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InvisibleGrumio

Registered: 10/18/19
Posts: 126
Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o] * 1
    #26492809 - 02/19/20 04:16 AM (4 years, 1 month ago)

Any input on these PE plates?

Started from a swab.

Only 1 plate showed any growth so transferred to 3 plates then took 3 samples from the best two plates and below is the result.

Are these good for grain now or should I transfer some more?

Plate 1:


Plate 2:


Plate 3:


Thanks

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InvisibleHobbyist
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Registered: 08/15/10
Posts: 805
Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: PNat]
    #26492831 - 02/19/20 05:07 AM (4 years, 1 month ago)

Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.


--------------------
Everything i say is completely hypothetical...


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OfflinePNat
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Hobbyist]
    #26493110 - 02/19/20 09:35 AM (4 years, 1 month ago)

Quote:

Hobbyist said:
Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea!!!  I will definitely try that method and I was thinking about this all night.  Would wrapping a foil on the media lid take care of the sucking pressure?

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Grumio]
    #26493324 - 02/19/20 12:08 PM (4 years, 1 month ago)

Quote:

Grumio said:
Any input on these PE plates?

Started from a swab.

Only 1 plate showed any growth so transferred to 3 plates then took 3 samples from the best two plates and below is the result.

Are these good for grain now or should I transfer some more?

Plate 1:


Plate 2:


Plate 3:


Thanks




They are probably fine . They dont look GREAT, but personally I've never gotten great looking pe myc on agar. I know other people do all the time, might just be the genetics I've worked with. That said, they've always fruited fine

But it really depends on your goal. If you are fine with MS, go for it

Quote:

Hobbyist said:
Quote:

PNat said:

How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea, assuming the seal fails while it still has positive pressure. And if it doesnt, just subtract a minute or so from the wait time

Also you'll probably have to factor in the volume of water and maybe ambient temp

Quote:

PNat said:
Quote:

Hobbyist said:
Quote:

PNat said:
Quote:

c10h12n2o said:

WARNING: this is a little bit less than kosher, but i will provide the reasoning every guide says to let the PC zero out on its own. naturally the pressure falls once you kill the heat, and once it hits zero it goes below zero, then eventually back up. in my early work with agar, i had some contamination due to opening the PC when the pressure was negative, which led to sucking contams into my agar. what i like to do now is a little impatient, but i have not not seen any problems from it in the 15k to 20k plates i have done with this method. so here is what i like to do: i carefully watch the pressure as it falls, then right as it gets to zero, right before it crosses the line, i pop the vent. this means there is a small hiss of positive pressure being released from the valve (which is good, because it means nothing was sucked in), with less and less pressure as you get closer to zero, with negative pressure building if you wait too long (which sucks in contams). so right before zero, i put on protective gloves/mits, pop the valve, open the PC, and immediately tighten the lids on the media bottles and remove them

lids tightened and bottles removed from PC


c10




How do we do this with a PC that does not have a pressure gauge?





Just an idea..  You could time how long it takes for your pressure cooker to go from your usual sterilization pressure(15,16,17, etc) to 0 by starting your timer when you remove it from the heat and then stopping it when you hear the seal fall(the seal is officially called an Air Vent Cover Lock). 

Then in the future you can just walk into the kitchen and 2 minutes before it's due to fall and then remove the weight when it does.

Frankly though, simply listening for the seal to fall and immediately going to remove the weight is probably sufficient.




That's a great idea!!!  I will definitely try that method and I was thinking about this all night.  Would wrapping a foil on the media lid take care of the sucking pressure?




The whole point of that trick is to NOT have any negative pressure, you want to do it while it's got positive pressure so that you dont have to worry about anything getting sucked in. That said, I still use foil, but it's mostly to prevent stuff from getting too wet


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

Edited by c10h12n2o (02/19/20 12:14 PM)

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InvisibleJohnnieYen
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26495598 - 02/20/20 04:40 PM (4 years, 1 month ago)

I also want to say thanks to you c10 for this guide. Getting back into the hobby and getting back into agar. Building a new still air box today as my former was just too small and cumbersome.
Just got 500 plates delivered and a media bottle.
I started some spores on no pour pastys a few weeks ago so i have some transfers to do, plus start some new plates with the streaking technique.


--------------------
[center

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: JohnnieYen]
    #26495633 - 02/20/20 04:59 PM (4 years, 1 month ago)

Happy to I could be helpful! Let me know if you have any questions about anything


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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OfflineSockadin
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26504912 - 02/26/20 01:51 PM (4 years, 1 month ago)

C10 this was a great write up. Also found some really useful information here. I love the idea of using colored plates based on culture varieties.

