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Offlineblack strat
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Check out my agar, please?
    #27035826 - 11/12/20 09:17 PM (14 days, 2 hours ago)

Absolute noob dropping in..

Firstly, hi.  I thought I was being super careful with everything; I followed PGT's agar tek video on YT.  I PC'd my two media bottles (containing the agar) for 30+ minutes @ 15psi.  I used ketchup cups.  I made a SAB.  I wiped EVERYTHING down with iso, multiple times thru-out.  I wore gloves.  I flamed my syringes until red. 

To be completely honest, I don't even know what I'm looking at here.  It's been about 48hrs, and I'm wondering if any of these look like they're supposed to, or even not terrible enough to just throw away and start over.  I used 4 different syringes/4 different strains.  Is it even too early to tell?  I can definitely be more patient, as long as someone with some real knowledge tells me to "be more patient".  I really thought I was careful enough, but maybe I fucked up somewhere.  Should I have sterilized the syringes?  I saw a video, I think it was PGT, where he wrapped a syringe in foil, elevated it off water, and PC'd it.  Maybe I should've done that?  I even iso'd the ketchup cups, although most people think they're clean enough from the package.  I dunno man.  Any feedback is appreciated.  Thanks.







oops, almost forgot - my lamp came in today too :]


Edited by black strat (11/12/20 09:56 PM)


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Offlineludwigs32
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Re: Check out my agar, please? [Re: black strat]
    #27035832 - 11/12/20 09:20 PM (14 days, 2 hours ago)

i dont know squat but mine didnt do anything for almost a week. spore to agar


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Offlinescarabaeus
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Re: Check out my agar, please? [Re: black strat]
    #27035835 - 11/12/20 09:21 PM (14 days, 2 hours ago)

Patience grasshopper. Confucious say let time march on.


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OfflineGrenik
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Re: Check out my agar, please? [Re: scarabaeus]
    #27035998 - 11/12/20 11:56 PM (14 days, 15 minutes ago)

How much from a syringe did you put onto each agar plate?


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Offlinestar_bit
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Re: Check out my agar, please? [Re: Grenik]
    #27036128 - 11/13/20 01:43 AM (13 days, 22 hours ago)

I'm not an expert, but they are looking a bit slimy (bacterial maybe?)


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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27036170 - 11/13/20 02:12 AM (13 days, 21 hours ago)

Quote:

Grenik said:
How much from a syringe did you put onto each agar plate?



7-12 I really aimed for one single drop.

on a few, which I think is apparent, more came out. a lot more. this was my first time so there was a bit of a learning curve. same with the darker agar - I tried to get fancy and make it 'purple', only to fuck it all up by making it closer to black lol. learning curve.

also, how should I be storing them? they're currently lid-side-up, in a closed cabinet. do they need light?  but obviously no air.... ....right?


Edited by black strat (11/13/20 02:15 AM)


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OfflinePhony Phone
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Re: Check out my agar, please? [Re: black strat]
    #27036399 - 11/13/20 07:10 AM (13 days, 17 hours ago)

They all look bacterial. I dont see any recognizable mycelium in them.
Check this thread and this thread.

If the syringes had spores in them, no, you shouldn't sterilize them or you would have killed the spores.

If you made a syringe out of spores, did you take care to:
- Drop the print as clean as possible on a foil? (Foil comes presterilized off the roll, open it in a sab to keep that way)

- Sterilize the syringe? You do that by boiling or pressure cooking some water, then you suck it with the syringe, 3 or so times and you leave the last water suck in the syringe to cool. You then use that water to make a spore syringe with your print.

Also did you:

Pressure cook the donor and receiving containers of the agar for 20-45 mins?

And a tip about sab: after you spray with soap water, let it sit for 30-40 min before using it, aerosolized particles fly through the air during that time and may become a vector of contamination.


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Offlinekarri0n
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Re: Check out my agar, please? [Re: Phony Phone] * 1
    #27037135 - 11/13/20 03:58 PM (13 days, 8 hours ago)

All of the ones that it look like the pooled liquid over them are bacterial. The ones with a single drop you will need to wait probably at least a week to know if it's mycelium, mold, or bacteria. If it grows quickly it's probably mold.

Remember that spore syringes probably have quite a bit of bacteria in them, and if there is water for that bacteria to move around, it will get everywhere. Agar works for isolating mycelium from bacteria because mycelium can move, but bacteria can't, so long as there is no water and you aren't moving the plates around.

Spores take time to germinate, as well. So there will be probably 48 or so hours that nothing "good" can possibly be growing. Once they germinate, then the mycelium will start growing but still, it tends to be slower than bacteria or mold.

I never had any luck with syringe directly to agar. It's recommended  an inoculation loop, or what I use are sterile swabs.

You can take q-tips, wrap in neat little foil packages, and PC them to make sterile swabs. I did this in a jar with a standard modified myco lid.

You can also buy them from medical places or mushroom supply places, or get them from a doctor's office.

You can also rip the plastic off a bread tie, wrap it once around a toothpick, then twist the rest of it into a long handle to make an inoculation loop that you can flame.

Put a drop of spore solution onto a sterile swab and make a neat zig zag over the plate, or

Put a 1cc of spore solution into a sterile shotglass, flame your inoculation loop, dip it in the spores, and make a clean zig zag across the plate.

See these photos. When you make the zig zag, then you can separate the bacterial snots from white, organized mycelium. The bacteria was not able to move across the plate, but the mycelium was, and so I took a transfer from it.



If you wait a week or so and let the plates sit, you might see some healthy mycelium that grows away from bacterial areas. The ones that have water over the whole plate are going to turn completely into boogers most likely.


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Offlineblack strat
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Re: Check out my agar, please? [Re: karri0n]
    #27037232 - 11/13/20 04:56 PM (13 days, 7 hours ago)

Quote:

karri0n said:
All of the ones that it look like the pooled liquid over them are bacterial. The ones with a single drop you will need to wait probably at least a week to know if it's mycelium, mold, or bacteria. If it grows quickly it's probably mold.

Remember that spore syringes probably have quite a bit of bacteria in them, and if there is water for that bacteria to move around, it will get everywhere. Agar works for isolating mycelium from bacteria because mycelium can move, but bacteria can't, so long as there is no water and you aren't moving the plates around.

