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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Using Agar to Raise the Thai-tanic (Closed)
#26608450 - 04/17/20 03:49 PM (3 years, 9 months ago) |
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I first grew mushrooms in 2015. I had no idea what I was doing. For my first grow, I injected an MS syringe of a cubensis strain called Thai-Tanic into 9 half-pint commercially-purchased BRF jars. I birthed the cakes, dunked and rolled and fruited them.

I got three flushes from 9 cakes that yielded a mere 65 dry grams.
I also collected spore prints.

Back to the present. As part of a separate ps. cyanescens project, I recently began working with agar for the first time. Since I can’t yet use the plates for the other project, I’m going to use them in my attempt to raise the Thai-tanic!
I assume my spores are contaminated because I’ve unwisely opened the foil and shown the prints to numerous people over the years. I plan to clean up the mess with agar to agar transfers and hopefully isolate some hearty mycelium.
Then, I’ll inoculate three quart jars (2/3’s full) of rye berries and mix the resultant spawn to CVG substrate in a Sterilite 32 quart monotub. I’ll also make a master plate and slant for future use.
This is an account of my second voyage aboard the Thai-tanic. Hopefully, this time around, it will be smoother sailing and I can produce much more than 65 grams of fruit.
For completeness, I’m including my agar preparation, based upon Bod’s Comprehensive Agar Tek.
Materials:
Malt extract agar (MEA). Pressure cooker. 70% isopropyl alcohol. 100 mm plastic, sterilized petri plates. 1000 ml Pyrex media storage bottle. One pint or quart mason jar with lid. Aluminum foil. 20 mm x 125 mm test tubes with screw caps. Wooden coffee-stirring sticks. 10 ml syringe. Scissors. Parafilm. Infrared thermometer. Inoculation loop. Torch. Tongs. Forceps.
4/13/20: I put on a face mask and gloves. I wiped the gloves with iso and I sprayed my sleeves with iso.
I sanitized my SAB with iso.
With iso, I wiped a sealed bag of 10 petri plates and put it in the SAB. I wiped scissors with iso and used them to cut off the bottom of the bag. I left the bag on for now.
In addition to the 10 plates, I decided to make two slants. I worked with the ratio of 5 grams of MEA per 100 ml of water.
Because I didn’t want to buy an autoclavable test tube rack, a mason jar will suffice to hold the test tubes vertically in the pressure cooker. I filled the mason jar with one inch of water.
I weighed 15 grams of MEA and, using a funnel, poured it into the media storage bottle.
I cut two coffee-stirrers into segments just over 2.5 inches long.
I heated a small pan of water until it began boiling.
I threw the stirrers into the pan and put on the lid.
After about 10 minutes, I turned off the heat and removed the lid to verify that the stirrers had sunk to the bottom of the pan, indicating that they were saturated with water. The stirrers need to be saturated so that they sink to the bottom of the agar in the slant, instead of floating on top of it. I left them in the pan for now.
I heated another pan of water to nearly boiling and, using a funnel, poured the water into the media bottle until the level reached 300 ml.
I screwed the lid onto the bottle of shook the shit out of it to ensure the MEA was dissolved.
Using forceps, I removed the stirrers from the pan and inserted one into each test tube.
I removed the cap from the media bottle and tilted the bottle so that the agar was closer to the mouth of the bottle.
I inserted the syringe into the bottle, sucked up 7 ml of agar and injected it into one test tube. I repeated the procedure with the other test tube.
I put the lid on the media bottle and screwed it on half-way to allow pressure to escape. I covered the lid with aluminum foil
I put the caps on the test tubes and screwed them on half-way. I covered the caps with aluminum foil.
I placed the test tubes into the mason jar. The lower ends of the tubes were submerged in the water. I put the lid on the mason jar and screwed it on half-way. I covered the lid with aluminum foil.
I put the PC rack into the PC and poured in 3 quarts of water. I put the media bottle and the mason jar into the PC, lowering them into the water at an angle to prevent air bubbles from getting trapped under their bases.
I put on the lid and turned the stove on to high.
I let the PC vent steam/air for 10 minutes and then I put on the regulator.
I ran the PC at 15 psi for 25 minutes.
I removed the PC from the stove and let it cool until the pressure gauge read 0 and the air vent/cover lock was down.
I took off the lid and, using pot holders, removed the bottle and jar.
Using tongs, I removed the slants from the mason jar. Through the foil, I screwed down the lids of the tubes. Then I removed the foil and tightened the lids again to be sure.
Both tubes displayed a ring of sediment in the position where the top of the agar was when the tubes were vertical in the PC. I must have accidentally drawn some sediment into the syringes and missed it. And then I missed it again when I injected the tubes. I swirled the agar around in the tubes in an attempt to dissolve the rings, but was unsuccessful.