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Sockadin]
    #26504919 - 02/26/20 01:53 PM (4 years, 1 month ago)

Thanks, I'm glad you found it helpful :smile:


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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OfflineInthepit
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26570393 - 04/01/20 04:53 AM (3 years, 11 months ago)

I could use some help to correct whatever I did to get this black mold.
My first guess is the from the water I was misting with.
Possibly I could avoid the black mold by misting with bleach?
:mushdance:   
A little history of the tubs; first they were too wet, haven't dialed in the proportions yet.
Then I took the lids and put them sideways, but I believe the tubs got too dry. Then I started misting heavily.
So basically over correction all around, noob style!

And I wonder how I got it, but there's lots of mold here in the tropics.
No one drinks the tap water here!


--------------------
:sporedrop: GLOSSARY  :sporedrop: ACROMYMS!   :sporedrop: GETTING STARTED :sporedrop:

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit]
    #26571577 - 04/01/20 04:26 PM (3 years, 11 months ago)

Have those tubs flushed yet? Or is this before the first flush?

At what point did the mold show up? I'd definitely clean that asap before it starts spreading spores


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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OfflineInthepit
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26571607 - 04/01/20 04:48 PM (3 years, 11 months ago)

Yes, 2nd and maybe 3ird. They were rather random looking compared to the first flush.
     

And the shoeboxes have been turned into a garden patch
And washed with soap and bleach.
I noticed the mold just recently.

But, in the future would your bleach mist be beneficial?
Bleach mister TEK formula

Thanks for your time! :hatsoff:


--------------------
:sporedrop: GLOSSARY  :sporedrop: ACROMYMS!   :sporedrop: GETTING STARTED :sporedrop:

Edited by Inthepit (04/01/20 04:56 PM)

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: Inthepit]
    #26571614 - 04/01/20 04:54 PM (3 years, 11 months ago)

No problem.  I think a lot of that is spores, but that one pic does look more like mold


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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OfflineHoneyBadger9321
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: c10h12n2o]
    #26652266 - 05/06/20 05:55 AM (3 years, 10 months ago)

When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
― Bill Hicks


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OfflineMrBovineJoni
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652281 - 05/06/20 06:10 AM (3 years, 10 months ago)

Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




I'm not entirely sure I understood your question but basically when microwaving you need to almost bring it back to boil (with some mixing,shaking in the middle to ensure consistent heating).  It does not warm up evenly so you have to shake/mix it a few times (lid closed).

Agar has to reach >85C before solid chunks start to melt and due to how microwave oven works it never is 100% even. Then at around 32-38 it starts setting again so you have a window between 60C(too hot to handle for most people, also you get more condensation but other than visibility  that it is fine to have it) and 35C to pour it.

For my 1L media bottle (about 800ml of agar in there) I do about 6min x 600W, shake,  followed by 3 min at 600W and then I repeat it in 30s-60s chunks until all chunks have melted and the liquid is uniform. I find that using maximum power (900W in my case) produces too localised heat in places where waves end up concentrating more so it can almost burn by accident.

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OfflineHoneyBadger9321
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652284 - 05/06/20 06:12 AM (3 years, 10 months ago)

Quote:

MrBovineJoni said:
Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




I'm not entirely sure I understood your question but basically when microwaving you need to almost bring it back to boil (with some mixing,shaking in the middle to ensure consistent heating).  It does not warm up evenly so you have to shake/mix it a few times (lid closed).

Agar has to reach >85C before solid chunks start to melt and due to how microwave oven works it never is 100% even. Then at around 32-38 it starts setting again so you have a window between 60C(too hot to handle for most people, also you get more condensation but other than visibility  that it is fine to have it) and 35C to pour it.

For my 1L media bottle (about 800ml of agar in there) I do about 6min x 600W, shake,  followed by 3 min at 600W and then I repeat it in 30s-60s chunks until all chunks have melted and the liquid is uniform. I find that using maximum power (900W in my case) produces too localised heat in places where waves end up concentrating more so it can almost burn by accident.




Wow, beautiful. Thank you it helps a lot.


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
― Bill Hicks


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OfflineHoneyBadger9321
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652285 - 05/06/20 06:14 AM (3 years, 10 months ago)

The microwaving takes up a lot of the work time. Is it possible to just skip this step. The microwaving is to achive crystal clear agar, correct?


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
― Bill Hicks


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OfflineMrBovineJoni
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652297 - 05/06/20 06:23 AM (3 years, 10 months ago)

Quote:

HoneyBadger9321 said:
The microwaving takes up a lot of the work time. Is it possible to just skip this step. The microwaving is to achive crystal clear agar, correct?



Sorry, I might have misunderstood your question.


So there are 2 steps where microwave is used in this TEK.

