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OfflineMushroomMommy
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Registered: 08/21/23
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HELP!!! Used suspish agar plates and think I’m just growing contam * 1
    #28511872 - 10/20/23 02:05 PM (3 months, 6 days ago)

Alright, sooooo my friend had bought 2 of these mold test kits a bit ago and never used them so we thought it might be fun to put some unverified LC on them and see what happened.

They looked like this:


We followed the directions on the box except added some additional sterile practice of wearing gloves, using iso70, SAB etc. I was a little skeptical of them as the agar came as a liquid in a bottle that didn’t have a safety seal but, whatever.

She wanted to try her hand at playing mad scientist so I gave her the reins and she got a little excited with the LC and put probably around 1cc in each petri. Afterward she put tape only on 2 sides of the dishes and we put them in the zip bags they came in after sanitizing the bags with iso.

I don’t have a photo from right after we made the dishes but on the surface of the agar, you could see small, opaque pieces of I’m guessing mycelium, similar to what you would see inside of the LC syringe.

I put them on my mushroom shelf and must have bumped them at some point bc when I checked them the next day there was some spilled liquid in the bag. I gloved up and took them out of the bags and taped all the way around the dishes and put them back.

The next day the little opaque bits were growing sort of and in petri #1 there was some white in the center which I assumed meant they were clean.

Day 2
Dish 1:


Day 5
Dish 1:


Dish 2, which has mostly looked the same since Day 2:
This photo is from the bottom of plate bc there’s a label on other side of bag


Day 6:
Dish 1


Dish 2 top view


Is that possibly even mycelium or am I growing covid-20????? I’ve now moved them away from the tubs

Help mushroom wizards!!! 😨


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🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


Edited by MushroomMommy (10/20/23 02:18 PM)


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Invisiblehellofresh
Strangler


Registered: 09/22/23
Posts: 215
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28511890 - 10/20/23 02:12 PM (3 months, 6 days ago)

It looks bacterial. From dish 1 you could try to transfer the white fluffy stuff to another plate and see if it grows out more cleanly.

When you opened the plates did you put your hand over the dish?


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Don't panic


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OfflineMushroomMommy
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Registered: 08/21/23
Posts: 230
Loc: La Luna
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: hellofresh]
    #28511901 - 10/20/23 02:20 PM (3 months, 6 days ago)

Quote:

hellofresh said:
It looks bacterial. From dish 1 you could try to transfer the white fluffy stuff to another plate and see if it grows out more cleanly.

When you opened the plates did you put your hand over the dish?




Can you look again I’ve added/updated a couple photos since you replied I think.

So you think that fluffy white is good stuff and the opaque agar colored booger stuff is bad? Should I trash plate 2?

I told her not to put hand over and from what I could tell standing off to side of SAB, she was mostly careful not to put her hand over the plates.


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🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


Edited by MushroomMommy (10/20/23 02:27 PM)


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OfflineKinoko314
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Registered: 12/16/22
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy] * 2
    #28511915 - 10/20/23 02:30 PM (3 months, 6 days ago)

The boogers are bacteria colonies.  Dish 2 is especially bad.  I think you're free of mold, but the LC must be bacterial.


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OfflineMushroomMommy
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Registered: 08/21/23
Posts: 230
Loc: La Luna
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Kinoko314]
    #28511932 - 10/20/23 02:42 PM (3 months, 6 days ago)

Quote:

Kinoko314 said:
The boogers are bacteria colonies.  Dish 2 is especially bad.  I think you're free of mold, but the LC must be bacterial.




Hiiiiii Kinoko!

Oh noooo if that’s the case, isn’t the white fluff probably bacteria too, since it grew over the boogers? Ref: Compare dish 1 pic on day 2 to the one on day 6>>the booger cluster along the side of the dish on day 2, is now white fuzz blobs where the boogers once were. 🥴

If it IS bacterial, would this have definitely come from the syringe liquid and not from something we did? Like, is it worth putting a couple drops on a verified sterile prepoured LMEA plate I just got? Is there any way to still use the LC or purify it or is repeated myc transfer from plate to plate the only way to clean it up.

The LC came from a Shroomery approved list vendor 🙁


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🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


Edited by MushroomMommy (10/21/23 05:20 PM)


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Invisiblehellofresh
Strangler


Registered: 09/22/23
Posts: 215
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy] * 1
    #28511937 - 10/20/23 02:44 PM (3 months, 6 days ago)

If you are going to do anything do it with plate 1.

Here is how I would try to make 3 transfers to the middle of new plates. Just the tips of the triangles into the growth.


