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OfflineFrrrunkis
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Syringe to agar - need advice after 1 week
    #26858221 - 08/02/20 06:34 AM (3 years, 5 months ago)

Hello! 1 week ago I've prepared and PCd my first agar (8g LME, 8g agar agar, 400 ml water). I've poured 10 plates into oven-sterilized (250C) glass petris in a SAB, closed and let them rest for an hour, then used an inoculation loop to streak the plates with solution from a cubensis syringe (ordered online, can't vouch for quality). I've then wrapped the plates with food cling-wrap and put them away in a clear box. Results below.

1
2
3
4
5
6
7
8
9
10

This is my first go at this, so I can't really judge. I can't see anything that'd resemble a nice concentric string-like growth ("rhizomorphic" is the word, I think?), on the other hand two plates - 7 and 9 - have a white fluffy growth. I don't know how to tell if that's cubensis or something else (mold?). Should I attempt to transfer anything?

Main things I could've done differently:
1. I was nervous about plates getting contaminated while cooling off inside the SAB and I didn't wait very long for them to cool off, so I think that's where most of my condensation came from. Maybe I can relax for a bit longer next time.
2. I forgot to shake my syringe before squirting the solution onto an empty plate. I don't know how important this is, but - intuitively - shaking could give me a more predictable result, correct?
3. The cling-wrap I used was pretty terrible and it tore easily, so I ended up working with 1 glove off just for wrapping (sprayed my hand with iso, but still), plus I think I overcompensated and wrapped the plates too much, possibly hurting gas exchange? I now bought some parafilm and will be using that to wrap.

Also, is it ok to store the syringe for further attempts? I've wiped it with iso and flamed the needle, then re-capped it and put back in the fridge.


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OfflineAablmd82
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26858314 - 08/02/20 07:52 AM (3 years, 5 months ago)

Spores store indefinitely when refrigerated, so you’re good on that end. IMO they don’t look good, but don’t throw 7 or 9 out yet. Could be there’s just too many mycelial networks and some might break through.

Spores sink and collect to form tiny clumps inside the syringe, so if you never shook the syringe that could’ve made it inconsistent. I would just skip the transfer for 7 and 9 and do another batch of jars.


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Clone -> agar -> UnBODified monotub (first attempt, left unattended a week before harvesting)


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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: Aablmd82]
    #26858331 - 08/02/20 08:00 AM (3 years, 5 months ago)

i’d take a transfer or two from plate #7. nothing wrong with tomentose growth, just doesn’t look as pretty as rhizomorphic growth

i’ve never worked with true petris so i can’t offer advice there, but yeah shaking the syringe probably woulda helped a bit. i’ve only done spore syringe to agar a few times but i’ve never been a fan. it’s way easier to work with prints on agar IME


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Aablmd82]
    #26858354 - 08/02/20 08:11 AM (3 years, 5 months ago)

I'll try and take better pictures of 7 and 9 later today. Right now I'm not sure if they're mold or cubensis. I've seen pictures of cobweb mold that look like 7, but I can't really tell.

In any case, what's the end game for agar if I want to use it to inoculate grain spawn? Should I wait for the growth to pin, which would be the ultimate indication of whether it's cubensis or something else?


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Invisiblesandman420
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26858369 - 08/02/20 08:20 AM (3 years, 5 months ago)

plate 7 maybe. :eek:

Plate 9 Ive never seen such loose fibrous mycelium on healthy cubensis. This is also pretty considerable growth for 7 days after ss to agar.


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26858849 - 08/02/20 12:57 PM (3 years, 5 months ago)

Better pictures:

9
9
7
7


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OfflineKyngwhatt
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26859061 - 08/02/20 02:30 PM (3 years, 5 months ago)

Bump Those are all good examples of cobweb Right??
8 and 9 the dense white is the only myc i see. somebody let me know if i'm completely wrong.
I recently learned about water agar with no nutrients and am in the process of using them to remedy this exact situation.