I placed the tubes in the standard, nearly horizontal slant position and allowed the agar to solidify. The sediment rings remain in the tubes. Hopefully they won’t impair the colonization of the slant.
Through the foil, I tightened the lid of the media bottle and removed the foil. I put the bottle in my SAB to let it cool.
Over the next 20 minutes or so, I checked the bottle’s temperature with my handy infrared thermometer. When the bottle reached 120 F, I removed the bag from the stack of plates and stood it, upright in a corner of the SAB. Because I feared holding 10 plates with one hand, I separated the plates into two five-plate stacks.
I removed the lid from the bottle and put the lid out of the way. I held the bottle with my left hand. With my right hand, I gripped the stack of plates. I filled the plates from the bottom up, in standard plate-pouring fashion. I then placed one five-plate stack onto the other one.
I was delighted to discover that I prepared the precise amount of agar. None was left over in the bottle.
I removed the bottle and cap from the SAB. Before the agar had a chance to completely solidify, I immediately cleaned the bottle and cap with hot water and dish soap. I cleaned up everything else and let the agar cool for an hour.
After re-sanitizing myself, I put 10, pre-cut one inch strips of Parafilm into the SAB. I wrapped each dish with Parafilm and re-stacked them. I tried putting the plastic bag over the stack, but its opening kept sticking to the Parafilm, so I couldn’t do it.
I lifted the SAB off of the plates and put the stack on a shelf.
I examined the slants and saw condensation inside the tubes. Later, the condensation had fallen onto the agar. I re-sanitized myself and the SAB. I put the tubes into the SAB. Holding a tube upside down, I removed the lid and turned the lid upside down. I gently shook the tube and a drop of water fall out. I screwed the lid on. I repeated this procedure with the other tube. I laid the tubes horizontally on a shelf.
And then I laid myself horizontally in bed and crashed.
4/14/20: The slants still appear overly moist inside. And they’re not going to dry out with the lids screwed on tight. So today, I unscrewed the lids and wrapped the lid/tube juncture with Parafilm. To ensure that I knew how much I was unscrewing each cap and to ensure that both caps were equally unscrewed, I did so like this:
With the stirrer side of the slant toward me, I marked the cap with a dab of my wife’s nail polish directly above the stirrer to establish the cap’s orientation.
I placed the slant into the SAB, holding it with the stirrer side of the slant away from me. I unscrewed the cap 180 degrees (one half turn), so that the nail polish dot on the cap was now visually above the stirrer from my perspective. I wrapped the lid/tube juncture with Parafilm. I repeated the procedure with the other slant.
The nail polish dots weren’t visible after I wrapped the slants. But that doesn’t matter. They served their purpose while I was unscrewing the caps. I know that the caps of both slants are now equally unscrewed. Let the evaporation can begin.
4/15/20: The temperature in my grow room, aka the “mushroom”, ranges from 66 degrees overnight to 77 during the day. The humidity is around 40%. I had the stacked plates and slants on top of a high bookshelf near a heating vent. When the heat comes on, it can reach well over 80 degrees up there. I worried that the higher temperatures might delay the desired evaporation of the condensation on the three plates and the water in the slants.
So I moved the slants and plates to the base of our fireplace in the living room, where the temperature ranges from 58 degrees overnight to 70 during the day, and there is a slight breeze from a window. Hopefully, this placement will facilitate evaporation.
I know there’s a lot of debate about stacking plates upside down, so the agar is on top, to prevent condensation or facilitate its evaporation. I never understood the logic of that practice, so I stacked my plates right-side up. In other words, the agar is on the bottom.
4/17/20: I was pleased to see that condensation is gone from all plates and there is visibly less water in the slants. But I also noticed that there is a small amount of fine sediment scattered on the bottom of the agar in all of the plates. There is also a small amount of sediment suspended in the slants.
Apparently, I didn’t shake the agar well enough before pouring or I didn’t shake it between pours, and I should have. Or perhaps I used too much MEA.
Is this a potential problem? Please chime in and tell me your informed opinions.
4/17/20: I put on a face mask and gloves. I wiped the gloves with iso and I sprayed my sleeves with iso.
I sanitized my SAB with iso.
I wiped the foil containing my prints with iso and put it in the SAB. I wiped a plate with iso and put it in the SAB. I put a pre-cut piece of 1” Parafilm in the SAB. I removed the Parafilm from the plate and set it off to the side.
I unwrapped the foil, exposing the prints.
I wiped the handle and shaft of my inoculation loop with iso I fired up my handy, hands-free torch.

