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Re: AGAR ENVY! (Anything and All things agar!) [Re: MetalPro]
    #28938535 -

Looks like mold from a dirty syringe to me unless you somehow introduced it into the culture.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: PBJ710]
    #28938558 -

I agree. I doubt if there is any cube mycelium on that plate.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: ambigu]
    #28938661 -

Thanks for the confirmation, I had my suspicions.  Any opinion on the leading edge of the spore plant? To me it looks like the T4 plate.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: MetalPro]
    #28938747 -

I don't see healthy mycelium on any of those 3 plates. You're just cleaning up a mold culture IMO.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: PBJ710]
    #28938762 -

Dumpster time 😎


--------------------
A Love Letter to New Growers
The 7 Edicts of Mushroom Cultivation as Decreed by Smellyhobbit

:kodama:


Anyone can get a canopy. But can you get what you want?

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Smellyhobbit]
    #28938786 -

This is some agar I made that didn't show any growth after a month so I used it and have some odd growth for setting a pin on it. Is this from the excess moisture in the plates? I poured them hot, live and learn. Looks like mycelium? just popping up weird.

Edited by Fr3nzy (09/07/24 05:58 PM)

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Fr3nzy] * 1
    #28938882 -

Very suspicious of anything not growing from the pin, unless you rolled it around a lot.


--------------------
LAGM 2026 grow log | My library/trade list

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Fr3nzy] * 2
    #28938895 -

Fr3nzy said:
This is some agar I made that didn't show any growth after a month so I used it and have some odd growth for setting a pin on it. Is this from the excess moisture in the plates? I poured them hot, live and learn. Looks like mycelium? just popping up weird.


All things aside I would take 3 new transfers from ~ 10 o-clock to a new plate

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Re: AGAR ENVY! (Anything and All things agar!) [Re: CocaineBuffet]
    #28939583 -

heyoo agers!

getting back into the game and eager to use my barely used flow hood to up my former-multi-spore game.

got 60 holy Grail dishes, three strains from a Trusted, and some quick questions:

does anyone hold transfers at a higher temp than RT?

Hydrating spores from prints: smart or just make sure you're not making dry agar?


--------------------
Laminar Breeze

Filter: 24x24x5.875 Koch
Pre: Dupont Ultimate 9600 14x20x1
Blower: 1TDT2

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Re: AGAR ENVY! (Anything and All things agar!) [Re: haniblecter] * 1
    #28939598 -

No need to hydrate the spores, just smash them in the agar and they'll germ if they're viable. You can run your plates hotter if you want them to be faster but temperature swings will cause heavy condensation. Heat also will make things more prone to contamination. There's no such thing as a 100% clean dish or grain spawn but letting them colonize at a cooler temperature will give them a better chance.

I would advise against the higher temperature and run everything at room temperature except once in fruiting conditions for certain species, if you wish to do so.

Patience will become your best friend.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: PhenoDreamers] * 1
    #28939705 -

Thanks!

had great success with TiT heaters for my MS innoc jars. was curious if i could shave some time with it.

baby steps at first, i guess


--------------------
Laminar Breeze

Filter: 24x24x5.875 Koch
Pre: Dupont Ultimate 9600 14x20x1
Blower: 1TDT2

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Re: AGAR ENVY! (Anything and All things agar!) [Re: PhenoDreamers]
    #28939758 -

This is my first ever post, so hello everyone! :laugh:

I inoculated 10 plates with p. cubensis using a spore syringe from an online retailer a week ago, and I'm wondering if someone can confirm my thoughts about the 2 plates below. My goal is to get a few plates clean before moving to grain, so I'm looking at doing some transfers to clean everything up before doing that. I don't have the eye for what's healthy and good to transfer yet (or if it's even what I think it is), so here I am.

Not sure if it matters since the agar is clearly feeding whatever's growing, but I made and poured the agar myself. The agar is 450ml water, 9g agar-agar, 8g light malt extract, and a few drops of blue gel food coloring.




I'm thinking on the plate labeled 9-1 1, the growth on the right side might be the mycelium I want, which I say only because it's where I initially started dragging the inoculation loop across the agar and it's white. If that seems right, should I wait a little bit to do a transfer for it to grow more, or is it large enough to go ahead and transfer from the outer edge? I'm not sure about the denser growth at 11 o'clock of the main growth, so I'd probably do a transfer from 6 or 7 o'clock of the main growth.

On the plate labeled 9-1 6 I would like to do a transfer from the topmost growth, maybe around 10 o'clock if anyone agrees that it looks like the right kind of mycelium. I assume the grey/brown at 6 o'clock is contamination of some kind.