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Whatt


Edited by Kyngwhatt (08/02/20 02:32 PM)


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Invisiblesandman420
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Re: Syringe to agar - need advice after 1 week [Re: Kyngwhatt]
    #26859124 - 08/02/20 03:12 PM (3 years, 5 months ago)

None of these look like plates I would consider taking forward.

I would start over and forget about these they all look off. 7 looks almost right but I really dont think so.


--------------------
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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: Kyngwhatt]
    #26859150 - 08/02/20 03:24 PM (3 years, 5 months ago)

it kinda does look like cobweb mold. i’m not 100% sure of it, but it does look similar to a plate in this thread https://www.shroomery.org/forums/showflat.php/Number/21648576


--------------------
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===================================
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Offlinejcm4620
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Re: Syringe to agar - need advice after 1 week [Re: Kyngwhatt]
    #26859164 - 08/02/20 03:31 PM (3 years, 5 months ago)

i wouldnt use any of them i even see a pink hue in one of them. if its cubensis it should look more like this





spore syringes to agar really suck but at least u tried to do it instead of just wasting your grain. this pic shows why u go to agar first u can see the myc is surrounded by bacteria but i was able to make the transfer from the area shown. all syringes are dirty to some extent. thats cuz all spores are dirty to some extent. unless the mushrooms were fruited invitro or in a 100% sterile environment and then swabbed or printed in a 100% sterile environment the syringe will contain whatever the environment the fruit/spores were in contained. and it dont really matter where the syringe came from. this one came from prolly the absolutely best vendor there is. of coarse there is better vendors than others but thats cuz they get their prints from ppl who try limit the amount of contams as much as possible and also prepare their syringes as clean as possible.



--------------------
PANAEOLUS FRUITING MADE SIMPLE



Edited by jcm4620 (08/02/20 03:32 PM)


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26859242 - 08/02/20 04:33 PM (3 years, 5 months ago)

Thank you all! @jcm4620, that's some precise cutting if you were able to make that work!

I've just finished my SAB work for tonight. I've made two transfers each from 7 and 9, we'll see how that works out. I've also started 5 new plates from the syringe. I made sure to shake it well this time and I was able to see black spots/strands in the liquid I dabbed my loop in, so hopefully I'll get more hits.

This was only my 2nd SAB session and first time I've ever done transfers. One question, when cutting small triangular wedges the mycelium kind of "melts away" or disappears when touched with the blade. Is this something to worry about? Should I cut bigger wedges to make sure there's a lot of visible white stuff left? I've made sure to cool off the blade repeatedly in clear agar before cutting, so hopefully I haven't killed off the mycelium.

I also need new plates, luckily I've found a supplier of sterile vented plastic petris. One less thing to worry about. :smile:


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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26859836 - 08/02/20 10:13 PM (3 years, 5 months ago)

i have the myc “disappear” sometimes too. i’ve just started to take slightly larger wedges so the myc stays on some of the agar wedge, but i think the myc colonizes the agar itself a little so i don’t know that it’s necessarily an issue. i just like seeing myc on the wedge for my own peace of mind

i used to dip my scalpel in clean agar first too but i honestly don’t think that it’s necessary. i’ve lately just been cutting straight into the agar w it hot & haven’t noticed any detriment to the mycelium’s health


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: hazyhorse]
    #26859977 - 08/03/20 02:21 AM (3 years, 5 months ago)

Thanks.

By the way, is there a rough timeframe for when to expect usable cubensis growth on agar? I've seen 2 weeks mentioned somewhere.


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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26860815 - 08/03/20 12:58 PM (3 years, 5 months ago)

yeah two weeks is probably a good estimate. maybe more or less depending on how aggressive the culture is. you typically want at least 50% of the plate covered to use for grains, though i’m sure you could get away with less if it’s clean & you’re impatient


Edited by hazyhorse (08/03/20 12:59 PM)


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OfflineMTManiac
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Re: Syringe to agar - need advice after 1 week [Re: hazyhorse]
    #26860858 - 08/03/20 01:12 PM (3 years, 5 months ago)

Quote:

hazyhorse said:
yeah two weeks is probably a good estimate. maybe more or less depending on how aggressive the culture is. you typically want at least 50% of the plate covered to use for grains, though i’m sure you could get away with less if it’s clean & you’re impatient




Thank you! I've been wondering about mss-agar timeline


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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: MTManiac]
    #26860914 - 08/03/20 01:43 PM (3 years, 5 months ago)

no worries!! it’s all a crapshoot anyways with time. the best thing to keep in mind is that it’ll happen at the mycelium’s pace & not how fast YOU want it to


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===================================
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===================================

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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: hazyhorse]
    #26861106 - 08/03/20 03:18 PM (3 years, 5 months ago)

Thank you.

