial GT syringe, took 4 weeks to show any growth besides bacterial looking initial watermarks on one of three plates, others were tossed at that point. by then brf cakes we also noc'd same time were ready to birth, same GT syringe again, it took 6 weeks to fully colonize. shred 4 cakes to shoebox and finally getting fruits, printing one now.
never used the plate, i see it has a couple knots now, will clone pin if one grows
backside:
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AyePlus
Stony Danza



Registered: 12/18/14
Posts: 3,393
Loc: Fairfield, Connecticut
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Re: Bacteria galore, forge on or start over? [Re: TeaforTwo]
#26379304 - 12/13/19 01:47 PM (4 years, 1 month ago) |
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Yeah thats what agar is for and its totally possible to clean up but that can be hard for someone who just started, Super bacterial spores suck to work with, and a print would probably be superior to a syringe, I misspoke. I’ve never actually used a SS only prints.
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SpeckOnEarth
Doo-Gooder


Registered: 11/21/19
Posts: 13
Loc: Alta California
Last seen: 2 years, 20 days
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Re: Bacteria galore, forge on or start over? [Re: TeaforTwo] 1
#26379305 - 12/13/19 01:48 PM (4 years, 1 month ago) |
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Thank you all so very much for your thoughts! So much knowledge here, this website (and you all) is a godsend. Feeling really encouraged and this whole agar thing just made this venture tremendously more intriguing. However I better look further into bacteria affecting yield. So many variables.
Warm regards!
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The Mycologist
Explorer

Registered: 05/06/16
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Re: Bacteria galore, forge on or start over? [Re: SpeckOnEarth]
#26379326 - 12/13/19 02:03 PM (4 years, 1 month ago) |
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Its just hard to tell when stuff is clean sometimes.
To clarify myself, If you have clean inoculate then your yields will be great.
Bacteria mixed with your inoculate can cause problems.
-------------------- "That you are here—that life exists, and identity; That the powerful play goes on, and you will contribute a verse.” ― Walt Whitman, Leaves of Grass

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SpeckOnEarth
Doo-Gooder


Registered: 11/21/19
Posts: 13
Loc: Alta California
Last seen: 2 years, 20 days
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Re: Bacteria galore, forge on or start over? [Re: The Mycologist]
#26379535 - 12/13/19 03:43 PM (4 years, 1 month ago) |
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Got it. I figure this is a good thing to face as a beginner. I’m going to power through and make as many transfers as possible to get to what I’m hoping for if any of the good guys show up in any of my plates.
Since there’s plenty of volume left in my spore syringe, I do want to start 10 more plates with better inoculation loop technique. I want to try what Growtech advises, making them less streaky.
At the very least I can learn a lot. Best case scenario, as mentioned above, with success I won’t have to do the spore syringe again.
Thanks again all. I’ll post updates in case anyone is interested in the outcome.
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PopaCap
Convicted mycophile


Registered: 07/23/18
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Re: Bacteria galore, forge on or start over? [Re: SpeckOnEarth]
#26379549 - 12/13/19 03:51 PM (4 years, 1 month ago) |
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No need for a loop. Just a couple drops on the plate and then tilt it to spread em’ out.
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AyePlus
Stony Danza



Registered: 12/18/14
Posts: 3,393
Loc: Fairfield, Connecticut
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Re: Bacteria galore, forge on or start over? [Re: PopaCap]
#26379558 - 12/13/19 03:54 PM (4 years, 1 month ago) |
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IMO Your best bet with a dirty syringe is making brf cakes, then you can clone after it fruits or transfer a small piece back to agar, in the second case you can make a shallow cake and use it like agar its harder for bacteria to travel across the 3d surface of brf + verm than the flat wet surface of agar.
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SpeckOnEarth
Doo-Gooder


Registered: 11/21/19
Posts: 13
Loc: Alta California
Last seen: 2 years, 20 days
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Re: Bacteria galore, forge on or start over? [Re: AyePlus]
#26379609 - 12/13/19 04:09 PM (4 years, 1 month ago) |
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Ah thanks Popacap, I’ll do some plates that way too.
I actually bought a pair of those syringes AyePlus, so I’ll do the bed cakes with that second syringe as you advise.
Thanks!!
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StygianKnight
A Mushroom

Registered: 03/12/12
Posts: 2,717
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Re: Bacteria galore, forge on or start over? [Re: SpeckOnEarth]
#26381039 - 12/14/19 10:08 AM (4 years, 1 month ago) |
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As an example here’s a tissue clone that brought along some bacteria with and is now speeding away from the bad area.
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AyePlus
Stony Danza



Registered: 12/18/14
Posts: 3,393
Loc: Fairfield, Connecticut
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Re: Bacteria galore, forge on or start over? [Re: StygianKnight] 2
#26381088 - 12/14/19 10:39 AM (4 years, 1 month ago) |
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Some bacteria is easier to see and get away from than others,
Some are sneaky little fuckers and nothing you do seems to help the myc win out.

Maybe I should do some brf plates for these, but tbh I have enough other shit on my plate that they will probably fall by the wayside.. sometimes throwing in the towel is the best move lol
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YogiBear



Registered: 08/24/19
Posts: 845
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Re: Bacteria galore, forge on or start over? [Re: AyePlus]
#26381273 - 12/14/19 12:50 PM (4 years, 1 month ago) |
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🍄
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CocaineBuffet
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Re: Bacteria galore, forge on or start over? [Re: YogiBear]
#26381279 - 12/14/19 12:54 PM (4 years, 1 month ago) |
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PM me if you want me to send you a spore print. I only have a few but since you are just starting out I think it will save you some time instead of using the syringe.
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YogiBear



Registered: 08/24/19
Posts: 845
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Re: Bacteria galore, forge on or start over? [Re: StygianKnight]
#26382633 - 12/15/19 04:24 AM (4 years, 1 month ago) |
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Quote:
StygianKnight said: As an example here’s a tissue clone that brought along some bacteria with and is now speeding away from the bad area.

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