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Offlineblueconfusion
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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19251753 - 12/09/13 06:33 PM (10 years, 2 months ago)

and how and when do you mix it in i know some antibiotics are heat sensitive


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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19251766 - 12/09/13 06:35 PM (10 years, 2 months ago)

This will be my first go with it so I'm just doing research now. Something tells me that a little goes a long way. Think about the size of most peoples fish tanks, then look at the plate. I do know that it cannot be PC'd so it needs to be added as the agar is cooling and gently swirled in.


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Re: Just say NO to LC and YES to PDYA [Re: Pastywhyte]
    #19251796 - 12/09/13 06:38 PM (10 years, 2 months ago)

really want to know the results i may have to conduct my own experiments this weekend i have a Snapple jar just sitting waiting to be used and some used dishes that are just calling to be my test monkeys :super:
i had tried with some other antibiotic a couple weeks ago but totally messed it up and i don't think it was the right stuff saw tetracycline almost grabbed it for round two but i wanted to research and see if anyone else had done it, and how they went about it.  what better time then here and now to find out


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Offlineblueconfusion
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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19251846 - 12/09/13 06:45 PM (10 years, 2 months ago)

After autoclaving, and when the agar has cooled enough that it’s not too hot to touch (about 1 to 1.5hrs), add antibiotics as follows:
Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plate with a single red line on the side.
Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml.  Mark the plates with a single green line on the side.
Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml.  Mark the plates with a single black line on the side.
Chloramphenicol – add 1ml chloramphenicol stock (at 25mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plates with a single purple line on the side.


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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19251967 - 12/09/13 07:08 PM (10 years, 2 months ago)

holy shit dude all that trouble, when you just need to add some water to a fully colonized grain jar, shake and extract the juice enough to inoculate 100's of jars, GLC ftw

simple and fool proof


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19251977 - 12/09/13 07:10 PM (10 years, 2 months ago)

Simple, but prone to contamination.


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19251985 - 12/09/13 07:11 PM (10 years, 2 months ago)

Quote:

blueconfusion said:
After autoclaving, and when the agar has cooled enough that it’s not too hot to touch (about 1 to 1.5hrs), add antibiotics as follows:
Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plate with a single red line on the side.
Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml.  Mark the plates with a single green line on the side.
Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml.  Mark the plates with a single black line on the side.
Chloramphenicol – add 1ml chloramphenicol stock (at 25mg/ml) per liter of agar to obtain a final concentration of 100ug/ml.  Mark the plates with a single purple line on the side.




Good stuff

Quote:

loki44 said:
holy shit dude all that trouble, when you just need to add some water to a fully colonized grain jar, shake and extract the juice enough to inoculate 100's of jars, GLC ftw

simple and fool proof




Well I am working with wild spores and clones and many of them are not as fast as cubensis. So antibiotic is good. As for regular PDA or PDYA its as easy as making jello :shrug: A GLC is not guaranteed either. You could have just inoculated 100's of jars with mold. Agar FTW.


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19252002 - 12/09/13 07:14 PM (10 years, 2 months ago)

Hey blue,

What's your motivation for using the snapple bottles vs a liquor bottle?

Just to have a pre-measured amount of agar for your petri?

Nice write up :thumbup:


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Re: Just say NO to LC and YES to PDYA [Re: SpitballJedi]
    #19252029 - 12/09/13 07:19 PM (10 years, 2 months ago)

Quote:

SpitballJedi said:
Simple, but prone to contamination.




no, key word here FULLY colonized, the problem is people get impatient and go ahead without letting the mycelium take over 100% of the grains and beyond, when you see all the grains white as snow and the mycelium crawling all over the glass because there's no more grain that's when is ready, not before

you don't really need all that trouble and expense if you are using MS, now if you want isolates agar is a must but for most people who are not in the comercial side but just wanna do a couple monos here and there GLC is the way to go


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Re: Just say NO to LC and YES to PDYA [Re: loki44] * 1
    #19252059 - 12/09/13 07:27 PM (10 years, 2 months ago)

Your grains are not going to be 100% sterile. The dormant spores or endospores will eventually germinate. It doesn't matter if your spawn is 100% colonized, it will still happen.

