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Fuzzy's First Agar Adventure


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#1 fuzzyfunguy

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Posted 23 February 2017 - 11:01 AM

This is my first sincere take at agar, I've worked with it in the past before but I always rushed it and never tried to truly isolate a good strain. Now I am in a position to take my time and do it right. Any tips or feedback is surely appreciated as I don't really know what I am doing.

I innoculated 3 plates from a B+ spore print and a week later got some tomentose (Is that how you spell it?) mycelium, check it out:
ca2db7bb9a2a728cb764a459265aa03e.jpg
ebad8bc5aaf64fb1de847a39acb9a9a6.jpg5914abf4bc62503ae7846a1d815472c8.jpgd152174681a8ad17eead3cd3272a9387.jpg

What points look most promising for a transfer? Should I go for the thickest part of the mycelium or more towards the outside, strongest looking tendrils?

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#2 Myc

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Posted 23 February 2017 - 11:11 AM

I tend to avoid the aerial mycelium and go for the fan shapes which are growing on the surface. 

 

Take a sample from the leading edge of the advancing mycelium. Get the smallest, tiny little snag you can manage - the size of the head of a pin or less if possible. I also try to grab from the area just before the mycelium becomes visible to the naked eye. 

Take several samples from each fan and observe growth. You will have a f-k ton of plates going and much work ahead of you in testing/further isolation. The refrigerator is your friend if you need to slow things down a bit. ;)

 

Nice work. 


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#3 fuzzyfunguy

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Posted 23 February 2017 - 11:47 AM

80c63c79506ee77fe0fae097aaf4f3c3.jpg
Yes, I am going to need to make a lot more plates!!! Good fun though, I am wondering what the best tool is to take small samples for transfers, a scalpel comes to mind but a sterilized set of tweezers seems like it would be more effiecient.

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#4 peacefrog

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Posted 23 February 2017 - 01:41 PM

I personally use an xacto knife. A good scalpel is hard to beat IMO. But I prefer to use the cheep ones from Walmart or somewhere similar. One could use tweezers, I have never tried it, but as long as you are able to flame sterilize, you should be good. The one issue that might arise, would be how clean and precise your transfers would be. But who knows, the only way to see is to try.

When doing agar to agar transfers, I cut, like Myc stated, very tiny pieces and just barely knudge the wedge onto the knife, not stabbed. Then with a very quick and deliberate circular twisting motion onto the new agar surface, and it comes off really fast and accurately placed. The new plate is only opened up for a second at most per transfer and the top is back on. You should try the tweezers for the heck of it, it appears you have a lot good growth to play with.

Good job on those agar dishes!

Edited by peacefrog, 23 February 2017 - 01:43 PM.

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#5 Arathu

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Posted 23 February 2017 - 01:48 PM

Scalpel or Xacto (I use Xacto because they don't melt like cheap plastic scalpels when flaming and I can change blades on the cheap with the 100x box of blades) The above advice sounds spot on. The small front of an aggressive rhizome is what I go after...............

 

Good job!

 

A


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#6 Arathu

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Posted 23 February 2017 - 02:07 PM

rhizomes_001.jpg

Looking for some aggressive rhizomes........

 

rhizomes_002.jpg

Hmmmmm....what have we here????? :biggrin:

 

Perhaps........if I'm seeing what I think I'm seeing here......if so I'd be transferring some stuff from the leading edge of the "rope"

 

More plates indeed..........

 

A


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#7 fuzzyfunguy

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Posted 24 February 2017 - 12:01 AM

I did some transfers earlier with a scalpel, it took me a couple tries to get a technique down but it started flowing soon after, I tried taking as small of pieces as possible on the very edge of the growth. Interesting you point that out Arathu, I didn't notice that before but it looks like ropiness! I will check that plate out closer to see if it is.

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#8 CatsAndBats

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Posted 24 February 2017 - 12:09 AM

On this thread like flies on dung! Just give it a little poke fuzzy!
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#9 425nm

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Posted 24 February 2017 - 12:15 AM

What are people using to transfer spore to a plate? I have been trying to use an inoculation loop to seemingly no success. I'm beginning to wonder if the spore just aren't sticking to the nichrome wire I used to make the damn thing :/



#10 Myc

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Posted 24 February 2017 - 09:58 AM

What are people using to transfer spore to a plate? I have been trying to use an inoculation loop to seemingly no success. I'm beginning to wonder if the spore just aren't sticking to the nichrome wire I used to make the damn thing :/

 

I use a scalpel or exacto knife for that too. I once had some disposable inoculation loops and noticed that it helps to wet the loop from the moisture on the lid of the plate - then try to grab some spores from your print. 

