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#1 darkzen15

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Posted 10 February 2019 - 03:32 PM

Hey guys, just started my first set of no pour agar plates yesterday. 4 plates started the entire pouring and pressure cooking phase went very well with little condensation.

Inoculated all 4 plates through a SHIP in a clean SAB. Needle flame sterilized between each plate.

Only issue is that i seem to have squirted a little too much spore solution. Some plates look quite wet.

We will see the outcome soon i guess. How long do agar plates normally take to grow out from a multispore solution?

As an update, no growth at all in my pf tek jars after 12 days.
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#2 RutgerHauer

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Posted 10 February 2019 - 03:47 PM

Hi! Good to hear you are getting into agar - I am not experienced in this myself, but will follow anyway for future agar works.

 

So, you are using a multispore solution for inoculating. Could take a while to see significant growth, depending on the strain. I have some different strains germinating and the Colombian strain seems to be a bit slow - showing just a little growth after 11 days. So, if the same goes for your PF jars, it might be growing, but just not showing yet!


Edited by RutgerHauer, 10 February 2019 - 03:50 PM.


#3 Deleena24

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Posted 10 February 2019 - 03:48 PM

Do you mind posting which Tek you used?
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#4 peacefrog

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Posted 10 February 2019 - 04:26 PM

Yea spore syringes can a [email protected]&$$ sometimes when inoculating agar. You really only want to use a drop or two. But we have all been there and done that lol. You go to push on the plunger, attempting to just barely push out a drop, and bam you inadvertently pushed too hard and it shoots out more than that.

They may be fine, just give it a few days before worrying too much about it. Like said above, germination could take several days.

Post some pics if you can.

Good luck and good vibes on your first agar attempt (agar work is one part of cultivation I love the most).

Edited by peacefrog, 10 February 2019 - 04:30 PM.

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#5 darkzen15

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Posted 10 February 2019 - 04:46 PM

Do you mind posting which Tek you used?

 

I used a variation of Pastywhytes no pour agar tek. Instead of glad mini rounds, I just used small PP5 tupperware containers and I used stiffened felt for the filter. 


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#6 darkzen15

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Posted 10 February 2019 - 04:47 PM

Hi! Good to hear you are getting into agar - I am not experienced in this myself, but will follow anyway for future agar works.

 

So, you are using a multispore solution for inoculating. Could take a while to see significant growth, depending on the strain. I have some different strains germinating and the Colombian strain seems to be a bit slow - showing just a little growth after 11 days. So, if the same goes for your PF jars, it might be growing, but just not showing yet!

 

Yeh my PFtek cakes are at 12 days so far with no sign of anything at all, which I guess is a mixed blessing as at least I do not have contamination yet. I used the same syringes on the agar plates too.



#7 darkzen15

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Posted 10 February 2019 - 04:48 PM

Yea spore syringes can a [email protected]&$$ sometimes when inoculating agar. You really only want to use a drop or two. But we have all been there and done that lol. You go to push on the plunger, attempting to just barely push out a drop, and bam you inadvertently pushed too hard and it shoots out more than that.

They may be fine, just give it a few days before worrying too much about it. Like said above, germination could take several days.

Post some pics if you can.

Good luck and good vibes on your first agar attempt (agar work is one part of cultivation I love the most).

 

Yeh it was really crappy, I was waiting to see the droplet emerging from the tip of the syringe but instead it was spraying a fine stream off the to the side. 



#8 Billcoz

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Posted 11 February 2019 - 12:25 AM

Yeah, Iv'e had the same issue trying to squirt a drop or two, and it clogs a little, and then a whole load pops out all at once, but for me they have always germinated, so you should be fine. If you get no growth in a week or so on the agar plates, and a week or so more for your already 12 day old pf cakes, then I'd be wondering about the spore solution, but for now all you can do is be patient, good luck!



#9 darkzen15

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Posted 11 February 2019 - 01:48 AM

Yeah, Iv'e had the same issue trying to squirt a drop or two, and it clogs a little, and then a whole load pops out all at once, but for me they have always germinated, so you should be fine. If you get no growth in a week or so on the agar plates, and a week or so more for your already 12 day old pf cakes, then I'd be wondering about the spore solution, but for now all you can do is be patient, good luck!


Yeh I made these spore syringes myself from 2 year old spores. I can see purple flecks in the syringe but I am not sure of their viability

#10 deemesis

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Posted 11 February 2019 - 05:35 AM

I store my agars upside down to get rid of any condensation, but I don't know if that would work after a MSS noc. How do you have yours stored?


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#11 darkzen15

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Posted 11 February 2019 - 05:42 AM

I store my agars upside down to get rid of any condensation, but I don't know if that would work after a MSS noc. How do you have yours stored?


Right side up. When I did microbiological work with proper Petri dishes, we stored them upside down in an incubator or a flow hood but if I did that with my no pour dishes, I'd soak the GE filter

#12 deemesis

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Posted 11 February 2019 - 05:49 AM

That is a problem for sure. But since you have 4 you might try turning one of them upside down, and never turning it right side up until you're ready to use it, just to see.



#13 RutgerHauer

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Posted 11 February 2019 - 10:05 AM

 

Yeh my PFtek cakes are at 12 days so far with no sign of anything at all, which I guess is a mixed blessing as at least I do not have contamination yet. I used the same syringes on the agar plates too.

 

 

You are definitely right on that! If there is at least no contamination visible, that's a good sign the issue probably isn't your sterile procedures.



#14 sandman

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Posted 11 February 2019 - 10:21 AM

You shouldn't use a SHIP on a agar no pour lid. You need to be able to see the needle tip really good so you don't blast the hell out of it with spore solution. You don;t have a good connection to what you are doing through a SHIP. The still air box will allow you to remove the lid and inoculate. 