As for identifying if it's actually cubensis, would you wait for the mycelium to pin?


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OfflineKyngwhatt
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26861197 - 08/03/20 04:25 PM (3 years, 5 months ago)


is an example of what you have going on, myc with cobweb



is an example of cobweb after it gets old enough and starts to sporulate



I would take a cut from the green section and transfer it to agar with no nutrients to try and let it outrun the cob and

I didn't really read the thread so sorry if i'm misunderstanding. if your myc is disappearing you are probably transferring the cobweb.
If i'm completely misunderstanding someone let me know because i'm trying to work through this situation. I cannot start over because i only had one melmac swab



--------------------


Whatt


Edited by Kyngwhatt (08/03/20 05:11 PM)


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Kyngwhatt]
    #26861988 - 08/04/20 05:00 AM (3 years, 5 months ago)

Yeah I did just that, I took a cut from the "green" area on your photo edit, hopefully it'll work. If not - no biggie, something else will work eventually. :smile:

One thing I don't quite get - what do you mean by "no nutrients"? I transfer to the same LME agar. Would anything (good or bad) grow without nutrients?


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OfflineKyngwhatt
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26862775 - 08/04/20 02:41 PM (3 years, 5 months ago)

Quote:

Frrrunkis said:
what do you mean by "no nutrients"?




this guy explains it well


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Whatt


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Kyngwhatt]
    #26878747 - 08/13/20 02:44 PM (3 years, 5 months ago)

Behold, round two. I've got many more of these now, but I'm only showing the interesting ones. I've started labelling them slightly better too. Excuse the condensation, I'm still not good at pouring at the right temperature. :/

So, remember 7 and 9 from the first batch? Here they are today!

Adam and his 2 kids:

I'm pretty sure they're not cubes. Cobweb mold?

Eve and her 2 kids (please appreciate the unintended vagina):

Same story as with Adam. All of these plates grew super fast too. I'm leaving them all to turn into whatever.

So, these are fresh from that same syringe as the first round, but I gave it a good shake this time. I made 8 of these, of which 3 are looking promising:

I think I'll try and make transfers (grain spawn material) from all 3 of these, at least 5 transfers each. Is it ok to transfer now (in 2-3 days), or should I wait longer?

I also got this piece of modern art. Any takers?


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Offlineverum subsequentis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26878764 - 08/13/20 02:53 PM (3 years, 5 months ago)

Contam bonanza. I see nothing worth saving in these pics. I believe you have a contaminated syringe and need to work on your sterile tek.


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: verum subsequentis]
    #26878789 - 08/13/20 03:15 PM (3 years, 5 months ago)

Ouch. Aren't all 3 white growths in the 3rd row cube mycelium? It's hard to take a photo through the condensation, but it looks white and fluffy, plus they all formed around black dots (spore clumps?) from the syringe.

As for the syringe and sterile tek, I'm sure you're right. I want to try and salvage what I can though.


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26883505 - 08/16/20 01:48 PM (3 years, 5 months ago)

Ok, I'd really appreciate another look. I hope these photos provide more detail.

02/08//3


02/08//5


02/08//4


I'm not sure which of the spots/discolorations on agar are normal (condensation spots/streaks, agar or malt extract clumps) and which are contaminants.

Looking closely, there seem to be sections where I should be able to cut just the white mycelium.



Edited by Frrrunkis (08/16/20 01:56 PM)


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Offlinedirtybirdx
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26883535 - 08/16/20 02:06 PM (3 years, 5 months ago)

Yea man I’d be afraid to continue with any of those...