Otherwise, I just scrape up a little pile as best I can with a knife and spread in a habitual pattern like a "Z" or something similar which you can recognize as your own. 


Edited by Myc, 24 February 2017 - 09:58 AM.

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#11 Jeepster

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Posted 24 February 2017 - 11:23 AM

Great thread! WARNING: Agar work is highly addictive.
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#12 CatsAndBats

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Posted 24 February 2017 - 01:04 PM


What are people using to transfer spore to a plate? I have been trying to use an inoculation loop to seemingly no success. I'm beginning to wonder if the spore just aren't sticking to the nichrome wire I used to make the damn thing :/


I use a scalpel or exacto knife for that too. I once had some disposable inoculation loops and noticed that it helps to wet the loop from the moisture on the lid of the plate - then try to grab some spores from your print.
Otherwise, I just scrape up a little pile as best I can with a knife and spread in a habitual pattern like a "Z" or something similar which you can recognize as your own.

Can you give any more details or hints on how to achieve the pile and then make a pattern with the spores?
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#13 425nm

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Posted 24 February 2017 - 02:12 PM

I do believe he is referring to using a streaking pattern something along the lines of this:

http://www.personal....hics/streak.gif

Which works great for bacteria and I have been attempting to use for fungi. However as stated earlier I'm not getting germination so I have yet to determine if it works as well. I suppose its a little redundant since you can just pick from the leading edge.
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#14 Arathu

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Posted 24 February 2017 - 04:26 PM

I flame modified stainless dental picks, then cool in target agar plate/jar with a stab motion on the edge of the plate (surely that gives stuff for spores to stick too methinks).....then grab spores from the edge of the print and streak my pattern on the plate......yes it's lifting the lid twice, so what, IME it works pretty well with laminar flow and even SAB........I'm not real worried about contamination on agar (within reason of course).........repeat until done or collapse......................agar makes choosing what we want and expanding/exploring that a distinct possibility.......... 

 

Great stuff right here fuzz..............keep going...................

 

 

post-33755-138195103478.jpg

 

 

@425nm some of the implements of destruction..........

 

post-33755-138195100035.jpg

 

Just what I found to work anyway...........

 

 

A


Edited by Arathu, 24 February 2017 - 04:44 PM.

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#15 Myc

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Posted 24 February 2017 - 11:33 PM

@catattack,

 

"Can you give any more details or hints on how to achieve the pile and then make a pattern with the spores? "

 

Like Arathu, I use a laminar workstation but that wouldn't prevent this being done in a glovebox.

 

The little pile I described is accumulated by tilting the blade at and angle - say 30* - and gently scraping the surface of the foil or other material where the print was deposited. It doesn't look like much but it is quite a large number of spores. If you zoom in on the print in the first photo, you may notice the tiny little section from which spores were sampled. (a.k.a. "pile of spores")

Then I try to spread the spores by moving and altering the angle of the scalpel upon inoculation. The attempt is to deposit spores in as even a smear as possible. The attached photo shows the "v" pattern I chose. If you zoom in, you can see that it's not an exact science but it gets the job done. Just hold the scalpel like a stylus - calligraphy is good practice. I cut into the agar with the finish stroke but still deposited spores along the seam. 

 

Hope this helps. 

 

 

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  • 20170202_181047 - Copy.jpg
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#16 peacefrog

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Posted 25 February 2017 - 08:35 AM

^^Spot on advice^^.


When I'm streaking from a print, it's a very gental, quick scrape. I never look at the end of my loop, but I always pay attention to the print itself. You will see the spores "move" so to speak from the scrape if that makes sense. Then streak in any pattern you like. I mostly do a zig zag personally, just out of habit.

Or if working with old dehydrated spores, like I am now cleaning up, I will gently crumble the print over the agar and not use my loop. This is not the best course of action for several reasons, but IME, one may need a lot more spore deposits just to secure some growth if they over 3 or so years old. A last ditch attempt so to speak.

Edited by peacefrog, 25 February 2017 - 08:41 AM.