1) Initial preparation to cook agar in the bottle before pressure cooking. This ensures that the contents of bottle are uniform and agar is evenly mixed in water. This step is very important and not what I described in my previous post above. if you skip this step you might get permanently lumpy/chunky agar if some agar bits don't properly dissolve before PCing.

2) Reheating of already PCed and cooked agar bottle (stored in fridge) to use for pouring. This is what I described above.

Edited by MrBovineJoni (05/06/20 06:24 AM)

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OfflineHoneyBadger9321
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: MrBovineJoni]
    #26652620 - 05/06/20 09:56 AM (3 years, 10 months ago)

Right yes! I was referring to the first (before PC-ing). I just cant seem to dissolve it completely and chunks still remain when i filter it. Maybe i am not doing it long enough. I had a look at RRs agar videos and there he does not cook it before PC-ing. Takes a lot of time for me and seems that the results are the same, just a little bit cloudy agar. I might be wrong tho?


--------------------
“Today a young man on acid realized that all matter is merely energy condensed to a slow vibration, that we are all one consciousness experiencing itself subjectively, there is no such thing as death, life is only a dream, and we are the imagination of ourselves. Heres Tom with the Weather.”
― Bill Hicks


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OfflineMrBovineJoni
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321]
    #26652673 - 05/06/20 10:29 AM (3 years, 10 months ago)

Quote:

HoneyBadger9321 said:
Right yes! I was referring to the first (before PC-ing). I just cant seem to dissolve it completely and chunks still remain when i filter it. Maybe i am not doing it long enough. I had a look at RRs agar videos and there he does not cook it before PC-ing. Takes a lot of time for me and seems that the results are the same, just a little bit cloudy agar. I might be wrong tho?



I see, that makes sense. To be honest I'm not sure about that chunks/sediment in the agar as I have never had it happen for me but I have seen plenty of people have it in their agar. Perhaps it is the matter of TEK or just the quality of ingredients.

What I usually do ( I started basing my initial agar work on C10s guide) for my media bottle (1L - safe to use for up to 850ml of mixture, otherwise it might boil over):
a) weight dry ingredients. In my case it is 12g of LME (light malt extract) & 16g of agar agar powder for ~800ml of water.  Mix them.
b) carefully transfer dry mixture to the media bottle and add about 100g of water, tightly close the bottle and shake the living shit out it .
c) Add the rest of the water (so it makes ~800ml or so) and shake once more. There are bound to be some chunks and flecks floating (for me they seem to be from the LME powder not the agar. LME absorbs water from the air like crazy so it does tend to clump up.
d) (optional) Add a drop of food colouring (I like green but it is up to personal preference).
5) Put the bottle cap on (it should not be tight so some of the steam can escape) and place in a microwave. Blast it on max for a while and keep shaking every few minutes (lid tightened for that!).
6) I do this until all of agar has dissolved (the temperature of the agar here is close to boiling point so wear gloves). However, be careful once the agar starts bubbling in places (and close to being fully dissolved) - as the cap is not super tight at this point. Agar boils over incredibly quickly so if you turn away for 20s you can end up cleaning your microwave. The result is crystal clear agar for me except for some bubbles on top from all the shaking (it will disappear after PCing).

7) Done - put the foil over the cap and place in a PC.

Steps 1-6 don't take longer than a total of 15minutes for me.

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Invisiblec10h12n2o
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Re: c10's Agar Guide + Tips & Tricks (post #1000) [Re: HoneyBadger9321] * 1
    #26653033 - 05/06/20 02:09 PM (3 years, 10 months ago)

Quote:

HoneyBadger9321 said:
When I put my agar mixture into the microwave to disolve it down, it just gels up the agar and everything else is liquidy. So i cant really filter it. Am I not boiling/microwaving it enough? I do around 10min all together.




Before it boils i stir it with a stir rod, once or twice

Once it starts boiling i turn the power down to 50% , stir it, and try to maintain that boil for a minute or so. Super carefully,  because it boils over very easily

I wouldnt say these steps are mandatory, but it definitely seems to help get crystal clarity and perfect uniformity.

Sounds like you are just omittimg the intermittent stir. Also make sure you use cold or lukewarm water for the initial mix before boiling. If the water is too hot it will clump up

Whole process of measuring, mixing, boiling, straining, and foiling takes me about 10 minutes total for 1 liter of agar


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

C10's Agar Guide + Tips and Tricks | c10's Flow Hood Build Guide


"Partial knowledge is more triumphant than complete knowledge; it takes things to be simpler than they are, and so makes its theory more popular and convincing."

"Convictions are more dangerous enemies of truth than lies"
― Friedrich Nietzsche

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