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InvisibleKauaiOrca
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Agar Question (trying first time)
    #21730366 - 05/27/15 09:15 PM (8 years, 8 months ago)

I've had decent success in the past making liquid culture from purchased spore syringes, but am convinced I want to try agar, cloning and liquid inoculant.  I've read a number of threads on Agar, but am still unsure about a couple of things.  Any help anyone can give me would be hugely appreciated.  Here are my questions:

1. I would like to be able to put the Agar into either a petri dish or glad mini round (not sure what is best) and then into the PC so when it comes out, it is sterile and ready to use.  What is better for this, mini rounds or petri dishes and do I need a glass petri glass or are the plastic ones able to PC?

2. What is the simplest formula for Agar to pour into the petri dish or mini round that will go into a PC?  Simple and effective works for me.  The "most effective" if it's much more difficult isn't worth it for me. 

3. If I should be using Glad Mini Rounds for this, about how high should I pour the Agar mixture? 

4. Is it much better to use cloned tissue or just a drop of multi-spore from a purchased syringe to get an agar culture going? 

Thanks in advance for your help.  This site is fantastic!!!


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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OfflineMonkeyJesusFresco
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21730408 - 05/27/15 09:28 PM (8 years, 8 months ago)

1) not petri dish, it will boil over

2) :shrug: someone will chime in with their mashpotato-agar recipe soon enough :smile:

3) as high as you'd like, as long as it fills the entirety of the bottom of the dish, but too thick and it may be difficult to cleanly cut little pieces out...

4) I would recommend starting off with a drop from a syringe (or swipe from a print) and don't worry too much about "isolating" just yet, grow out the multispore and then clone from there, and then isolate more if so desired


Edited by MonkeyJesusFresco (05/27/15 09:28 PM)


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InvisibleMad Season
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Re: Agar Question (trying first time) [Re: MonkeyJesusFresco]
    #21730425 - 05/27/15 09:33 PM (8 years, 8 months ago)

Haha I don't use potatoes. For every 100 ml I do 2g agar and 2g light malt extract


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: Mad Season]
    #21730438 - 05/27/15 09:37 PM (8 years, 8 months ago)

Quote:

Mad Season said:
Haha I don't use potatoes. For every 100 ml I do 2g agar and 2g light malt extract




Thanks, I can do that! … About how many ml water would I need for one glad mini round?  Just trying to calculate how much I need to make to fill 8 mini rounds???


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisibleMad Season
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21730445 - 05/27/15 09:40 PM (8 years, 8 months ago)

Around 50 mls normally per dish

Start with half a liter and go from there. I'd ddefinitely use a folded piece of paper for weighing and pouring the dry malt extract. It's seriously sticky stuff


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


Edited by Mad Season (05/27/15 09:42 PM)


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OfflineMonkeyJesusFresco
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Re: Agar Question (trying first time) [Re: Mad Season]
    #21730454 - 05/27/15 09:44 PM (8 years, 8 months ago)

:super:


Edited by MonkeyJesusFresco (05/27/15 09:44 PM)


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: MonkeyJesusFresco]
    #21730485 - 05/27/15 09:53 PM (8 years, 8 months ago)

Quote:

MonkeyJesusFresco said:
:super:




Appreciate your advice!  Is cloned tissue more or less likely to generate a isolate that will fruit better than a multispore drop on the Agar?  I'm trying to determine the best way to get a really great isolate that produces the way I see some grows on this site.  I get decent results but never anything like the pictures I see here.


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21731945 - 05/28/15 10:09 AM (8 years, 8 months ago)

One other question … If I use the agar technique and get a totally clean culture, I can then suck up a little of it in a syringe, squirt it into a liquid culture jar (sterilized, 4% dex/LME) and grow out clean LC, right?  I like the technique of LC because I know how to do it and can grow lots of it better than having to blend LI every time.  Just sucking up the the clean LC into a syringe seems pretty easy. 

Is there any reason I shouldn't be thinking like that and should stick to LI with the blending of the Agar wedge tek? (that I've never tried, but seems to have a lot more steps)


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisibleMad Season
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21732129 - 05/28/15 11:08 AM (8 years, 8 months ago)

For me I put spores on agar and transfered until it was clean, then grew it out, took a favorable  clone and then isolated from there.

As for LC just take a clean agar wedge and drop it into an LC. Having a clean inoculation will make the chances of the LC being clean pretty high. Make a few lcs from wedges and test them out


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: Mad Season]
    #21794830 - 06/11/15 08:26 PM (8 years, 7 months ago)

Quote:

Mad Season said:
For me I put spores on agar and transfered until it was clean, then grew it out, took a favorable  clone and then isolated from there.

