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Offlineblack strat
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Check out my agar, please?
    #27035826 - 11/12/20 09:17 PM (15 days, 11 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 (15 days, 11 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 (15 days, 11 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 (15 days, 8 hours 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 (15 days, 7 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 (15 days, 6 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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OnlinePhony Phone
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Re: Check out my agar, please? [Re: black strat]
    #27036399 - 11/13/20 07:10 AM (15 days, 1 hour 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 (14 days, 16 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 (14 days, 15 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 (14 days, 15 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 (14 days, 14 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.

My Journal / Grow Log


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 (14 days, 12 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 (14 days, 12 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 (14 days, 11 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 (14 days, 10 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.


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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 (14 days, 9 hours 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 (14 days, 8 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 (12 days, 22 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 (12 days, 18 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.


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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 (12 days, 18 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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