Despite alcohol fumes hanging thick in the air like ripening moss, I flamed the loop and avoided self-immolation.
With my left hand, I lifted the lid off the plate, keeping the lid above the plate. With my right hand, I stuck the loop into the edge of the agar to cool the loop and to collect a bit of sticky agar.
I scraped the loop around on a portion of the print and picked up many more spores than I intended. Hey, it was my first time!
I swiped the loop across the center of the agar in a Z formation.
I put the lid on the plate.
I wrapped the plate with Parafilm and put it on a shelf in my “mushroom.”

And now I wait.
Stay onboard for updates.
I welcome your questions, comments and castigation.
Thanks,
Tom
Edited by tomcards (04/30/20 03:04 PM)
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26612275 - 04/19/20 07:54 AM (3 years, 9 months ago) |
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4/19/20: The Thai-tanic plate sits on a wooden table near half-pint jars of BRF for a separate cyanescens project. The table top is 26 inches off the ground. This morning, it was 64 degrees in the grow room. I was shocked to discover that there was condensation on the lid of the plate.
I checked the stack of poured, un-inoculated plates that sit inside a closed, floor level cabinet of my desk. The lid of the top plate had condensation on half of it. Damn! I thought the condensation issue was resolved.
The heater came on, the room warmed up and minutes later the condensation was gone from the lid of the Thai-tanic plate. Obviously, the cooler overnight temperature caused the condensation.
I picked up the plate and tilted it and a very thin layer of water ran across the surface of the agar. The water is invisible unless I tilt the plate.
Is this normal? Will the presence of this very thin layer of water impede or prevent mycelial growth?
Please chime in and offer your opinions and advise.
Maybe it is better to store the plates upside down (agar on top). I flipped the un-inoculated stack upside down. I'll check it for condensation in the morning.
The Thai-tanic plate is currently right side up. There is no sign of mycelium yet. I'd like to turn the plate upside down, but I worry that the water will fall onto the lid, taking the spores with it.
Should I turn the plate upside down? Please tell me what you think.
Thanks,
Tom
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bodhisatta 
Smurf real estate agent