As for the rest of the plates, none of them have any growth yet. I figured I would see something after a week, but I understand spores can also take a while longer before showing any growth. I also think I've read syringes in general aren't going to be clean, hence wanting to do a bunch of plates to try to get at least one or two plates of something viable.

Even if nothing from these 2 plates is good, I'm still having fun, so any input is going to be very much appreciated! :laugh:

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Re: AGAR ENVY! (Anything and All things agar!) [Re: OTTY] * 1
    #28939927 -

Are you using a SAB or a flow hood?

Both plates don't look promising. Are you able to obtain prints? I would urge you to get prints because spore syringes are kind of a gimmick to me.

I would start over. See how your plates do when left non-inoculated so we can rule out the agar being the cause of contams

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Re: AGAR ENVY! (Anything and All things agar!) [Re: OTTY]
    #28939936 -

OTTY said:
This is my first ever post, so hello everyone! :laugh:

I inoculated 10 plates with p. cubensis using a spore syringe from an online retailer a week ago, and I'm wondering if someone can confirm my thoughts about the 2 plates below. My goal is to get a few plates clean before moving to grain, so I'm looking at doing some transfers to clean everything up before doing that. I don't have the eye for what's healthy and good to transfer yet (or if it's even what I think it is), so here I am.

Not sure if it matters since the agar is clearly feeding whatever's growing, but I made and poured the agar myself. The agar is 450ml water, 9g agar-agar, 8g light malt extract, and a few drops of blue gel food coloring.




I'm thinking on the plate labeled 9-1 1, the growth on the right side might be the mycelium I want, which I say only because it's where I initially started dragging the inoculation loop across the agar and it's white. If that seems right, should I wait a little bit to do a transfer for it to grow more, or is it large enough to go ahead and transfer from the outer edge? I'm not sure about the denser growth at 11 o'clock of the main growth, so I'd probably do a transfer from 6 or 7 o'clock of the main growth.

On the plate labeled 9-1 6 I would like to do a transfer from the topmost growth, maybe around 10 o'clock if anyone agrees that it looks like the right kind of mycelium. I assume the grey/brown at 6 o'clock is contamination of some kind.

As for the rest of the plates, none of them have any growth yet. I figured I would see something after a week, but I understand spores can also take a while longer before showing any growth. I also think I've read syringes in general aren't going to be clean, hence wanting to do a bunch of plates to try to get at least one or two plates of something viable.

Even if nothing from these 2 plates is good, I'm still having fun, so any input is going to be very much appreciated! :laugh:


both are not worth using. first plate looks like mold second looks like mold ft maybe cubensis. pour plates wait a week or so for contaminants from pouring to develop use the plates select only colonies that look excellent. never settle for mediocrity it's a waste of time and effort. you really shouldn't have to "clean up" an indoor print unless it was disgustingly poorly made, only select the healthiest colonies after they've grown out onto the sterile surface and hopefully away from any random bacteria in proximity to the spores.

drop of spore suspension, drag it across the plate with a loop if you're into that, let it grow and organise a bit once it's germinated and select the fastest and most robust looking growth for subculturing. completely rhizo growth is seldom mold if that helps. if you need to go past t2 you're polishing a turd in my opinion. my t1 is for double checking the candidates taken are reasonably healthy and wedges from t1 go to grain. remember until you actually fruit a culture you know nothing about it except the traits of the genetic line it comes from. not all cubes are bred equally well unfortunately.

truly enmeshed bacteria is nearly undetectable sometimes until it goes to grain so don't worry about it. bacteria often possess flagella or adhere to the hyphae, so subculturing over and over to escape the perceived threat of unseen bacteria is a bit of a fool's errand that many fall into. it's not like we can verify that our cultures are truly axenic anyway, only that they don't present characteristics associated with acute bacteria growth.

may as well drop the food colouring too, it becomes denatured and makes the plate harder to see.


--------------------
The fool who persists in his folly will become wise.

Edited by phenyl (09/08/24 08:18 PM)

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Re: AGAR ENVY! (Anything and All things agar!) [Re: phenyl] * 1
    #28940002 -

Thanks for the responses!

I do work in a SAB, but I've only done it a handful of times over the last few weeks. I did a test run of making the agar and pouring it into a few plates about two weeks ago, just to try to get a bit of practice before wasting spores on the plates. I didn't do anything with those - just wrapped them and set them aside. One had growth and I pitched it, the remaining two didn't have anything since I checked a day ago. Here's a picture of one of those I just took (less food coloring, hence the pale color comparatively :P):




Here's another few of the same batch as my original post, just so you can see:









Either way, maybe it is my technique that needs work. I mixed and boiled the agar, put it in a 500ml media bottle, sterilized the bottle (vented the PC for 10 minutes, then ran at 15psi for 20 minutes, foil on the bottle), let the agar cool to around 125, poured it into my plates in my SAB, trying my hardest to make sure nothing except opening of the media bottle hovers over the plate. 