Personally I would make a control plate and do nothing just to see if it contaminates and try the lc again as well. Just open it enough to put the tip of the needle in and put a drop then move it around the plate and see what grows.

Anyways best of luck, if your lc contaminates the 3rd plate that you just stick the tip in it's trash.


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Don't panic


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OfflineKinoko314
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28511947 - 10/20/23 02:51 PM (3 months, 6 days ago)

The myc and the bacteria are pretty well intertwined.  It would be hard to get a clean transfer from it. 

There is myc there, for whatever that's worth.  I think the LC is too bacterial to use though.


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Invisiblehellofresh
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Registered: 09/22/23
Posts: 215
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Kinoko314]
    #28511956 - 10/20/23 02:58 PM (3 months, 6 days ago)

Your probably right it just reminds me of some of my early plates before I realized I was putting my hand over them 🤦. Good to get the agar time in.


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Don't panic


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OfflineMushroomMommy
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Registered: 08/21/23
Posts: 230
Loc: La Luna
Last seen: 2 hours, 45 minutes
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Kinoko314]
    #28512464 - 10/20/23 11:16 PM (3 months, 6 days ago)

Quote:

Kinoko314 said:
The myc and the bacteria are pretty well intertwined.  It would be hard to get a clean transfer from it. 

There is myc there, for whatever that's worth.  I think the LC is too bacterial to use though.




Is there any way to clean or still use the LC? Or no?


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🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


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OfflineKinoko314
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28512468 - 10/20/23 11:28 PM (3 months, 6 days ago)

Well, you could probably still use it for pf-tek.  The grainy structure of the vermiculite makes it hard for bacteria to spread across the cake.


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OfflineMushroomMommy
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Registered: 08/21/23
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Kinoko314]
    #28515194 - 10/23/23 11:42 AM (3 months, 3 days ago)

Quote:

Kinoko314 said:
Well, you could probably still use it for pf-tek.  The grainy structure of the vermiculite makes it hard for bacteria to spread across the cake.




Hmmm I’ve not done PF Tek before. I’ve got (3) pint sized sterilized rye grain jars prepared from another experiment I didn’t end up needing them for.

Could I inoculate the remaining 8cc into 2 or 3 of those jars and see what happens? Or should I just let the syringe sit longer in fridge to give the mycelium time to overtake the bacteria more?


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


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Invisiblehellofresh
Strangler


Registered: 09/22/23
Posts: 215
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28515465 - 10/23/23 04:21 PM (3 months, 3 days ago)

Hey, if your going to put the dirty lc to anything you should try pf tek.

If your up for a time sink/waste you could try to put it to agar again then try some transfers to clean it up. Kinoko314 is probably right it might be really hard to clean up and not worth the time.

Put the tiniest drop possible to agar by lifting enough to stick the needle tip into the middle of the plate. I had all sorts of weird problems with my first agar plates before I got my hand movements down.

Your other grow looks like it's almost finished so you could try to get spores or a clone on agar from it.

If your insistent on putting it to the rye I personally use .5-1ml per pint and 1-2ml per qt with clean lc. Look into top fruiting if you go that route but don't expect much or much help.

Personally I would just toss it and move on.


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OfflineKinoko314
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: hellofresh] * 1
    #28515474 - 10/23/23 04:30 PM (3 months, 3 days ago)

If you really want to try to clean it up you can transfer to water agar.  That's just agar with no nutrients in it at all.  Just water and agar.  The mycelium will get some nutrients from the transfer piece and grow out across surface, albeit very lightly.  You can then take a transfer from the leading edge back to nutritional agar.

You can try it that way if you really want to save it without pf-tek.


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OfflineWhiterabbit016
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Registered: 04/17/23
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Last seen: 4 days, 5 hours
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Kinoko314]
    #28515734 - 10/23/23 07:16 PM (3 months, 3 days ago)

Came here to say water agar. To get two dishes that bad, have to either have major gaps in sterile proc or bad lc.

Can try the pftek but I honestly think you’re going to waste a lot of time and effort and not get a lot out. Go water agar, will take 2-3 weeks longer but you’ll have much better outcome if you’re hellbent on saving this lc.


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InvisibleFungus Mountain
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Registered: 10/16/14
Posts: 1,188
Loc: Front row @ the Freakshow
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Whiterabbit016]
    #28515976 - 10/23/23 09:45 PM (3 months, 3 days ago)

You could try looking up 'Hot Pour' for agar. Basically you would try and clean up a bacterial plate by pouring ~140 degree agar over the the culture in the hope that the myc would recover faster than the bacteria. If/when that happens you could then grab a piece of the myc that recovered and broke thru to another plate.