Spores take time to germinate, as well. So there will be probably 48 or so hours that nothing "good" can possibly be growing. Once they germinate, then the mycelium will start growing but still, it tends to be slower than bacteria or mold.

I never had any luck with syringe directly to agar. It's recommended  an inoculation loop, or what I use are sterile swabs.

You can take q-tips, wrap in neat little foil packages, and PC them to make sterile swabs. I did this in a jar with a standard modified myco lid.

You can also buy them from medical places or mushroom supply places, or get them from a doctor's office.

You can also rip the plastic off a bread tie, wrap it once around a toothpick, then twist the rest of it into a long handle to make an inoculation loop that you can flame.

Put a drop of spore solution onto a sterile swab and make a neat zig zag over the plate, or

Put a 1cc of spore solution into a sterile shotglass, flame your inoculation loop, dip it in the spores, and make a clean zig zag across the plate.

See these photos. When you make the zig zag, then you can separate the bacterial snots from white, organized mycelium. The bacteria was not able to move across the plate, but the mycelium was, and so I took a transfer from it.



If you wait a week or so and let the plates sit, you might see some healthy mycelium that grows away from bacterial areas. The ones that have water over the whole plate are going to turn completely into boogers most likely.




wooooow, that was so much great info, thank you. jus got home from work, and yes, it's looking even more like snot than last night. there are one or two that haven't completely shit the bed. (more like I shit the bed.... anyway....)

I tracked everything you suggested I do, made perfect sense.

Question -- is the reason I can sterilize pretty much everything BUT the water filled syringes because in order to do so, I would need to PC them, which would kill the bacteria, but ALSO kill the spores in the process, too? Basically, it seems like I chose wrong when I chose syringes. They are from a vendor from this site, and everything came sterile-wrapped, etc., the whole 9... So that's what got me thinking, "why tf am I flaming these supposed completely sterile needles??" Seems like I'm jus doing an extra step for nothing.  In fact, by killing the spores near the tip, I'm almost going BACKWARDS in a sense.. yeah?

I'm thinking it's time to buy prints..? I have a shitload of product left, unfortunately. I bought 4 separate syringes/strains and didn't even break 1mL on any of them.  I am FOR SURE gonna do what you suggested.. jus gotta make more agar now.

Another question - the tap water in my city is fucking terrible. I don't drink it. it stains any dishes with white chalky residue. who tf knows what's all in it. so, for my agar mix, I was using bottled 'spring water'.  is this cool? is there better water I could use? distilled, etc..

And being there are so many minerals/chlorine/whatever from the tap, is it even ok to PC using that water?  after I PCd my media bottles, they too were left with white rez on them, and all over inside the actual PCer. I kept their caps on, but obviously loose. Maybe some shit got in those, too?

thanks again for your advice. I will use it!!


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Offlineblack strat
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Re: Check out my agar, please? [Re: Phony Phone]
    #27037237 - 11/13/20 05:00 PM (13 days, 7 hours ago)

Quote:

Phony Phone said:
They all look bacterial. I dont see any recognizable mycelium in them.
Check this thread and this thread.

If the syringes had spores in them, no, you shouldn't sterilize them or you would have killed the spores.

If you made a syringe out of spores, did you take care to:
- Drop the print as clean as possible on a foil? (Foil comes presterilized off the roll, open it in a sab to keep that way)

- Sterilize the syringe? You do that by boiling or pressure cooking some water, then you suck it with the syringe, 3 or so times and you leave the last water suck in the syringe to cool. You then use that water to make a spore syringe with your print.

Also did you:

Pressure cook the donor and receiving containers of the agar for 20-45 mins?

And a tip about sab: after you spray with soap water, let it sit for 30-40 min before using it, aerosolized particles fly through the air during that time and may become a vector of contamination.




I see one of my questions was essentially answered by ya before I asked it, thank you. But yes to your first scenario - I bought the syringes from a vendor here.

pieces are starting to connect and I'm learning from y'all's responses. very much appreciated!


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Offlinekarri0n
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Re: Check out my agar, please? [Re: black strat]
    #27037315 - 11/13/20 05:58 PM (13 days, 6 hours ago)

If you PC'd a spore syringe, the spores in there are now dead. It would be sterile though!

I wouldn't blame the vendor, this is a typical experience. I haven't had the pleasure of working directly from prints for cubensis but I do believe that's what most people move toward pretty quickly. Water is a pain in the ass and bacteria loves it. I took a spore swab from a wild shaggy mane, and put that to agar and got less bacteria than commercially produced, trusted spore syringes. I wouldn't buy more spores if I had 4 syringes with 9CC each though. You have enough spores for a decade of drug use there.



Most of the pros on here use tap water for stuff. I kinda think non-chlorinated would work  marginally better, but haven't tried and city water works for me. I use a brita filter for my family, pets, and plants, and this definitely removes chlorine if you are worried about it.

To get rid of the hard water deposits, use a tablespoon or two of white vinegar in your PC water.

I am not much further along than you are. Your experience is exactly what I did the first time around and  you can read my journal to see exactly that.

Swipe some spores and you will be in business my friend. Mush love.

https://www.shroomery.org/forums/showflat.php/Number/21922023


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

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Edited by karri0n (11/13/20 06:02 PM)


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OfflineGrenik
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Re: Check out my agar, please? [Re: karri0n]
    #27037525 - 11/13/20 08:23 PM (13 days, 3 hours ago)

Good attempt even if you do not get much (any) mycelium. Do not toss them yet. Let them grow and see what happens. You are doing the right thing by trying and asking Q's.

There are a lot of different ways to swab the plates from a syringe, but the picture below is how I hold the syringe. The syringe would have been shaken, held needle up and the barrel flicked with my finger, shaken some more, etc. to break up the spores in the syringe. All of this done outside of the still air box (SAB).

Of course it would have a needle on it, I would be wearing gloves, and both the syringe body and my gloves would have been wiped with alcohol. I use my thumb and forefinger to pull the plunger down. Trying to push the plunger down like you are giving someone a shot does not work for me.



In the SAB, I hold the syringe like in the picture and squeeze out a few drops onto a paper towel (or the towel the SAB is sitting on). If you flame sterilized the needle then you have to squeeze out a few drops until it stops hissing and then I squeeze out a few more drops onto the paper towel (or anyplace not on your agar).

