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OfflineSittin_On_A_Toilet
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Registered: 10/17/20
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First Agar Experience - Need Advice on Best Path
    #26989990 - 10/17/20 11:54 AM (3 years, 3 months ago)

I recently decided to get back into this world after a ~5 year break where I only tried BRF cakes from a spore syringe. This time I am starting with spore syringe to agar as I've had nothing but bad luck with contaminated syringes and agar work is very interesting to me.

Right now I have 12 wide mouth half pint mason jars filled a little over 1/4" with LME Agar cooling after 40mins in a PC at 15 PSI. I bought the premixed stuff for my first few experiments - Malt Extract Agar. The lids are high quality PP lids which have a syringe filter and inoculation port. I bought these as I thought I was going to start with an LC from syringe, but after more reading here decided that was a bad idea.

I also have 20 gamma sterilized petri dishes when I decide to get more serious with the agar work and try a strain isolation. But this first experiment I stuck to a 'no pour' tek. I plan on keeping all colonizing jars (agar/grain/other) in a temp controlled room at 80F.

Questions I have before moving forward:
- I would like to avoid opening sterilized jars to inoculate, even in my SAB. Can I inoculate through the port on my lid in a small area on the agar? Or should I absolutely use the wire loop zig zag method?
- My plan is to inoculate 5 agar jars, saving the rest in case I need to do transfers to avoid contams. Once I have at least 5 clean and fully colonized agar jars I want to start ~3 LME LCs (in same style jars) using agar wedges, and use the rest of the wedges to inoculate QT WBS jars. Does that sound like a good way to get my operation going?

Thanks in advance to everyone on this board. I wanted to jump in ASAP, but decided to be patient and read/gather materials for a month before starting. I'm 100% open to anyone's advice on how I should proceed would be greatly appreciated.


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OfflineSittin_On_A_Toilet
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Registered: 10/17/20
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Re: First Agar Experience - Need Advice on Best Path [Re: Sittin_On_A_Toilet]
    #27040220 - 11/15/20 09:28 AM (3 years, 3 months ago)

Sorry to bump my own question, just want to answer my own questions in case someone stumbles upon it.

I would like to avoid opening sterilized jars to inoculate, even in my SAB. Can I inoculate through the port on my lid in a small area on the agar? Or should I absolutely use the wire loop zig zag method?

I ended up trying it 3 ways going MS to Agar, the pros/cons are OPINIONS based on what I've read here and actually attempted:

1. Leave lid always sealed, MS syringe thru SHP, and drip a few MS drops on center of agar (as close as I could get, my innoc ports aren't centered)

    Pro's - Easiest way for an absolute beginner to get some agar going. If it contams it's from the MS (assuming you
    PC'd correctly).
    Con's - You have liquid on the surface of the jar now. Most advice I've read say to keep solution away from the edge
    of the glass and pooled liquid water in ANY substrate (except LC) increases chances of contam by a lot. This tek is not
    ideal if you want to try to isolate genetics, which shouldn't be a priority if you're starting with a spore syringe. You
    just need to get a bunch of healthy myc going from the syringe.


2. Open lid, 1 drop of MS on agar, use wire loop to drag liquid in a zig zag
    Pro's Advanced technique for separating healthy myc from contams. Very little liquid on surface. Less than 1 drop
    can be rolled around. Even if the syringe causes a lot of contamination you'll still have some healthy myc to pull from.
    You can also isolate sub species easier with it more spread out. The average consensus I've seen is there really isn't a
    point to attempt to isolate sub species until you have fruiting bodies. You don't know if the sub species you're isolating
    from a MS is just a really good colonizer or actually a good fruiter as well.


    Con's - This technique takes more time = more time with agar lids off = higher chance of contam if you're
    inexperienced. It looks easy on videos, but takes a bit of practice especially in a SAB.


3. Open lid, scoop a small ball of agar out of the center with a wire loop, then a few MS drops in the hole
    Pro's Very easy and fast to do. You could even scoop agar right under the innoc port, put lid back on, and innoc     
      through that. The liquid doesn't run around the surface of the agar when moved because it's in a hole.
    Con'sOverall there aren't many cons and this is a good middle ground between 1 and 2 if your goal is to start a
      healthy myc colony from a MS, this is not aimed to be used with more advanced teks.
You need a sterile environment,
      but not for long. It's easy to scoop out a ball in the center of the agar. It's easy to aim the spore syringe into the
      hole.