The subject has been debated many times. People with experience have mostly abandoned them and moved on to agar, which is what this thread is about AND the originator GLC has said he wished he'd never wrote the tek.

This is not my thread so, I will not say anything more. But, telling these guys on this thread to use GLC will get you nowhere because they already know the deal.


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Edited by SpitballJedi (12/09/13 07:30 PM)


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Re: Just say NO to LC and YES to PDYA [Re: Pastywhyte]
    #19252074 - 12/09/13 07:30 PM (10 years, 2 months ago)

Quote:

blueconfusion said:


Well I am working with wild spores and clones and many of them are not as fast as cubensis. So antibiotic is good. As for regular PDA or PDYA its as easy as making jello :shrug: A GLC is not guaranteed either. You could have just inoculated 100's of jars with mold. Agar FTW.




OP what other species are you working with?well if you are cloning and using wild spores you need dat dere agar


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19252075 - 12/09/13 07:30 PM (10 years, 2 months ago)

Quote:

loki44 said:
Quote:

SpitballJedi said:
Simple, but prone to contamination.




no, key word here FULLY colonized, the problem is people get impatient and go ahead without letting the mycelium take over 100% of the grains and beyond, when you see all the grains white as snow and the mycelium crawling all over the glass because there's no more grain that's when is ready, not before

you don't really need all that trouble and expense if you are using MS, now if you want isolates agar is a must but for most people who are not in the comercial side but just wanna do a couple monos here and there GLC is the way to go




So what about the center of the jar, where your ms inoculation deposited some sneaky trich, and you cant see from the side, and when you shake it up the mycelium is white. Your GLC still good? Not to mention spitballs point about the endospores, which I firmly believe in. Agar is not hard, and you don't need to be a "commercial" grower to enjoy it on a small scale. Frankly I find agar to be one of the most enjoyable aspects of the whole grow and I would probably have been bored already if it didn't exist.


Edited by Pastywhyte (12/09/13 07:39 PM)


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19252091 - 12/09/13 07:36 PM (10 years, 2 months ago)

Quote:

loki44 said:
Quote:

blueconfusion said:


Well I am working with wild spores and clones and many of them are not as fast as cubensis. So antibiotic is good. As for regular PDA or PDYA its as easy as making jello :shrug: A GLC is not guaranteed either. You could have just inoculated 100's of jars with mold. Agar FTW.




OP what other species are you working with?well if you are cloning and using wild spores you need dat dere agar




That was actually my quote. If you must know I'm working on some wild Aussie cubes, some p alleni, wild subs collected from several parts of the world, a wild print of hericium coralloides, I'm also gonna get a buddy to let me swab the gills of his wild reishi that he found, ps cyancesens, and a few others in the mix as well.


Edited by Pastywhyte (12/09/13 07:45 PM)


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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19252112 - 12/09/13 07:39 PM (10 years, 2 months ago)

Sounds fun. Liquid culture is a pain in the ass


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Re: Just say NO to LC and YES to PDYA [Re: kechlesurf]
    #19252128 - 12/09/13 07:43 PM (10 years, 2 months ago)

It is but I am starting to work with it for the sake of my mexicana and pan experiments. Still use a wedge to inoculate it of course.


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Re: Just say NO to LC and YES to PDYA [Re: PirateSwazey]
    #19252134 - 12/09/13 07:45 PM (10 years, 2 months ago)

Quote:

PirateSwazey said:
Hey blue,

What's your motivation for using the snapple bottles vs a liquor bottle?

Just to have a pre-measured amount of agar for your petri?