Then remove the agar container top (hold the edges and remove it so that your hand never is above the agar) and move the needle over the agar. Just the needle, not your hand, not the barrel of the syringe, just the tip of the needle.

Most times, there is a little liquid at the end of the needle - a drop that is just forming. If that is the case, then I squeeze the barrel with my pinky and ring finger and that is normally enough to get one drop to form and drop from the needle. If not, then pull the plunger with your thumb and index finger. It will be hard to get one drop, but you should not get more than a few drops.

Whenever possible I use spore prints, but if you are going to grow gourmets like Oyster then you will get mycelium syringes and you will use the process above. However, liquid culture syringes (mycelium syringes) tend to be much cleaner than spore syringes so they are easier to clean up on agar.


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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27037568 - 11/13/20 08:42 PM (13 days, 3 hours ago)

cool cool cool!  thanks man.

had a thought -- in theory, would this be a clean way to do inoculation from syringe to agar plate? ::

since foil comes sterilized in the roll, instead of PCing anything, could I unroll a bit in the SAB -> shoot some spore-water onto the foil from sterile syringe -> use sterilized loop to "dab/grab" some spores from foil surface -> then do the swipe like you guys described?

also karri0n, no sir, I did not PC the syringes, thankfully. I was jus trying to figure all the logic out by typing it.

and please, after the swipe, how do I store?? lid side up, or down? is a closed cabinet ok?

Grenik, for sure, gonna hang on to em for a little bit, see what happens, update you guys with new pics, and go from there. I was super bummed at work when I read some of these first comments confirming it's bacterial. but I'm over it, ready to crack at it again. I'll get this shit eventually hahah.


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OfflineTryna
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Re: Check out my agar, please? [Re: black strat]
    #27037613 - 11/13/20 09:03 PM (13 days, 3 hours ago)

Cool post here.


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Offlinekarri0n
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Re: Check out my agar, please? [Re: Tryna]
    #27037696 - 11/13/20 10:10 PM (13 days, 2 hours ago)

I like that syringe hold technique and may try it next time. They are a bitch to control. I have found better luck holding it in my fist and pushing the plunger with my thumb.... The way a killer would work a needle, not a doctor. This looks like it will offer more control.


I wouldn't try that foil thing. That sounds like too much of a mess and likely to introduce contamination.

I re-read Bod's agar tek and he also mentions dropping a drop of spore solution onto a sterilized loop instead of using a shotglass, that is probably easier. Read through the whole thing again before you do another sesh. https://www.shroomery.org/forums/showflat.php/Number/21922023

Flame the loop, cool it in the blank agar plate, drop spore solution onto the loop, then do the swipe.

Store it lid side up. Lid side down is for growing bacterial cultures.


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

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OfflineGrenik
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Re: Check out my agar, please? [Re: karri0n]
    #27037748 - 11/13/20 10:56 PM (13 days, 1 hour ago)

Good advice on transferring to the loop. I use the 90 mm petri dishes and I was not sure if you had enough room to use a loop on the condiment containers. I did a lot of plates syringe to loop to agar. I now am rather good at getting just one drop onto the agar, so I do a drop to agar and then use the loop to swab it around.

You can get clear, colored agar

You can read through these plates. I make sterile agar and then pour plates. I boil my agar before pouring. I boil it on the stove in a pan instead of the microwave. You can strain it through a cheesecloth if you are not using yeast and that will help clear it. These plates were not strained through cheesecloth.

For coloring I use gel food coloring instead of the liquid you may have used for icing and stuff. It adds a lot of color.

I make the agar a nice dark color when it is in the pot. A nice squirt, more than just a couple drops. When you pour a thin layer it looks lighter.

I do not think the clarity of the agar matters at all to the mycelium, but it may help you identify contaminants easier.

As you may be able to tell, I really like agar! I do not really care if I grow anything as long as I get to play with petri dishes :cool:


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Offlineblack strat
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Re: Check out my agar, please? [Re: karri0n]
    #27037855 - 11/14/20 12:24 AM (12 days, 23 hours ago)

Quote:

karri0n said:
I like that syringe hold technique and may try it next time. They are a bitch to control. I have found better luck holding it in my fist and pushing the plunger with my thumb.... The way a killer would work a needle, not a doctor. This looks like it will offer more control.


I wouldn't try that foil thing. That sounds like too much of a mess and likely to introduce contamination.

I re-read Bod's agar tek and he also mentions dropping a drop of spore solution onto a sterilized loop instead of using a shotglass, that is probably easier. Read through the whole thing again before you do another sesh. https://www.shroomery.org/forums/showflat.php/Number/21922023

Flame the loop, cool it in the blank agar plate, drop spore solution onto the loop, then do the swipe.

Store it lid side up. Lid side down is for growing bacterial cultures.




read it! great info! also read this one, from Stro:
https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1

phenomenal write-up.

(yeahh.. not gonna do the foil thing.. gonna go needle to loop to plate. simple.)


Edited by black strat (11/14/20 12:26 AM)


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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27040069 - 11/15/20 10:00 AM (11 days, 14 hours ago)

Quote:

Grenik said:








this was good, worked well for me man.

I had about 6 or 7 unused agar cups from the fridge, so I made a loop with my xacto and some wire, flamed that and my syringes, put a drop on the loop, and zig-zagged the cups, all inside my SAB w/ iso, gloves, etc..

Hopefully they turn out better than the last 13 did..

ONE out of the 13 though, looks like some mycelium is trying to grow away from the snot - I'll take a pic in a few days for you guys.

one thing I don't think I've read yet is how I should store them. karri0n said lid side up, I appreciate that, but what about light? dark cabinet, or somewhere out in the open?

thanks guys.


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Offlinekarri0n
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Re: Check out my agar, please? [Re: black strat]
    #27040460 - 11/15/20 02:16 PM (11 days, 9 hours ago)

Quote:

black strat said:

one thing I don't think I've read yet is how I should store them. karri0n said lid side up, I appreciate that, but what about light? dark cabinet, or somewhere out in the open?

thanks guys.




Light is beneficial for all stages of growth.

It's not like weed where light is needed for photosynthesis, they just like to know that the light exists. A 6500k CFL bulb or daylight from a nearby window is plenty for both the mycelium and the fruits. LED is not as good as CFL for cubes.