In the end I had 1 contam out of 10, and that was from leaving the seal off the jar... And all of them fully colonized their agar. I had 2 of the 10 that were extremely rhizomorphic, I'm now working on transferring those to real petri dishes. The other colonized agar will be used to colonize grain master jars.

- My plan is to inoculate 5 agar jars, saving the rest in case I need to do transfers to avoid contams. Once I have at least 5 clean and fully colonized agar jars I want to start ~3 LME LCs (in same style jars) using agar wedges, and use the rest of the wedges to inoculate QT WBS jars. Does that sound like a good way to get my operation going?

Skip the LCs until you have genetics you know are good (actually fruited) and want to save long term. Even then, if you're continually working with agar you never really have a reason to make a liquid culture, unless someone else has another opinion?

Thanks for reading


Edited by Sittin_On_A_Toilet (11/15/20 09:31 AM)


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OfflinePBJ710
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Re: First Agar Experience - Need Advice on Best Path [Re: Sittin_On_A_Toilet]
    #27040271 - 11/15/20 10:08 AM (3 years, 3 months ago)

Not sure this is a direct answer to your question, but maybe it can provide some insight and my opinion.

You should consider even the finest vendor made MS syringes to be inheritly contaminated to some degree until proven otherwise.  The lower the amount of liquid carrier that you can put on the agar, the lower the chances of a contamination getting placed there with the desired spores.  You want a very thin layer of the spores to cover the surface to encourage as many different inoculation points as possible - this will allow you to select from clean parts of the plate for future transfers much easier than if they are all intermingled in the middle of the plate.

My next batch of spores that I run on agar, I plan to use a drop on a sterile swab and swipe the dishes.  I think I've been bringing over some bacteria at times via agar and this may help with it.

Out of those options, I would probably use #2.  1 and 3 would be more difficult to get clean cultures out of since everything would be in the middle or under the SHP and mixed together.

Good luck!  It sounds like you're on a solid path to success with your choices so far and with the research you are doing.


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OfflineSittin_On_A_Toilet
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Registered: 10/17/20
Posts: 16
Last seen: 3 years, 1 month
Re: First Agar Experience - Need Advice on Best Path [Re: PBJ710]
    #27041023 - 11/15/20 06:07 PM (3 years, 2 months ago)

Quote:

You should consider even the finest vendor made MS syringes to be inheritly contaminated to some degree until proven otherwise.  The lower the amount of liquid carrier that you can put on the agar, the lower the chances of a contamination getting placed there with the desired spores.  You want a very thin layer of the spores to cover the surface to encourage as many different inoculation points as possible - this will allow you to select from clean parts of the plate for future transfers much easier than if they are all intermingled in the middle of the plate.




I 100% agree, even the best syringe makers can have contam. But if you're brand new to this, trying method #1 over 10 jars you should get AT LEAST 1 clean jar, even with a dirty syringe. But if you don't have good sterile technique down and try 2 or 3, you might end up contaminating your jars by simply opening them and fucking around too long. Maybe I'm overthinking things though....

My next agar attempt I'm going to mix my own and actually pour it when it hits the right temp after PC'ing. My first attempt at a 'Pour Agar' tek, I ran out of time so I let the agar solidify and re-heated in the microwave the next day to pour. But I didn't get it 100% melted and didn't notice until I was suited up in my clean room and ready to go. So I said fuck it and poured some chunky plates. I also learned that day that parafilm doesn't work very well when the plates have hot agar in them... they melt like crazy. But these plates are for practicing sub species isolation. I'll try for real when I have actual fruiting bodies to start with a clone.


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OfflineFrozen Jedi
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Registered: 10/17/20
Posts: 26
Last seen: 3 years, 9 days
Re: First Agar Experience - Need Advice on Best Path [Re: Sittin_On_A_Toilet]
    #27041137 - 11/15/20 07:27 PM (3 years, 2 months ago)

Good stuff man! looks like we're rowin the same boat! thanks for the links! stay in touch!


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