Nice write up :thumbup:



Liquor bottles don't fit in my PC and thank you


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Re: Just say NO to LC and YES to PDYA [Re: SpitballJedi]
    #19252151 - 12/09/13 07:48 PM (10 years, 2 months ago)

Quote:

SpitballJedi said:
Your grains are not going to be 100% sterile. The dormant spores or endospores will eventually germinate. It doesn't matter if your spawn is 100% colonized, it will still happen.

The subject has been debated many times. People with experience have mostly abandoned them and moved on to agar, which is what this thread is about AND the originator GLC has said he wished he'd never wrote the tek.

This is not my thread so, I will not say anything more. But, telling these guys on this thread to use GLC will get you nowhere because they already know the deal.




Do you realize that those dormant spores or whatever nasties are gonna still be in your grain  when you spawn to bulk? I'm not trying to convince anyone. Most experienced growers in this site grow at fairly large scale and it would be  a disaster to lose an entire crop because of bad spawn  so agar is understandable in their particular circumstances. Once you have more to lose, makes sense to go the extra step to avoid contamination, but just because they do it doesn't mean is necessary for a successful grow


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Re: Just say NO to LC and YES to PDYA [Re: blueconfusion]
    #19252174 - 12/09/13 07:51 PM (10 years, 2 months ago)

i will also be getting some wild spores, this was my interest in the antibiotic agar, other than that i prefer agar for all the points made by spitball and pasty.  i am no where near a commercial grower i do this for fun and am simply trying to provide the info to those new growers that think an LC or GLC is the way to go.  I have done both and both have contaminated on me.  if my agar is contaminated i don't have to throw the whole thing away i can simply isolate good tissue away from the contamination easy as that!  how you gonna save a GLC or LC?


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19252187 - 12/09/13 07:53 PM (10 years, 2 months ago)

Quote:

loki44 said:
Quote:

SpitballJedi said:
Your grains are not going to be 100% sterile. The dormant spores or endospores will eventually germinate. It doesn't matter if your spawn is 100% colonized, it will still happen.

The subject has been debated many times. People with experience have mostly abandoned them and moved on to agar, which is what this thread is about AND the originator GLC has said he wished he'd never wrote the tek.

This is not my thread so, I will not say anything more. But, telling these guys on this thread to use GLC will get you nowhere because they already know the deal.




Do you realize that those dormant spores or whatever nasties are gonna still be in your grain  when you spawn to bulk? I'm not trying to convince anyone. Most experienced growers in this site grow at fairly large scale and it would be  a disaster to lose an entire crop because of bad spawn  so agar is understandable in their particular circumstances. Once you have more to lose, makes sense to go the extra step to avoid contamination, but just because they do it doesn't mean is necessary for a successful grow



yes but there is a difference between spawning to a properly pasteurized bulk with dormant spores than propagating those dormant spores by moving them to a sterilized grain


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Re: Just say NO to LC and YES to PDYA [Re: loki44]
    #19252195 - 12/09/13 07:55 PM (10 years, 2 months ago)

Quote:

loki44 said:
Most experienced growers in this site grow at fairly large scale and it would be  a disaster to lose an entire crop because of bad spawn  so agar is understandable in their particular circumstances. Once you have more to lose, makes sense to go the extra step to avoid contamination, but just because they do it doesn't mean is necessary for a successful grow




Most of them also grow more than one variety/species at a time as well so they would need different cultures anyway. Agar just makes sense for them, however I would say for the average cultivator who just wants a tub of good shrooms once a year agar is just as worthwhile. That way he can have a good culture slanted up and when he is ready he can just grow it without having to worry about things like, poor yield, or bunk potency or all the other crap that you can end up with doing ms. Oh and his one grow a year is probably pretty important to him so he got just as much to lose if it ends up in the toilet cause he did a GLC.

Really if I have no choice but to do an LC, I would take an LC inoculated with a clean agar wedge over a GLC any day :shrug:


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