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#15 darkzen15

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Posted 11 February 2019 - 02:43 PM

You shouldn't use a SHIP on a agar no pour lid. You need to be able to see the needle tip really good so you don't blast the hell out of it with spore solution. You don;t have a good connection to what you are doing through a SHIP. The still air box will allow you to remove the lid and inoculate. 

 

Yeh my house has a real bad mould problem that I can't identify the source of. Like fruits and bread will go off in about 4 days and have visible mould growth. Never had that issue in any other house I have lived in. I did use a SAB but I wanted to take extra precautions to limit the exposure of any media to the air. 

 

Once I build my confidence levels up, I will move back to open lid agar teks. Still not looking forward to working with a pour agar tek though, without a lam flow hood, that SAB is gonna fog up something chronic.

 

Anyway, at the very least, this hobby is developing (or testing) my patience.



#16 darkzen15

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Posted 11 February 2019 - 02:44 PM

 

 

Yeh my PFtek cakes are at 12 days so far with no sign of anything at all, which I guess is a mixed blessing as at least I do not have contamination yet. I used the same syringes on the agar plates too.

 

 

You are definitely right on that! If there is at least no contamination visible, that's a good sign the issue probably isn't your sterile procedures.

 

 

Yeh my money is on the spores being dead or I simply did not scrape enough into the solution that made up the syringes.



#17 Billcoz

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Posted 12 February 2019 - 03:03 AM

 

Hey man, there is a way to get no condensation at all in the no-pour plates, don't put the lid on them after filling em until the agar is cool, then carefully wipe the condensation off the inside glass. After you PC them, bring the temps down slowly and leave em in there until it's completely cool. Check out these, made with non-toxic water soluble highlighter marker ink for color-IMG_20190103_023155.jpg IMG_20190115_181007.jpg IMG_20190109_034927.jpg

After inoculating/transferring to a plate I put em in ziplock baggies and tighten the ring down, making it so much easier to see sectors, it cab be a bitch with mini round or jar lids. Some air gets trapped in, they have not been opened since transferring and seem to have enough O2 to grow quickly, 2 weeks or less to colonize the plate from live culture/myc tissue, less than 3 weeks from dry spores. Good luck.


Edited by Billcoz, 12 February 2019 - 03:04 AM.


#18 darkzen15

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Posted 12 February 2019 - 04:40 AM


Hey man, there is a way to get no condensation at all in the no-pour plates, don't put the lid on them after filling em until the agar is cool, then carefully wipe the condensation off the inside glass. After you PC them, bring the temps down slowly and leave em in there until it's completely cool. Check out these, made with non-toxic water soluble highlighter marker ink for color- IMG_20190103_023155.jpg IMG_20190115_181007.jpg IMG_20190109_034927.jpg
After inoculating/transferring to a plate I put em in ziplock baggies and tighten the ring down, making it so much easier to see sectors, it cab be a bitch with mini round or jar lids. Some air gets trapped in, they have not been opened since transferring and seem to have enough O2 to grow quickly, 2 weeks or less to colonize the plate from live culture/myc tissue, less than 3 weeks from dry spores. Good luck.
That's actually brilliant. I will wait for the agar to cool and then wipe the condensation off before pressure cooking next time. Do you know if that has had any effect on your contam rates?

I won't be doing that much sector isolation. If i confirm a pure mycelium plate I will be doing mycelium flurries and replating until senescence sets in or I get hit by a contam.

I am only growing for microdosing personally so I am not too worried about big flushes. I just want to consistently churn out some chronic tek style grows to keep the psilocybin content fresh.

Edited by darkzen15, 12 February 2019 - 04:44 AM.


#19 Billcoz

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Posted 12 February 2019 - 07:06 PM

 

 

 

Hey man, there is a way to get no condensation at all in the no-pour plates, don't put the lid on them after filling em until the agar is cool, then carefully wipe the condensation off the inside glass. After you PC them, bring the temps down slowly and leave em in there until it's completely cool. Check out these, made with non-toxic water soluble highlighter marker ink for color- IMG_20190103_023155.jpg IMG_20190115_181007.jpg IMG_20190109_034927.jpg
After inoculating/transferring to a plate I put em in ziplock baggies and tighten the ring down, making it so much easier to see sectors, it cab be a bitch with mini round or jar lids. Some air gets trapped in, they have not been opened since transferring and seem to have enough O2 to grow quickly, 2 weeks or less to colonize the plate from live culture/myc tissue, less than 3 weeks from dry spores. Good luck.
That's actually brilliant. I will wait for the agar to cool and then wipe the condensation off before pressure cooking next time. Do you know if that has had any effect on your contam rates?

I won't be doing that much sector isolation. If i confirm a pure mycelium plate I will be doing mycelium flurries and replating until senescence sets in or I get hit by a contam.

I am only growing for microdosing personally so I am not too worried about big flushes. I just want to consistently churn out some chronic tek style grows to keep the psilocybin content fresh.

 

I have not had any contams in agar dishes, though I've only done about 35 so far(knocking on wood now). In couple 3 month old ones, the myc actually ate up most of the agar in the dish, leaving only a tiny layer at the bottom of the myc disc. Just be clean with your SAB work, you should be fine.

 

Good thinking as far as using the agar to make sure you got a clean culture, and for quicker colonizing of grains etc, it really speeds it up.


Edited by Billcoz, 12 February 2019 - 07:08 PM.


#20 Billcoz

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Posted 12 February 2019 - 07:13 PM

Oh, by myc "flurries", do you mean your making a liquid inoculent, or your using the myc for you microdosing? I just have never heard of the latter before, sounds doable. 

 

If you meant that you making syringes with live culture from your agar plates, that is a great way to speed up colonizing.






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