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#17 CatsAndBats

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Posted 25 February 2017 - 09:12 AM

I do believe he is referring to using a streaking pattern something along the lines of this:

http://www.personal....hics/streak.gif

Which works great for bacteria and I have been attempting to use for fungi. However as stated earlier I'm not getting germination so I have yet to determine if it works as well. I suppose its a little redundant since you can just pick from the leading edge.

 

 

I flame modified stainless dental picks, then cool in target agar plate/jar with a stab motion on the edge of the plate (surely that gives stuff for spores to stick too methinks).....then grab spores from the edge of the print and streak my pattern on the plate......yes it's lifting the lid twice, so what, IME it works pretty well with laminar flow and even SAB........I'm not real worried about contamination on agar (within reason of course).........repeat until done or collapse......................agar makes choosing what we want and expanding/exploring that a distinct possibility.......... 

 

Great stuff right here fuzz..............keep going...................

 

 

post-33755-138195103478.jpg

 

 

@425nm some of the implements of destruction..........

 

post-33755-138195100035.jpg

 

Just what I found to work anyway...........

 

 

A

 

 

@catattack,

 

"Can you give any more details or hints on how to achieve the pile and then make a pattern with the spores? "

 

Like Arathu, I use a laminar workstation but that wouldn't prevent this being done in a glovebox.

 

The little pile I described is accumulated by tilting the blade at and angle - say 30* - and gently scraping the surface of the foil or other material where the print was deposited. It doesn't look like much but it is quite a large number of spores. If you zoom in on the print in the first photo, you may notice the tiny little section from which spores were sampled. (a.k.a. "pile of spores")

Then I try to spread the spores by moving and altering the angle of the scalpel upon inoculation. The attempt is to deposit spores in as even a smear as possible. The attached photo shows the "v" pattern I chose. If you zoom in, you can see that it's not an exact science but it gets the job done. Just hold the scalpel like a stylus - calligraphy is good practice. I cut into the agar with the finish stroke but still deposited spores along the seam. 

 

Hope this helps. 

 

 

^^Spot on advice^^.


When I'm streaking from a print, it's a very gental, quick scrape. I never look at the end of my loop, but I always pay attention to the print itself. You will see the spores "move" so to speak from the scrape if that makes sense. Then streak in any pattern you like. I mostly do a zig zag personally, just out of habit.

Or if working with old dehydrated spores, like I am now cleaning up, I will gently crumble the print over the agar and not use my loop. This is not the best course of action for several reasons, but IME, one may need a lot more spore deposits just to secure some growth if they over 3 or so years old. A last ditch attempt so to speak.

 

 

 

Good stuff guys, I'm just trying to glean more hints/tips/knowledge from my compatriots!

 

I might have to use the 'last ditch' method to revive some older fellas. I'll look. :tongue:


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#18 BigTexas

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Posted 25 February 2017 - 09:17 AM

I use a clay carving kit from harbor frieght, has scalpel like knives and scrapers, perfect for agar work it was $4 and I can flame sterilize them. 

image_17438.jpg


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#19 CatsAndBats

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Posted 25 February 2017 - 09:23 AM

I use a clay carving kit from harbor frieght, has scalpel like knives and scrapers, perfect for agar work it was $4 and I can flame sterilize them. 

attachicon.gifimage_17438.jpg

 

 

I use dental tools like arathu:

 

https://mycotopia.ne...r-30/?p=1304089

 

I just keep mine submerged in 91% iso, I should probably add a few drops of jetdry or citrus to act as a surfactant to help the alcohol to penetrate microfractures/cracks in the tools.


Edited by catattack, 25 February 2017 - 09:25 AM.

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#20 Myc

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Posted 25 February 2017 - 10:46 AM

If you notice, there is just the edge of a shot glass in one of the photos - LOL

 

I keep the tools standing in alcohol until I'm ready to flame sterilize. 

Then I rest them on the edges of the shot glass to cool in the airstream. 

Great minds all think alike. 

Also, I use a soldering torch for flame sterilization. It gets that metal red-hot pretty quick. And if you've shaken off all the alcohol, you're less likely to set the plastic scalpel handles on fire. 

 

I'm impressed by the clay working tool set. Never seen anything like it. Thanks for sharing.........everyone........and welcome to the party BigTexas. You're fitting right in already. 

 

For my last tip and trick:

I too, use surgical scalpels. I re-use them over and over. The blades get smutty over time.

The smut can be polished off with a scotch-brite pad and some comet. It leaves the blades shiny and new in case you forget to remove them from the alcohol bath one day and come back to find them corroded or rusty. 


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