For the plates, Put them on a shelf somewhere that you are less likely to think about moving them and looking at them and they won't be disturbed. Just the normal light in your house is fine for now.


--------------------
Anything written above is just as likely to be accurate as it is to be corrected by someone smarter than me two posts from now.

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Edited by karri0n (11/15/20 02:19 PM)


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Offlinekarri0n
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Re: Check out my agar, please? [Re: karri0n]
    #27040519 - 11/15/20 02:46 PM (11 days, 9 hours ago)

I tried that syringe hold technique today to swipe some spores with a loop as well.

I love that hold, it works much better than anything I have tried so far.

I do think I prefer the sterile swab to the loop though. The loop was tougher to control. I will make one with stiffer wire next time.

Keep us updated on how this works out man! :mushroom2::mushroom2::mushroom2:


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OfflineGrenik
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Re: Check out my agar, please? [Re: karri0n]
    #27040920 - 11/15/20 06:53 PM (11 days, 5 hours ago)

Nice to see you getting a work process that works for you. Just to set some expectations...not every drop will have spores in it. I normally do 4 plates from a syringe and 1 or 2 normally have no growth.  No contamination, no mycelium, just a blank plate. But on the plates that do grow, I always get a good area I can transfer from.

Good luck to you!


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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27041053 - 11/15/20 08:23 PM (11 days, 3 hours ago)

Quote:

Grenik said:
Nice to see you getting a work process that works for you. Just to set some expectations...not every drop will have spores in it. I normally do 4 plates from a syringe and 1 or 2 normally have no growth.  No contamination, no mycelium, just a blank plate. But on the plates that do grow, I always get a good area I can transfer from.

Good luck to you!




yes! this is what really had me thinking last night. I couldn't imagine EVERY drop having spores, so doing needle -> loop -> plate just didn't seem totally guaranteed in my mind. glad you confirmed and gave realistic expectations.

I know I said a few days for a pic lol but I wanna post it now.



just based on my 'luck' so far, this isn't mycelium, is it? :sad:

karri0n, took your advice - with the new plates I'm leaving them where they are now, undisturbed. but I do see the zigzags are already little bacteria-boulevards.. I was a lot more careful this time around too, so I dunno men....

I DO have a couple of prints coming in the mail now though. I wanna try everything lol. this shit is so cool.

thanks again bros.

oh, super amateur question - for an iso spray bottle -- do y'all dilute with water? any ballpark-ratio you could give me please?

and next session i am DEFINITELY gonna try the sterile swab way. just bought 300 cheapy qtips, gonna wrap in foil n PC.... is there a tek for it, or do ya jus sorta make little foil packages n elevate them in PCer, and bada-bing, sterile swabs?

:mushroom2:


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Re: Check out my agar, please? [Re: black strat]
    #27041064 - 11/15/20 08:33 PM (11 days, 3 hours ago)

I think there is a good chance that you have something salvageable. I would make a transfer where I have indicated and try growing it out to see what happens.



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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27041120 - 11/15/20 09:11 PM (11 days, 3 hours ago)

really?? ok, will do, thanks brother.

damn, so that's not mold, huh? it's quite fuzzy.  then again, I don't know jack shit. hah :thumbup:


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Re: Check out my agar, please? [Re: black strat]
    #27041211 - 11/15/20 10:30 PM (11 days, 1 hour ago)

Quote:

black strat said:
is there a tek for it, or do ya jus sorta make little foil packages n elevate them in PCer, and bada-bing, sterile swabs?

:mushroom2:




I didn't see a tek when I did it, just a description. I stuck em in a jar with 4 micropore-covered GE holes, just because I had it sitting around from pf tek. Then PC'd for 20 mins. Then I did see a tek today which basically mirrored the process I used:

https://www.shroomery.org/forums/showflat.php/Number/22556356/fpart/all/vc/1


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Edited by karri0n (11/15/20 10:31 PM)


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Re: Check out my agar, please? [Re: karri0n]
    #27042782 - 11/16/20 09:30 PM (10 days, 2 hours ago)

good evening fellas.

jus PCed 400ml of new agar, waiting for it to come down n hit that sweet temp to pour, fuck, I think Bods tek says 110F? whatever, gotta go look.  wrapped some qtips too.

anyway, please forgive my obsessive questioning, but are we sure this is myc? and not some devious fuckin fuzzy white mold?  and if we're set to go, do you concur with my red marks on where to slice from? not so much on the smaller plate, I think that one should grow a little more.. but at least from the bigger, more occupied plate?

(6 pics, but we're looking at jus 2 separate plates here)



thanks.


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Re: Check out my agar, please? [Re: black strat]
    #27043007 - 11/16/20 11:28 PM (10 days, 43 minutes ago)

Are all of your plates from the same source?

I am still a newbie, I just swabbed my 550th plate tonight. I feel confident that you are in good shape and it is worth transferring. I thought from the beginning you just needed time, but I am still learning also.

I find that mycelium grows with that leading edge that you have in your pics. I find contams to have a smoother, more uniform leading edge. I am sure it is not a perfect way to identify, but that growth looks good to me.  I will try and post some magnified shots so you can see the difference, but it may be a few days before I get to it. Also, mycelium seems lighter and cotton like while contams are more matted and dense.

Still, I am always happy when mushrooms grow or my plates become rhizomorphic. That is the only way I really know.:confused:

I like your choice of transfer choices. I would try the ? location for practice. On the plate with 4 red marks, I would also try the 6:00 position. You could drop the 3:00 one if you only want to do 4. But your original choices are also good spots, so your choice.


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Re: Check out my agar, please? [Re: black strat]
    #27043029 - 11/16/20 11:37 PM (10 days, 34 minutes ago)

It looks to me like you have the right idea, i.e. always transfer AWAY from the contamination. It is obvious to you by now (and everyone else  lol), that you have a bacterial mess. I have salvaged plates just as bad by transfer though, so I say give it a shot.

I always have a jar of anti-bacterial agar on the shelf just for this kind of situation. It consists of the usual agar recipe with the addition of gentamycin sulfate (g sulfate is the anti- bacterial component). It is available as an agar supplement from one of the Shroomery vendors (I can't remember which one at the moment). It is a bit of a pain to mix if you are not familiar with this kind of thing but definitely doable. Premixed anti-bact. agar is available from Paul Stamets' company but I don't do business with him because I think his business practices suck -but that is a topic for another thread.