Hot Pouring Agar


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“Until they became conscious they will never rebel, and until after they have rebelled they cannot become conscious.”
George Orwell, 1984

"If you can't explain it simply, you don't understand it well enough."
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OfflineMushroomMommy
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Registered: 08/21/23
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: Fungus Mountain]
    #28516130 - 10/24/23 03:27 AM (3 months, 2 days ago)

Alright alright, I’ll toss them and test the syringe again with 1-2 drops on a pre-poured agar plate (bought some LMEA color plates from a highly rated seller on Etsy). I only used these plates from the mold kit because my friend had brought them over and no longer needed them.

I have since purchased the materials to make my own LMEA plates, but am waiting on some replacement Petri Dishes, as the ones that I received had a hole in the packaging, thus rendering them no longer sterile.

I’ll set up the SAB and sterilize everything up to my standards and do it myself this time.

I think I mentioned this previously, but the last time, I let my friend do it and also let her convince me that we didn’t need to sanitize the SAB to the extent that I do for my grain inoculation procedure (which she had observed the day before) probably because she just didn’t want to sit through the process again. I can be a bit anal and I’ve got a whole, somewhat lengthy routine 😝. Not to toot my own horn, but maybe my over-the-top, meticulous behavior is what has gotten me midway through my first grow without contam or issues—so, maybe it’s worth something!

:rollsafe:

Or maybe it’s just dumb luck.


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


Edited by MushroomMommy (10/24/23 03:27 AM)


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OfflineSupaThaRipper
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28516248 - 10/24/23 07:19 AM (3 months, 2 days ago)

I agree with white rabbit on using water agar. With water agar, the mycelium will grow faster than the contaminants. Also the principle of a SAB is that actual “still air” is your line of defense and not sterility. However I still sanitize my SAB as well with h202. I use h202 over 70% iso. H202 can do everything ISO does PLUS kill mold. I have exclusively begun to use only H202 rather than ISO


Edited by SupaThaRipper (10/24/23 07:20 AM)


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OfflineMushroomMommy
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Registered: 08/21/23
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Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: SupaThaRipper]
    #28517012 - 10/24/23 06:53 PM (3 months, 2 days ago)

Pour hot agar over the top of the current dish(s)? Should I try it as an experiment or trash them? Here’s a photo of them right now, thinking if there’s bacteria in there I should probably just toss and not let it go airborne, I just feel bad for the mycelium because you can see at least in dish 1, she’s trying real hard to win the battle

:sadanddisappointed:




Update: Dish 1 is sitting on a shelf above my washing machine, inching its way closer to the trash and Dish 2 is in the wheelie bin outside. Currently fighting off getting sick from one of my kids who is currently not holding back on sneezing directly into my face, so handling a dish full of bacteria is giving big ick factor


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


Edited by MushroomMommy (10/24/23 07:01 PM)


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Invisiblehellofresh
Strangler


Registered: 09/22/23
Posts: 215
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: MushroomMommy]
    #28517030 - 10/24/23 07:12 PM (3 months, 2 days ago)

If your up for the work you could try some of these plates with some no pour water agar and try some transfers like the guys above said or use one plate for water agar transfers and hot pour on the other. Either way you need more plates.

Agar is seriously easier than it seems once you do it a few times and it saves lots of money.

:dancingshroom:


Edit:fix link...


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Don't panic


Edited by hellofresh (10/24/23 07:21 PM)


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OfflineMushroomMommy
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Registered: 08/21/23
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Last seen: 2 hours, 45 minutes
Re: HELP!!! Used suspish agar plates and think I’m just growing contam [Re: SupaThaRipper]
    #28517043 - 10/24/23 07:23 PM (3 months, 2 days ago)

Quote:

SupaThaRipper said:I still sanitize my SAB as well with h202. I use h202 over 70% iso. H202 can do everything ISO does PLUS kill mold. I have exclusively begun to use only H202 rather than ISO




As I understand it, h2o2 is just as effective but slower. Needs to sit for ~10m to disinfect whereas iso70 takes like 30s, but obviously h2o2 is less harsh to breathe. Hmmmmm interesting! I’ll knock up a spray bottle for things that I don’t need a quick disinfect on


--------------------
🅃 🄴 🄰 🄼    🄲 🄻 🄸 🄽 🄶 🅆 🅁 🄰 🄿

ABCD stands for Always Be Collecting Data

“I don't know anything about anything, but I know enough about everything to know that you don't know what the fuck you're talking about”


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