;(

There is a bit of healthy mycelium surrounded in a few places but it wouldn’t be worth it in my opinion.


Did you make/use a still air box?  Even the first 50 plates I made never grew anything that funky.


You might be bummed about shroomies being pushed back, but this is quality learning for you.  When you see the healthy white rhizo mycelium, you’ll feel much better.

A lot of molds are white.  Cobweb mold which is very common shows up a lot with us and I’ve probably even thrown away good cultures being unsure and not wanting to take the risk.

That’s why rhizomorphic growth is so coveted — it’s so easy to see whether it’s contaminated or not.  Gives you more of an assured feeling.

Toss them, make your still air box, pour some more plates, and give it another go!  👍


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Re: Syringe to agar - need advice after 1 week [Re: dirtybirdx] * 1
    #26883546 - 08/16/20 02:18 PM (3 years, 5 months ago)

Also, you said you weren’t sure what you were seeing, so

- you have bacterial contamination around the entire outer edge of every plate.  It’s the whitish yellowish film near the sides.

That’s not necessarily a plate-killer, but the fact that it’s present in all three sounds like sterile technique, or potentially unsterilized agar.

- most of the white you see in the first two plates are mold.

- the ring on the third plate, and most of that growth, is mold.

- yellow spot is bacteria

- the only real mycelium I’m confident about is on the second plate, you see two small growths resembling hands - that’s what you want the whole growth to be.  And with none of the other stuff.

Hope this helps


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: dirtybirdx]
    #26883572 - 08/16/20 02:35 PM (3 years, 5 months ago)

Thanks for your input!

Yeah I use a SAB, as per Bod's tek. I've only worked in it 3 times so far (this was my 2nd agar pour/use), so I'm clumsy and my technique sucks, but I think I'm as prepared as possible. Gloves, iso, flame, loop, scalpel, the whole thing. I sterilize my agar in a PC for 30 mins. Glass petris are oven-sterilized at 250C for 30 mins. I've now bought sterile plastic petris to simplify the process.

Maybe this syringe is really just bad and I should get another one, or a spore print.

I guess I'll still try to transfer from these plates just to see what happens. I'll give my syringe another fresh go too, just to be sure. Maybe I'll get lucky. If not - at least I'm learning.

The "whitish yellowish film" on the outer edge is only there on 02/08//4. On the oher two that's actually the parafilm wrap (it reaches around the edge onto the top and bottom a bit, and it's semi-transparent, so it looks similar under the light). So hopefully at least my agar is clean.


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OfflineFrrrunkis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis] * 1
    #26969138 - 10/04/20 01:49 PM (3 years, 3 months ago)

I just wanted to thank you guys for the advice I've received here! I gave these plates a shot, did 2 more rounds of transfers and I've now got 7 oat jars going strong. Today I was going through all my plates to see what's staying and what's not, and - to my surprise - some of them are pinning!



I'm now much more confident that I can actually make this work.

Thanks again!


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Offlineverum subsequentis
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26969190 - 10/04/20 02:15 PM (3 years, 3 months ago)

congrats


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OfflineAtmozFear
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Re: Syringe to agar - need advice after 1 week [Re: Frrrunkis]
    #26969205 - 10/04/20 02:20 PM (3 years, 3 months ago)

I still have yet to see pins from an agar plate


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Offlineverum subsequentis
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Re: Syringe to agar - need advice after 1 week [Re: AtmozFear]
    #26969217 - 10/04/20 02:26 PM (3 years, 3 months ago)

It'll happen.


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Offlinehazyhorse
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Re: Syringe to agar - need advice after 1 week [Re: verum subsequentis]
    #26969994 - 10/05/20 12:18 AM (3 years, 3 months ago)

just forget abt em long enough & they pop up lol when i get lazy it starts to happen

congrats on the agar


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you're not the first to set foot here, just another
===================================
i love glass petris & you can too!!
posts i constantly refer back to
new to mushroom cultivation?? read this!!
===================================

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