The above paragraph was not targeted specifically for you, but rather just as some general info for whoever may read this. You may still be able to salvage and be  a winner using what you currently have to work with. Good luck! :mushroom2:


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Re: Check out my agar, please? [Re: scarabaeus]
    #27043041 - 11/16/20 11:44 PM (10 days, 27 minutes ago)

Oh yeah! I forgot to mention. If in the future you have a mold contamination rather than a bacterial one, in my experience it is better to just start over. A mold that is turning colors (usually green) means that it is making spores, and spores float around and get everywhere. At least bacteria doesn't make a 'contamination fountain' like a sporulating mold will.


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Re: Check out my agar, please? [Re: scarabaeus]
    #27043044 - 11/16/20 11:45 PM (10 days, 26 minutes ago)

You can get it here. You can also buy plates from several sources with it already added to the agar.

How did plates 8 and 12 do from the original post?


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Re: Check out my agar, please? [Re: Grenik]
    #27043057 - 11/16/20 11:56 PM (10 days, 15 minutes ago)

Ah yes, shroom supply. I was kinda thinking it was them but wasn't sure. BTW, I have ordered stuff from them in the past and have found them to be quick and reliable. Thanks Grenik :thumbup:


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Re: Check out my agar, please? [Re: scarabaeus]
    #27043097 - 11/17/20 12:43 AM (9 days, 23 hours ago)

you guys are the shit. I'm really digging this community so far. there is SO much information on this site, it's kinda fuckin absurd.

Grenik, yes sir, the substance in question is in fact 'light, and cotton-like' by the looks of it! almost wispy hah. also, when you say 'from the same source', what do ya mean exactly? I poured all 13 of those together, yes, and inoculated the same time, but I used 4 different syringes. I don't know why, I wanted to try em all and see what would happen. there's GT, Dancing Tiger, Brazil, and Mexican Dutch King. the 2 I'm transferring from are both Dancing Tiger. I poured 30 fresh cups of agar tonight, so tomorrow I'll transfer these. thanks for the advice brother.

scar, I had no idea that stuff existed, I will definitely be ordering some. and yeah, it is a mess lol, but it's my first time and fuck it, maybe what better way to start off than trying to see if I can climb my way out of this mess, ya know? to be completely honest, I actually am having a good time with this shit. yeah, of course it'd be awesome if this go-around actually pans out for me, but it's been fun so far either way. and thank you for your input as well man!!

:mushroom2: :mushroom2: :mushroom2: :mushroom2:


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Re: Check out my agar, please? [Re: Grenik]
    #27043100 - 11/17/20 12:47 AM (9 days, 23 hours ago)

Quote:

Grenik said:
You can get it here. You can also buy plates from several sources with it already added to the agar.

How did plates 8 and 12 do from the original post?




hmm, honestly not too sure which is which anymore exactly :/ most of them are ONLY snot still. the 2 above I guess are showing promise, and I'd say there are another maybe 2 or 3 that I'm holding out jus a little bit of hope for, and they're jus running a little late to the party.


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Re: Check out my agar, please? [Re: black strat]
    #27043367 - 11/17/20 08:56 AM (9 days, 15 hours ago)

If that is mycelium you need to transfer asap.


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Re: Check out my agar, please? [Re: IdiotCircusBoy]
    #27044150 - 11/17/20 06:17 PM (9 days, 5 hours ago)

I really hope that's not mold.

I have some growth that looks just like that on one of my plates and I Identified it as mold because it grew straight from the edge and quickly, and it has that hairy "brillo" look.

I do my photos up through the bottom of the plate since my no-pours don't have great clarity and are tough to see on camera through the tops and sides, but I will explain what I am talking about best I can.



This grew on this plate 1 day after inoculating my plates, starting from the far left edge in the first pic and from the bottom in the second. The two little circles or "bubbles" are new, and might be bacteria, or even mushroom mycelium that germinated under the mold colony.



Side view:


Note how brillo, wispy, hairy it looks, and also how quickly it grew. It appeared as a tiny spot in the corner, two days before the mycelium in the following pics appeared multiple places, and has consumed almost the whole plate, while the mycelium has the one larger colony but is mostly moving slower.



Side view:


The mycelium is also cottony and fluffy. However it seems to be thicker, and doesn't have a brillo hairy appearance moving in wild directions. I also notice that cubensis mycelium tends to eat the food coloring out of the agar, turning it to its natural brown color(I used grainwater, it will be white if you used flour) and mold doesn't.

I really hope I was able to detail the difference in appearance here. I don't know that what you have is mold or mycelium, but I wish you the best of luck and just hope I can help.

The biggest way to tell for me is speed of growth, because cubensis mycelium appearance really can be pretty variable. Mycelium is slower than mold.


Edited by karri0n (11/17/20 06:32 PM)


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Offlineblack strat
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Re: Check out my agar, please? [Re: karri0n]
    #27044441 - 11/17/20 09:17 PM (9 days, 2 hours ago)

fuck....

man, I just really really do not know. I did the transfers a few hours ago.  all I can say for a fact is that at the very outermost edges of my 'white stuff', it begins to straighten out. I have no clue if that means anything. also, it always seems to be growing away from the bacteria. damnit, who the fuck knows. I don't even know how I am supposed to tell.


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Re: Check out my agar, please? [Re: black strat]
    #27044454 - 11/17/20 09:26 PM (9 days, 2 hours ago)

yeah, now that I'm looking online at pics, I think it's cobweb mold. awesome.

basically just wasted 15 cups of agar carefully transferring different pieces of cobweb mold. wow. I am saaaalty.

edit: ok, so here is one of my newer cups, I swiped with an inoc loop a few days ago..


if what I had earlier is cobweb, then this most certainly is too. it's growing/looking EXACTLY how those others did at first when they started. I don't fuckin get it. how can none.. out of like almost 40 cups.. none of them give me even a hint of myc??

fuck. I'm done for the night. this is such a bummer.

edit again: jus read that putting peroxide on cobweb mold eats at it like wet cotton candy. dunno how accurate that it. swabbed some peroxide on the original cups I took the transfers from.... nothing happened. jus sat there kinda. who knows. who the fuck knows lol.


