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OfflineWinterOwned
Fighter of the Nightman
I'm a teapot User Gallery
Registered: 08/30/08
Posts: 149
Last seen: 1 year, 3 months
Need some agar encouragement please.
    #26510984 - 03/01/20 08:14 AM (3 years, 10 months ago)

Do y’all agree this looks like blue mold? It’s hard to see because I added too much gypsum but this does NOT look good to me. I must have done something wrong. I outran contaminants and ended up with this beautiful agar dish. Sterilized my grain properly.

Something must have gone wrong between there and my SAB, maybe in the SAB. I had a hard time slicing the agar with a (sterilized) scalpel and getting the wedges out. I did my best not to lift the lid up all the way but you kind of got to a little bit to get the wedge out and into the jar. I feel like this agar experience was all a huge waste of time.

Previous to this, I just inoculated grain jars directly from the spores from the manufacturer. A couple of my jars turned out not contaminated, and I grain to grained them to new jars.

Any ideas what I did wrong, what I can do better or words of encouragement would be appreciated.





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OfflineWinterOwned
Fighter of the Nightman
I'm a teapot User Gallery
Registered: 08/30/08
Posts: 149
Last seen: 1 year, 3 months
Re: Need some agar encouragement please. [Re: WinterOwned]
    #26511247 - 03/01/20 11:27 AM (3 years, 10 months ago)

I’ve lost 4 of seven jars. The same rate I had without agar.


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Invisiblemurderlabz
RIP Stoneman
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Registered: 05/18/19
Posts: 551
Loc: The Multiverse
Re: Need some agar encouragement please. [Re: WinterOwned]
    #26511553 - 03/01/20 03:05 PM (3 years, 10 months ago)

You agar work ilooks great, see the slime your oats are leaving on the glass? They are too wet letting bacteria run crazy.

Can you walk us through your prep?

What is your grain prep?
What type of lids are you using?
"hard time cutting agar" what is your agar recipe?


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Offlinesusurrador
Psychedelic Cowboy


Registered: 03/31/19
Posts: 1,432
Loc: SW US Flag
Last seen: 10 months, 6 hours
Re: Need some agar encouragement please. [Re: murderlabz]
    #26513866 - 03/03/20 01:51 AM (3 years, 10 months ago)

In your SAB- if you're hovering the lid over the dish while you're cutting and taking you're wedge for the grain jar, that may be where something got in.

In the SAB particles will fall straight down if there aren't any air currents, which there shouldn't be in a still-air-box.

But the outside of the lid of your dish isn't sterile so if your clean scalpel passed under the non-sterile lid, baddies could have dropped in your dish/on your scalpel on your way in or out of the dish. Same for dropping the wedge into the jar. Lift the lid up and away. Then replace it exact opposite of how you removed it.

Using your thumb and middle finger on one hand and approaching from the sides (not from above at all) gently grab the petri lid and lift it up and away entirely from the dish. Do not hold the lid above the dish or your scalpel while making cuts and transfers. The SAB provides the barrier for falling contam particles.

It is ok to hold your lids upside down for a moment while you cut and transfer. Sometimes while holding the lid away and upside down, I'll stick my pinky out to stabilize the agar dish while I cut. If the air is still and settled in your SAB, there should be no particles falling hence why we can expose the petri without contaming it. Same is true for the lids of petris and jars.

What is your gypsum for?


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"If it doesn't work, you can always hit him with it."



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OfflineWinterOwned
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Registered: 08/30/08
Posts: 149
Last seen: 1 year, 3 months
Re: Need some agar encouragement please. [Re: susurrador]
    #26518155 - 03/05/20 07:58 AM (3 years, 10 months ago)

So I guess I’ll try setting the lid down instead of hovering it over the dish next time. I’m happy with the consistency of my agar but i just find it difficult to move around in box period. I’m using a glovebox which is completely airtight rather than sleeves.

The gypsum was part of the recipe for the tek I read but it seemed like so small of an amount I put more in. I’m not going to use it at all next time.

As far as prep, I’m soaking my rye for a day, bringing to a boil for ten minutes and then letting them steam off until I can set them on a pice of toilet paper without  leaving a wet mark. I sterilize in a PC for 60 or 90 minutes.

I’m a little confused on how long to pc. I have a book that calls for 20 minutes only but everyone here seems to say that’s inadequate.


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Offlinesusurrador
Psychedelic Cowboy


Registered: 03/31/19
Posts: 1,432
Loc: SW US Flag
Last seen: 10 months, 6 hours
Re: Need some agar encouragement please. [Re: WinterOwned]
    #26518388 - 03/05/20 10:56 AM (3 years, 10 months ago)

Yo, watch these.

Especially the Agar to Agar transfer vids.

Watch them many times.

Simple process but very important details!

https://www.shroomery.org/forums/showflat.php/Number/24337011/vc/1#24337011

Also, don't use a glove box! Make a SAB!


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"If it doesn't work, you can always hit him with it."



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Offlinethe_professor
Learner


Registered: 10/07/19
Posts: 4
Loc: GA
Last seen: 3 years, 10 months
Re: Need some agar encouragement please. [Re: susurrador] * 1
    #26518691 - 03/05/20 01:23 PM (3 years, 10 months ago)

Weed out the sterilization vectors for the grain. Next time you run jars through PC, leave one out and don't transfer to it. 

Took me a while to find out that my grains weren't the problem. Next, I had to work on my sterile techniques for my SAB work. What I found is that my grains were failing because my agar had bacteria even though it looked clean. It just took the bacteria longer to germinate than the myc. You want your myc to outpace it in the grain...

MS to grain is a crapshoot. That agar looks nice but I like my dishes to show less genetic difference before transferring. I would transfer those leading edges to three more plates (isolation) with updated sterile and SAB techniques and wait them out. I keep a few questionable dishes around during that transfer session to see if they go bad.

In the long run, don't give up. I had a revelation along my journey that I had to come to terms with. I was overdoing just about everything. I thought back then that if spraying ISO in the SAB is part of it, then I should spray more often. WRONG.

I really had to peel back everything and think about the room, air quality, SAB tools (racks, towels, etc), butane torch from an alcohol lamp, gloves to NO GLOVES (i don't wear gloves anymore), switch from diluted 90% ISO to straight-up 70%, wiping down the dishes priot to letting sit....and I am about 90% clean on my dishes now. Up from about 25%.

Don't give up!


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