Here's my setup:



When I use the SAB I wipe everything down inside and out with 70% isopropyl, wear gloves and even a facemask just in case I breathe something into the box. The towel is a new towel each time, and I dampen it before setting the work surface and SAB on it. I flame sterilize the tip of both my syringe and inoculation loop. There's no real draft in the area and no fans at all, and I even block the vents in the room to try to prevent the AC from blowing stuff around. 

I am realizing that I'm dumb as shit and deposited the spore solution into the inoculation loop directly with the syringe over a little waste cup I had for the syringe (they didn't touch or anything, and they were both flame sterilized before and after each plate) and not a drop onto the plate, then spreading it with the loop. Not sure why I did that.

This post by BOD is why I said what I said about syringes https://www.shroomery.org/forums/showflat.php/Number/21922023. They also mention doing 5 to 10 plates to get something viable, then transferring to another dish - that's what I meant by "cleaning it up". Maybe I misunderstood what they meant.

I'll ditch the food color until I get a good plate - that was probably dumb to start, I just liked the color of it I guess. Other than that and the  spore > loop > plate thing instead of spore > plate > loop, anything I could improve on?

Thanks again - sorry for the text wall.

Edit: Spelling.

Edited by OTTY (09/08/24 10:37 PM)

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Re: AGAR ENVY! (Anything and All things agar!) [Re: OTTY]
    #28940020 -

How soon do you wrap your plates after pouring? I’m pouring just above 100*f and still getting considerable condensation. Can’t see shit sometimes! I just got a ffu so I’m hoping that might help blow so of it prior to placing the lid.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Letsfly24]
    #28940026 -

I didn't wrap my test run until they were completely cooled, which was overnight. I didn't wrap the ones from my first inoculation attempt until after they were inoculated. Either way, both stayed in the SAB until they were ready to be dealt with.

I poured both batches around 125f, then let them cool in the SAB. For the ones I ended up inoculating, I slipped them back into their sleeve until I was back and ready to inoculate, so they sat in the SAB for a few days in their original packing sleeve.

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Letsfly24]
    #28940037 -

Letsfly24 said:
How soon do you wrap your plates after pouring? I’m pouring just above 100*f and still getting considerable condensation. Can’t see shit sometimes! I just got a ffu so I’m hoping that might help blow so of it prior to placing the lid.


If you have an FFU pour at 130 and leave them overnight. I pour in an SAB. I just poured 80 plates a couple days ago. Started pouring at 125Β° and left them overnight. Condensation was gone on all but 8 (top 1 of each stack of 10).

There is so much condensation inside the SAB by the time I'm done. More than in the plates. They still dry out.

Edit: pour your agar softer also. Helps a lot with condensation. I use 1% - 1.6% agar depending on the recipe.

Edited by Bigdogg (09/08/24 11:45 PM)

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Re: AGAR ENVY! (Anything and All things agar!) [Re: OTTY]
    #28940043 -

Not your contamination culprit, but 45 minutes is a safer sterilization time for 500 ml bottles.

I don't think loading spore solution onto the inoculation loop instead of the plate first was likely a problem either, though it may have meant both the flamed bits of metal stayed in still air longer after cooling than they would have using a different technique.

The best way IMO to germinate spores from a syringe is with a BRF puck. After it germinates, you can transfer a piece of mycelium to agar. Streaking agar with a drop from a syringe works fine, but definitely do more than one plate.

If you're getting contamination on blank plates, you have an issue with your pouring or handling technique. In a still air box, the usual culprits are hovering non-sterile things over open plates and failing to achieve still air (not waiting long enough for it to settle, making motions that are too abrupt, or silly shit like using a torch or spraying stuff inside the SAB while working in it).

I'd guess one of those is responsible for the satellite mold colony on your plate, but the main suspicious colony could be that or just an unlucky spore from your syringe.

Personally I'd skip the towel and just wipe down the table.


--------------------
LAGM 2026 grow log | My library/trade list

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Re: AGAR ENVY! (Anything and All things agar!) [Re: Bigdogg]
    #28940180 -

Bigdogg said:
Edit: pour your agar softer also. Helps a lot with condensation. I use 1% - 1.6% agar depending on the recipe.


Nice.. I didn't know that trick. I can't wait to try it with my next batch. Thanks!

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