Edited by black strat (11/17/20 10:25 PM)


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Re: Check out my agar, please? [Re: black strat]
    #27044562 - 11/17/20 10:43 PM (9 days, 1 hour ago)

Hmmm

You are probably putting too much material on the plate. One drop on the inoculation loop and spread it around. A typical inoculation loop holds 10 microliter of liquid.  So 1 mL would be enough for 100 plates. 1 drop is about 50 microliter, so 5x the amount you need. 

Mycelium grows and spreads. It may start slower than mold, but it normally spreads faster than contams. You want a very small sample and let the mycelium run, then transfer from the edges.

Let’s look at those transferred samples in a week. The original plates that you swabbed with peroxide will probably die, that does not prove cobweb, it proves peroxide is bad. 

Deep breath.

Switch to spore prints if the syringes concern you.


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Edited by Grenik (11/17/20 10:46 PM)


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Offlineblack strat
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Re: Check out my agar, please? [Re: Grenik]
    #27044637 - 11/17/20 11:50 PM (9 days, 21 minutes ago)

:tongue2:

I was dying reading that reddit post. especially because I AM that noob hahah. man, I feel dumb. but you know, the only reason I went with the ol cobweb theory is because shroomscout's right - it keeps getting repeated over and over, propagating that paranoia, then my stupid ass sees all that, and suddenly "wahh, I have cobweb!!" lol, oh boy.

alright. I'm good. gonna leave em alone like you said. and yeah man, my prints should be here any day now, can't wait. thanks bro.


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Re: Check out my agar, please? [Re: black strat]
    #27044679 - 11/18/20 12:53 AM (8 days, 23 hours ago)

Stay away from peroxide. It's toxic to any fungus, including mycelium.

Those swipe Plates still look like too much water to me.

Keep an eye on your swabbed plates and your transfer plates. Have patience.

As long as you are putting very, very small amounts of spore solution on the plate, and following sterile technique, you will get mycelium.

I saw you say something about being worried that not every drop will have spores. A drop will have thousands of spores. If you see them in the water, those are actually clumps. They're microscopic. To give you a hint of how many are in there, I aspirated sterile water back into the spore syringe after it was empty to rinse the last leavings out, put that on a BRF cake, and it germinated. This was just testing to see if there really were still spores in there.

Are these plates different varieties of cubes? You should see slightly different looking mycelium between varieties and between plates.

At least a few are going to have usable growth. You're gaining experience and understanding with every step, so just have patience and take heart in the fact that you're both learning and will see success.


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Re: Check out my agar, please? [Re: karri0n]
    #27045809 - 11/18/20 05:59 PM (8 days, 6 hours ago)

very very well said at the end, my brother. I notice my little bumps of disappointment and frustration are short-lived, and then I'm wanting to start messing around with this shit more and more. for example, my prints came in today. stoked. b+, gt, ecuador, & pes amazonian(?).  already got the sterile swabs and fresh plates ready to go. gonna go smallll this time hah. I'm listening!

hey, is there any good argument for using a loop over a sterile swab? entirely curious. I'm gonna swab em tonight tho.


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Re: Check out my agar, please? [Re: black strat]
    #27046242 - 11/18/20 09:28 PM (8 days, 2 hours ago)

I tried loop the other day after getting mold on one of the plates I did with the swab.

My thought was possibly that I introduced contam from the foil wrap which I didn't remove fully but only peeled down partially, and that a mood spore had landed on it at one of the times I opened the jar to take a swab out.

A solid metal wire that I flame the entire thing can't have that issue.

It will depend on the results, but I had an easier time controlling the sterile swabs than the loop. I see people using both.


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Re: Check out my agar, please? [Re: karri0n]
    #27046360 - 11/18/20 10:28 PM (8 days, 1 hour ago)

Can anyone tell me once the agar plates are fully colonized, do they go in the fridge or stay out in room temperature? I have not been able to find this simple answer anywhere, thank you! Also, this was my agar at 11 days, don't worry about the black, that is electrical tape as I find the dishes flimsy and more subject to contamns if I don't secure them.


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Re: Check out my agar, please? [Re: jake103]
    #27046364 - 11/18/20 10:29 PM (8 days, 1 hour ago)



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Re: Check out my agar, please? [Re: jake103]
    #27046379 - 11/18/20 10:42 PM (8 days, 1 hour ago)

I put plates I want to store in the refrigerator.  I have only removed one from cold storage and used it so I am not an expert.  Transfer before they get completely filled and then store cold.

Does your plate have condensation on the top? If so, put a warm glass or bowl of water on it to slightly heat the lid and get rid of the condensation.  Then take another picture and post it. Was it from a spore print?


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Re: Check out my agar, please? [Re: jake103]
    #27046482 - 11/18/20 11:29 PM (8 days, 43 minutes ago)

Quote:

jake103 said:





That colonization does not looks very good imo...
You should transfer it at least once to get some better growth.


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Re: Check out my agar, please? [Re: GrinchGrower]
    #27047355 - 11/19/20 03:31 PM (7 days, 8 hours ago)

Ummm...this is only at 11 days. And no, I did not have condensation on these particular dishes. I tried the hot water method suggested for some of the newer ones but it did not work. I have them incubating with my jars. The ones pictured are from a spore syringe.


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Re: Check out my agar, please? [Re: jake103]
    #27047358 - 11/19/20 03:31 PM (7 days, 8 hours ago)



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Re: Check out my agar, please? [Re: jake103]
    #27047399 - 11/19/20 04:13 PM (7 days, 7 hours ago)

Quote:

jake103 said:
Can anyone tell me once the agar plates are fully colonized, do they go in the fridge or stay out in room temperature? I have not been able to find this simple answer anywhere, thank you! Also, this was my agar at 11 days, don't worry about the black, that is electrical tape as I find the dishes flimsy and more subject to contamns if I don't secure them.




leave in room temp in a baggie during colonization
at 2/3 of colonization put in the refrigerator. dont let it get at 100% plate colonization or it gets messy


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Re: Check out my agar, please? [Re: Phony Phone]
    #27047932 - 11/19/20 09:11 PM (7 days, 3 hours ago)

OK, thank you! I have been too nervous to transfer them because I don't want to contamn and this is so far the best looking agar I have been able to colonize. All white, no other colors. I will put them in baggies as suggested and transfer soon. Also, what do you mean by they get messy?