As for LC just take a clean agar wedge and drop it into an LC. Having a clean inoculation will make the chances of the LC being clean pretty high. Make a few lcs from wedges and test them out




I made up some glad mini rounds with agar and put a couple drops of MS spore solution.  One of them has grown out some very puffy white stuff on top.  Looks almost like cotton balls growing about 1/3-1/2 an inch above the surface of the Agar.  Is that normal?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisibleMad Season
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21794844 - 06/11/15 08:30 PM (8 years, 7 months ago)

Pics would help but this is a certain culture I had that was super fuzzy. They look like pillows



--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: Mad Season]
    #21794883 - 06/11/15 08:39 PM (8 years, 7 months ago)

Quote:

Mad Season said:
Pics would help but this is a certain culture I had that was super fuzzy. They look like pillows






Here's a picture



--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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InvisibleMad Season
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21794893 - 06/11/15 08:41 PM (8 years, 7 months ago)

Looks good to me. Quite overgrown for spores but still mycelium.


--------------------
contam and car window art
How to shroomery like a pro! (Seriously, everyone read this!)
Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: Mad Season]
    #21794906 - 06/11/15 08:46 PM (8 years, 7 months ago)

Quote:

Mad Season said:
Looks good to me. Quite overgrown for spores but still mycelium.




Thanks. Really appreciate your help. First try with Agar. Did 6 mini rounds with different strains (TEXAS, Amazon, PE6, PE, ATL #7, South America) and this one has the most growth after about 4 days BY FAR.  The others don't show much colored growth on top but when I look at the agar from the bottom, I see string dark strands growing down into the agar on all the rest of them.  The TEXAS strain, though is really growing fast. 

Should I just grow out some of this puffy stuff on grain then take the biggest fruit, clone it and move it to Agar?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Offlinemushpunx
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21794923 - 06/11/15 08:50 PM (8 years, 7 months ago)

Quote:

KauaiOrca said:
Quote:

Mad Season said:
For me I put spores on agar and transfered until it was clean, then grew it out, took a favorable  clone and then isolated from there.

As for LC just take a clean agar wedge and drop it into an LC. Having a clean inoculation will make the chances of the LC being clean pretty high. Make a few lcs from wedges and test them out




I made up some glad mini rounds with agar and put a couple drops of MS spore solution.  One of them has grown out some very puffy white stuff on top.  Looks almost like cotton balls growing about 1/3-1/2 an inch above the surface of the Agar.  Is that normal?





Next time instead, drop your spore solution onto a sterilized loop or sterile cotton swab and swipe the agar with it.

It makes it easier to decipher clean germination growth because you know where you swiped, the liquid kinda rolls around and causes the fuzzy growth

Just do your best to take a healthy piece away from bacteria or any colonies, and it should look better on the next plate. But swiping definitly helps, cause a lot of nasties can hide under the germination when its all over the place :thumbup:


Dont go for the biggest fruit, go for fast growing, big healthy pin clusters. Big fruits can be related to environmentals, cloning big fruit doesnt always = big clones. Cloning clusters gives you good shot at big yeilds


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InvisibleKauaiOrca
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Re: Agar Question (trying first time) [Re: mushpunx]
    #21794945 - 06/11/15 08:54 PM (8 years, 7 months ago)

Quote:

mushpunx said:
Quote:

KauaiOrca said:
Quote:

Mad Season said:
For me I put spores on agar and transfered until it was clean, then grew it out, took a favorable  clone and then isolated from there.

As for LC just take a clean agar wedge and drop it into an LC. Having a clean inoculation will make the chances of the LC being clean pretty high. Make a few lcs from wedges and test them out




I made up some glad mini rounds with agar and put a couple drops of MS spore solution.  One of them has grown out some very puffy white stuff on top.  Looks almost like cotton balls growing about 1/3-1/2 an inch above the surface of the Agar.  Is that normal?





Next time instead, drop your spore solution onto a sterilized loop or sterile cotton swab and swipe the agar with it.

It makes it easier to decipher clean germination growth because you know where you swiped, the liquid kinda rolls around and causes the fuzzy growth

Just do your best to take a healthy piece away from bacteria or any colonies, and it should look better on the next plate. But swiping definitly helps, cause a lot of nasties can hide under the germination when its all over the place :thumbup:


Dont go for the biggest fruit, go for fast growing, big healthy pin clusters. Big fruits can be related to environmentals, cloning big fruit doesnt always = big clones. Cloning clusters gives you good shot at big yeilds




Thanks!  I'll do this.  Any thoughts on the other mini rounds with no growth on top but when I look at the agar from under and hold the mini round to the light, I see dark strands moving through the Agar.  Is that Mycellium or something else?


--------------------
"The universe is endless, limitless and infinite.  Any effort to define it's boundaries is an attempt to overcome ignorance.  We are physical, mental and spiritual beings ... there is no beginning and there is no end.  There is only memory.  Our repeated loss of memory experiences create the illusion of beginnings and ends.  Immortality is the ability to retain full memory through all consciousness transformations.  Loss of memory is man's greatest curse and, in very real terms, death."

-- Ancient Taoist Master


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Offlinemushpunx
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Re: Agar Question (trying first time) [Re: KauaiOrca]
    #21795049 - 06/11/15 09:22 PM (8 years, 7 months ago)

Sometimes its hard to get germination on agar
Could be, just give them some more time :shrug:


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