Registered: 04/30/13
Posts: 61,889
Loc: Milky way
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26612291 - 04/19/20 08:01 AM (3 years, 9 months ago) |
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Agar dishes are 96% water. You're not going to completely avoid condensation. Don't worry about it lol
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: bodhisatta]
#26612346 - 04/19/20 08:18 AM (3 years, 9 months ago) |
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Bod,
I'm thrilled and honored by your response! Thank you for your reassurance! I'll leave the plate right side up.
Respectfully,
Tom
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26624819 - 04/24/20 01:49 PM (3 years, 9 months ago) |
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It's been one week since I inoculated the agar with the Thai-tanic spores. There is no sign of life.
Well, the spores are five years old. I'll give it another week and see what happens.
On 4/23, three days after I inoculated the agar with the Thai-tanic spores, I inoculated another plate of the same agar with fresh spores of another strain. On 4/26, mycelium appeared on the plate. So the agar isn't the problem.
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poisoned
untitled



Registered: 04/17/13
Posts: 1,738
Loc: Yurop
Last seen: 1 year, 3 months
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26624821 - 04/24/20 01:50 PM (3 years, 9 months ago) |
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With 5 yo spores, it will probably take >2 weeks.
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: poisoned]
#26638768 - 04/30/20 11:03 AM (3 years, 8 months ago) |
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Well, it's 4/30 and there are still no signs of life. Nada. Zip. Zero. Zilch. Bupkiss.
I have two other projects in progress, which are doing quite well. I'm through with my attempt to raise the Thai-tanic. Unfortunately, for now, it will remain sunken.
Unless one of you wants to try to resurrect it.
On one piece of foil, I have half of a 1" print and an intact 3/4" print. I you live in the US and you want it, PM your address to me and it's yours.
Yes, that's right. I'm offering old, sketchy, possibly dead spores. But at least I'm honest about it. And it's better than simply throwing them away, which I will do if no one responds within 72 hours.
Thanks,
Tom
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Sunny Skies
Cluster Head


Registered: 05/03/17
Posts: 421
Loc: my house
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26638779 - 04/30/20 11:10 AM (3 years, 8 months ago) |
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Try again mate! I got 4 year old PESA spores going after 2 tries...the others ..nothing
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: Sunny Skies]
#26638879 - 04/30/20 11:59 AM (3 years, 8 months ago) |
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Sunny,
Thanks for the encouragement, but honestly, I'm done with this project.
Let me know if you want to give it a go.
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Sunny Skies
Cluster Head


Registered: 05/03/17
Posts: 421
Loc: my house
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: tomcards]
#26638900 - 04/30/20 12:13 PM (3 years, 8 months ago) |
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You have access to syringes where as I do not..Maybe you could rehydrate the spores making your own..I shall have to pass on this and I do appreciate your offer.
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pureshrooming
feels like a stranger


Registered: 05/28/18
Posts: 321
Last seen: 15 days, 19 hours
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: Sunny Skies]
#26638968 - 04/30/20 01:05 PM (3 years, 8 months ago) |
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When I started agar work I was in a very similar situation. I had couple year old dirty af B+ prints that I was beating my head against the wall trying to get clean. I finally broke down and ordered from a trusted supplier and spent a month living/breathing agar. I'd suggest holding onto your prints and get some successes under your belt then come back. It feels really good to conquer what seemed hopeless at one point.
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (in progress) [Re: pureshrooming]
#26639021 - 04/30/20 01:24 PM (3 years, 8 months ago) |
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Pureshrooming,
I'm already having success with my other agar project. I'm using the same agar, but with fresh spores.

I did my first transfers today.

I'm simply tired of trying to raise the Thai-tanic.
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tomcards



Registered: 04/24/15
Posts: 427
Loc: San Francisco
Last seen: 10 months, 19 days
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Re: Using Agar to Raise the Thai-tanic (Closed and still sunken.) [Re: tomcards]
#26639260 - 04/30/20 03:01 PM (3 years, 8 months ago) |
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Guys,
I posted my offer on the Trade forum.
To eliminate redundancy, I'm signing off from this thread, which I will edit as "Closed".
Thanks for your support!
Tom
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