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OfflinePhony Phone
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Re: Check out my agar, please? [Re: jake103]
    #27048021 - 11/19/20 10:20 PM (7 days, 1 hour ago)

at full colonization the mycelium tends to reach for the plate walls and crawl out


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Re: Check out my agar, please? [Re: Phony Phone]
    #27048205 - 11/20/20 01:43 AM (6 days, 22 hours ago)

48 hrs since first transfer....



can't even tell you how appreciative I am of you guys offering opinions and kind words..

I jus reeeally am curious to hear what maybe a trusted cultivator, or someone who's been doin this shit for a good while thinks about these plates..

I'm a slightly skeptical person as it is, but I jus can't say out of all the pics I've seen on this site of mycelium, that my plates look similar to theirs. I'm not seeing that 'ropey', uniformed length, and fan-like shape that basically all yalls tend to have. lol please tell me I'm wrong.


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Re: Check out my agar, please? [Re: black strat]
    #27048693 - 11/20/20 12:14 PM (6 days, 11 hours ago)

Dude, that is not myc.  Pitch them and start over.


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Re: Check out my agar, please? [Re: CosmicKramer]
    #27048833 - 11/20/20 01:41 PM (6 days, 10 hours ago)

lol yeah, sounds about right.


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Re: Check out my agar, please? [Re: black strat]
    #27048835 - 11/20/20 01:43 PM (6 days, 10 hours ago)

Quote:

black strat said: I'm not seeing that 'ropey', uniformed length, and fan-like shape that basically all yalls tend to have. lol please tell me I'm wrong.





You're talking about rhizomorphic growth, I call the shape lightning roots. From spores there can be a hundred of those rhizomes crawling all over one another so it just looks like a mess. Once you isolate strains out by taking very small tissue samples on transfers, they tend to become more rhizomorphic.

Myc can take lots of forms. The common three are rhizomorphic, cottony, and leathery. Cubensis can make all three.

Not even all cube strains turn rhizomorphic. You can have a tomentose strain that produces killer fruits and a rhizo strain that never fruits at all, from the same spores from one fruit. USUALLY, rhizomorphic growth is preferred I believe due to its colonization speed and possibly ease of identification.

I really can't say either way with those. It hasn't turned green or gray or pink so that's definitely a plus. It looks wiry, bit also fluffy. I kinda thought mold from the start but I can very easily be wrong and don't want you to throw it out on my bad advice.

You could smell one as a sacrificial plate. Mold smells like mold, mycelium smells like mushrooms. You might not even smell anything and just waste a plate.


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Re: Check out my agar, please? [Re: karri0n]
    #27049571 - 11/20/20 10:52 PM (6 days, 1 hour ago)

Yeah, unfortunately they do not look like what I was hoping.  They do not look like mycelium. I am sorry that my advice pushed you to waste some plates and time. We have all gone through it though. I think these are probably a lost cause.

Again, sorry that I was wrong on the mycelium call. Hopefully you got some practice on technique and are still exited to try more plates!


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Re: Check out my agar, please? [Re: Grenik]
    #27049730 - 11/21/20 01:46 AM (5 days, 22 hours ago)

Jus opened one to sacrifice - lmfao, smelled like the wet sock you find behind the washing machine after 2 weeks.

man, do I feel like an ass....

then I opened another. it smelled like mushrooms, and mold. then another, and same thing.

is it possible there is myc in some of these, trying to compete with mold? I mean, they obviously can and do grow together.

and just for my own curiousity, what is that stuff growing out on the edge? the stuff actually growing IN the new agar, it looks like.



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Re: Check out my agar, please? [Re: Grenik]
    #27049735 - 11/21/20 02:02 AM (5 days, 22 hours ago)

Quote:

Grenik said:
Yeah, unfortunately they do not look like what I was hoping.  They do not look like mycelium. I am sorry that my advice pushed you to waste some plates and time. We have all gone through it though. I think these are probably a lost cause.

Again, sorry that I was wrong on the mycelium call. Hopefully you got some practice on technique and are still exited to try more plates!




whaaat dude?  hell no, do not apologize. everything is about intentions, and yours were absolutely coming from a good place.  I still very much appreciate your input.

what's got me down a little and kinda bummed, is I see TC's commenting all the time on other noob posts, throwing seasoned advice at them, definitively telling em if they got myc or not, and I still feel like I'm poking around in the dark over here man.  I really do put a decent effort into taking the best, clearest pics that I possibly can with the phone I have, because I feel like if I'm gonna ask you guys for your help, I wanna make it as easy as possible for you to check out what I got. but, ah, fuck it, that's neither here nor there. and yep, I'm sure as shit gonna keep pushing on!  what else am I gonna do hahah?


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Re: Check out my agar, please? [Re: karri0n]
    #27049953 - 11/21/20 09:07 AM (5 days, 15 hours ago)

I've never used those ketchup cups before, but I can't imagine they are sterile or easy to make small transfers out of.  Maybe buy some plates and do a streak.  The plates aren't expensive.  I think karri0n had the right idea.  I've done and had success with shot glass approach.  My only difference is I cool the loop in receiving dish before dipping into shot glass.

 
Quote:

karri0n said:
All of the ones that it look like the pooled liquid over them are bacterial. The ones with a single drop you will need to wait probably at least a week to know if it's mycelium, mold, or bacteria. If it grows quickly it's probably mold.

Remember that spore syringes probably have quite a bit of bacteria in them, and if there is water for that bacteria to move around, it will get everywhere. Agar works for isolating mycelium from bacteria because mycelium can move, but bacteria can't, so long as there is no water and you aren't moving the plates around.

Spores take time to germinate, as well. So there will be probably 48 or so hours that nothing "good" can possibly be growing. Once they germinate, then the mycelium will start growing but still, it tends to be slower than bacteria or mold.

I never had any luck with syringe directly to agar. It's recommended  an inoculation loop, or what I use are sterile swabs.

You can take q-tips, wrap in neat little foil packages, and PC them to make sterile swabs. I did this in a jar with a standard modified myco lid.

You can also buy them from medical places or mushroom supply places, or get them from a doctor's office.

You can also rip the plastic off a bread tie, wrap it once around a toothpick, then twist the rest of it into a long handle to make an inoculation loop that you can flame.

