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Agar questions sigh i know


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#1 stonnerkidd

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Posted 19 November 2021 - 06:18 AM

Hey guys im going to attempt to do agar from syringe spores. I wish to do spore swabs too but i already have syringes spores.
So i already have a little home made still air box with holes for my arm.
I know to sterlize the whole area and whip everything down. Spray around the dish?
And do i need that perfilm for the agar plates? Do they come with it most sites?
Do i need them? Or can i use plastic baggies each one lol.
Big questions is when i use the syringe do i use the needle? Wnd just flame the needle like i would and open and drop a couple drop? Or can i just twist the cap off and squeeze a couple drops out. When i flame needle for 5-10 seconds and let cool for about same??? Dont wanna burn any spores.
Any tips be cool. Turn off all fans and mask. Lmk any tips. If this goes well ima order more plates and swabs
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#2 HrVanker

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Posted 19 November 2021 - 06:25 AM

Prints and swabs are great for agar. Syringes aren't so good, but I've done it. My syringes resulted in bacterial spots on the plates, but I was able to isolate the healthy mycelium. Just a single drop per plate should be fine.

Alternatively, you could do up some PF jars and grow out the spores from the syringe. Then clone a fruit or two, and take some prints which will be easier to work w/ on agar.

When I work in my SAB, I spray and wipe everything down w/ isopropyl. You can probably be fine using plastic bags for your plates, they are cheaper than para-film and are reusable. So that's a plus. I use jars, but others who use plates may have some better input.

Edited by HrVanker, 19 November 2021 - 06:27 AM.

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#3 Myc

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Posted 19 November 2021 - 10:40 AM

I use plates and always use Parafilm. It's worth every penny and a box will last for years. The film is also good for other stuff like masking for painting scale models. Perfect for grafting experiments or making repairs to broken branches in the cannabis garden.

 

Plastic bags will allow the plate to lose moisture. But I use them for control plates and such.

The ziploc bags are UV sterilized and are clean until you open the bag - where atmosphere (and particles) enter the bag. Unless this is done in a sterile airstream or glovebox - your bag is contaminated. Period.

They can work in a pinch but they cannot be re-used unless re-sterilized.

 

I've never had a problem going from spore solution (syringe) to agar. Shake the syringe and place a drop of solution on your plate. Place the lid on the plate, wrap with Parafilm, and manipulate the plate so as to distribute moisture evenly across the surface.

If you get contaminants - simply transfer healthy tissue to a fresh plate.


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#4 Salty117

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Posted 19 November 2021 - 12:52 PM

I don't recall where I read this suggestion, but I read somewhere of someone mentioning that they use the flame-sterilized syringe tip to dig up a little crater in the center of the agar plate to contain the drop. I tried this and it worked fine, but I like the idea more of spreading the spores around to spread out the colonies. I also have a problem with my plates solidifying at an angle due to my house being old and having settled ALOT over the last 80 years, so I have been considering using an inoculation loop to spread the spores, so they don't end up pooling in one area.

 

Also, not to be a stickler I was just pointed this out when I was first learning this process, when you spray down your SAB and equipment with alcohol spray, that is sanitizing, not sterilizing. Sterilization takes place through flame exposure, or under 15psi in a pressure cooker/autoclave.

 

When I flame sterilize the syringe tip, I try to get as much of the metal part glowing hot and just be careful not to set the plastic part on fire. Blowing it out also means having to re-sterilize. I wait about 10+ish seconds to try to begin inoculating but I also use a laminar flow hood that will help cool it down faster. When I still used my SAB, I would have a small condiment cup that I would shoot a very very small amount of spore solution into before inoculating in order to cool the syringe tip quicker. To some that may be wasteful, but at least I knew the spores that did get onto the agar plate weren't going to be sterilized by hot metal on their way out.


Edited by Salty117, 19 November 2021 - 01:52 PM.

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#5 Myc

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Posted 19 November 2021 - 06:57 PM

Re: Using Parafilm

 

Go to this article:

https://mycotopia.ne...s-banzai-style/

and scroll down to posts #8 and #10 which detail the proper use of the film. It takes some practice. I also use the Type M film detailed in the tutorial.

 

If you read the whole thread I'm sure you'll find it enlightening, I still use those techniques to this day. -- Pouring agar with a 60cc syringe so as to meter the amount of agar per vessel is pure genius gold. Less messy and the syringes can be re-sterilized in a Pressure Cooker for further use.

Detailing how to even boil agar is pure genius gold.

Just do yourself a favor and take a few minutes to check out the link.


Edited by Myc, 19 November 2021 - 07:00 PM.

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#6 Arathu

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Posted 19 November 2021 - 10:05 PM

And folks like Myc are the attendants and assistants in the New Library......virtual Alexandria no doubt.....

 

Pull up a beanbag and a hookah..... 

 

There are so many gems here you won't be able read and look at it all.....

 

banzai is very much responsible for some of my debauchery......hah! Great link! 

 

 

A


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#7 Myc

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Posted 20 November 2021 - 10:29 AM

I don't promote 'mazon but check this out:

https://www.amazon.c...h/dp/B07C2LKDFJ

 

A 250 foot roll for $27 USD.

You can wrap more than 5000 plates with this amount of material. I suspect this would last a lifetime for most folks.


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#8 Arathu

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Posted 20 November 2021 - 10:48 AM

I highly recommend learning to use lab film and also good solid temperature control of stored and incubating pitri plates.....

 

Plates inverted, literally turned upside down, help with condensation too but can be troublesome if clone tissue is a large mass.....but if a culture takes it will be stuck to agar in short order......

 

The use of lab film on plates makes this easy peasy...Agar is a very useful technique and skill set to acquire and develop, well worth the time and investment......IMHO

 

Good price on the film by the way.... :biggrin:

 

As with everything I've gone way overboard and already bought a couple lifetimes worth.....but if I start selling my gourmet species and strains that could change quickly... 

 

A


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#9 Myc

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Posted 20 November 2021 - 10:54 AM

That's what happened for me.

I learned this fun hobby and figured out aseptic technique.

 

Now, I get to put these skills to use in a commercial setting - where I get paid.

All of that attention to detail now pays dividends.

Just goes to show that you never know where you'll be in a couple of years.


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#10 MushLuvR

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Posted 20 November 2021 - 11:50 AM

That's Awesome @Myc,.. I have that same 250 ft box of Parafilm and have done at least 100 plates and SO much left.  I know you can use Saran Wrap, but this stuff is Gold and once you get the feel of how to stretch it without snapping, it's Easy.  AGAR work is definitely a skillset, and the right pouring temperature is the Key.  ALL worth it(no replacement for practical experience), and I also have goals of possibly going further like you @Myc.  Time will Tell, I'm just enjoying my work at this point(Eating FINE Edibles)... Plottin, Planning... AND Perfecting.  If I can be this successful in a SAB(anyone can with experience), there is NO stopping Me... with the right tools(LFH).  MUSH Life!

 

MushL


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#11 Sidestreet

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Posted 20 November 2021 - 01:13 PM

 

Alternatively, you could do up some PF jars and grow out the spores from the syringe. Then clone a fruit or two, and take some prints which will be easier to work w/ on agar.

 

I would go this route.  Gets you right to a desirable fruiting strain by cloning.


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