Put a drop of spore solution onto a sterile swab and make a neat zig zag over the plate, or

Put a 1cc of spore solution into a sterile shotglass, flame your inoculation loop, dip it in the spores, and make a clean zig zag across the plate.

See these photos. When you make the zig zag, then you can separate the bacterial snots from white, organized mycelium. The bacteria was not able to move across the plate, but the mycelium was, and so I took a transfer from it.



If you wait a week or so and let the plates sit, you might see some healthy mycelium that grows away from bacterial areas. The ones that have water over the whole plate are going to turn completely into boogers most likely.




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Re: Check out my agar, please? [Re: IdiotCircusBoy]
    #27050005 - 11/21/20 10:00 AM (5 days, 14 hours ago)

yeah, I ordered an 8 pack of those Holy Grail jawns off amazon. says driver is out for delivery right now.

if nothing else, the ketchup cups have been a cheap way for me to just dip my toes in and start playing around with agar. I'm enjoying it. I did a print swipe with a sterile swab about 60 or so hours ago. so far none of those are showing signs of anything. which I count as a small win, because up until those, every one of my cups has been just an extremely sloppy bacterial orgy from the 70s, you know, the kind where there is just complete and utter disregard for any semblance of conservative or selective love-making -- I mean everything is fucking everything. you know that scene from the movie Caligula? yeah like that, only on a microscopic level.


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Re: Check out my agar, please? [Re: black strat]
    #27050064 - 11/21/20 10:44 AM (5 days, 13 hours ago)

Quote:

black strat said:
Jus opened one to sacrifice - lmfao, smelled like the wet sock you find behind the washing machine after 2 weeks.

man, do I feel like an ass....

then I opened another. it smelled like mushrooms, and mold. then another, and same thing.

is it possible there is myc in some of these, trying to compete with mold? I mean, they obviously can and do grow together.

and just for my own curiousity, what is that stuff growing out on the edge? the stuff actually growing IN the new agar, it looks like.






I was personally thinking the same thing. Some of your plates seem to have short hair along with the long hair and I think the short hair could be myc. If it continues to grow like that and consumes the food coloring I'd bet it's mycelium.

The problem is that mold might have sporulated all over everything in the plate, or it might not have.


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Re: Check out my agar, please? [Re: black strat]
    #27050376 - 11/21/20 02:09 PM (5 days, 10 hours ago)

Best of luck with those things.  I'm assuming you don't need wrap those since the lid screws on.  Just found the below post from about a year ago by Mr. Alien.  Agar is pretty cool cause you feel like a scientist. 

https://www.shroomery.org/forums/showflat.php/Number/26374264#26374264

Quote:

black strat said:
sloppy bacterial orgy



---Best band name ever---


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Re: Check out my agar, please? [Re: IdiotCircusBoy]
    #27053200 - 11/23/20 12:46 AM (3 days, 23 hours ago)

Quote:

black strat said:
yeah, I ordered an 8 pack of those Holy Grail jawns off amazon. says driver is out for delivery right now.

if nothing else, the ketchup cups have been a cheap way for me to just dip my toes in and start playing around with agar. I'm enjoying it. I did a print swipe with a sterile swab about 60 or so hours ago. so far none of those are showing signs of anything. which I count as a small win, because up until those, every one of my cups has been just an extremely sloppy bacterial orgy from the 70s, you know, the kind where there is just complete and utter disregard for any semblance of conservative or selective love-making -- I mean everything is fucking everything. you know that scene from the movie Caligula? yeah like that, only on a microscopic level.




Hey you probably have some germination by now, eh?

And have you had a chance to try out the holy grail containers? I recommend the "defective lid" vendor that sells 20 for cheaper, they are the same ones and the lids fit on both, I bought 8 then 20 and am gonna be getting 20 more.

let me know how you like the feel of the twist containers vs those ketchup cups. I've been considering trying the ketchup cups since it's a lot easier to get a shitload of them and they seemed pretty convenient. It would be nice to have just like 100 plates I could swipe/transfer/clone/whatever to, whenever I want, and not have to worry about them so much.


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Re: Check out my agar, please? [Re: karri0n]
    #27053215 - 11/23/20 01:24 AM (3 days, 22 hours ago)

Quote:

karri0n said:
Quote:

black strat said:
yeah, I ordered an 8 pack of those Holy Grail jawns off amazon. says driver is out for delivery right now.

if nothing else, the ketchup cups have been a cheap way for me to just dip my toes in and start playing around with agar. I'm enjoying it. I did a print swipe with a sterile swab about 60 or so hours ago. so far none of those are showing signs of anything. which I count as a small win, because up until those, every one of my cups has been just an extremely sloppy bacterial orgy from the 70s, you know, the kind where there is just complete and utter disregard for any semblance of conservative or selective love-making -- I mean everything is fucking everything. you know that scene from the movie Caligula? yeah like that, only on a microscopic level.




Hey you probably have some germination by now, eh?

And have you had a chance to try out the holy grail containers? I recommend the "defective lid" vendor that sells 20 for cheaper, they are the same ones and the lids fit on both, I bought 8 then 20 and am gonna be getting 20 more.

let me know how you like the feel of the twist containers vs those ketchup cups. I've been considering trying the ketchup cups since it's a lot easier to get a shitload of them and they seemed pretty convenient. It would be nice to have just like 100 plates I could swipe/transfer/clone/whatever to, whenever I want, and not have to worry about them so much.




hey bro, NO, I don't see ANYthing yet.... I am pretty content with peeking at them here and there, and not seeing any bacteria or mold, but the impatience in me gets triggered when I'm not seeing any myc either.. ah well, playing the long game, it seems they are.. little fuckers. print to swab to plate is the way to go tho for me, no doubt.

so no, I haven't used the new plates yet, I'll probably cook up a batch of agar sometime this week, and use both the new ones, and remaining ketchup cups. tbh, I couldn't really tell ya if they're the way to go yet. I'm such a noob and don't have much to compare anything to yet hah. but like I said, the cups I inoc'd 4-5 days ago are still contam free, so they must be sufficient on some level, ya know?

I talked to a couple TCs here and they both said the same thing -- I should use the rest of my syringes on good ol brf cakes. that's my next project I'm lookin forward to.

I'll remember that about the "defective" ones -- thats a fuckin steal if they work the same as the others. cheers!


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