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Cookin' in the kitchen of love - Cybele's agar log


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#1 cybele

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Posted 10 September 2019 - 12:29 AM

So I have decided to start a thread for all of my agar work. I would like to have a single place so I do not have to start multiple threads for every question or hijack anyone else's threads with pestering. It will give me a place to keep up with my experiments and isolation's. Whilst having an amazing community to critique my work at the same time! Criticism, and advice is completely welcome! I have been working with these amazing life giver's for too long to have not gotten into agar. I see a lot of newer members jumping right into agar, and bulk! I commend you all. The wise does at once what the fool does at last.

 

So.. at last.

 

 

This should be quite the journey. I will be starting by using the no pour technique and doing my transfers in a SAB. I have many hours inside of my SAB doing my best to perfect my aseptic technique, and agar should allow me to see where I sit with my skill set is inside of it.

 

I am starting with the following:

 

Panaeolus Cambodginiensis - in a spore syringe

Panaeolus Cyanescens (Alabama) - spore print *Gifted from a great Pan expert, and a generous individual from here on topia

Golden teacher - currently growing - The plan is to transfer a clone to some jars, and collect some prints for future plates/jars

 

The following prints are two years old, and have been kept in a book bag in the bottom of a closet. A true test to cleaning up an old dusty print.

 

Golden Teacher - Spore prints from a previous grow

Koh Samui - Spore prints from a previous grow

 

I'm currently cookin' up the following:

 

Paul Stamet's MEA from TMC

I split the recipes in half for my 500ml Erlenmeyer Flask

 

  • 10 grams of tan malt
  • 1 gram of nutritional yeast
  • 10 grams of agar
  • 0.5 grams of activated charcoal *Not included in the traditional TMC MEA recipe (can my AC users weigh in here on the amount used - pun intended :tongue: )

 

I also have some potatoes boiling on the stove so I can start making some PDY while the MEA is in the pressure cooker. Also following the Paul Stamet's recipe from TMC

 

(PDY) Potato Dextrose Yeast Agar

  • the filtered, extracted broth from boiling 300 grams of sliced potatoes in 1L of water for 1 hour * adapted for a 500ml flask
  • 5 grams dextrose sugar
  • 1 grams yeast (optional)
  • 10 grams of Agar

 

I have some pictures coming, along with the exact brands, and prices of the supplies. I am an aviation technician by trade so lets talk tools! I would love to see how, and why everyone uses what they use!

 

Lesson number one, and always number one. Experience is the best teacher. It seems I dumped the agar powder in too fast. I should have slowly let it slide in and stirred it along. Instead my measured amount went right down the funnel causing some clumps. So I am betty crocker shakin' and stirrin' the fuck out of this hahaha.

 

 

Thank you all for visiting my piece of this great pie.


Edited by cybele, 10 September 2019 - 02:33 AM.

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#2 cybele

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Posted 12 September 2019 - 11:28 AM

Potato Dextrose Agar (PDY)

This time I mixed the agar in the room temperature potato water, and it did not clump. It mixed in extremely well, and after the PC phase it gelled up perfectly.

I added the dextrose, and the yeast while it was on the burner.
Then I added one drop of blue food coloring.

CF86-E0-D7-0002-4-B61-9-D20-4-D1-F91-B36

F9-D0-B219-F32-C-4-DE0-904-B-F224-AAEE58

Edited by cybele, 12 September 2019 - 11:43 AM.

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#3 sandman

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Posted 12 September 2019 - 11:42 AM

I like to add agar powder and other dry ingredients to cool water in a pot and then bring to a simmer. Simmer for about 10 minutes while stirring often. The agar can be clumpy and just not mixed up well if you skip the step of simmering/stirring before putting in your flask.

 

I know adding pictures is difficult and well hidden on this site. Click more reply options yellow button at bottom right and then you can attach the pics with a link at the bottom left of the post.

 

edit: you edited a lot in the few minutes that it took for me to write that post haha. Nice.


Edited by sandman, 12 September 2019 - 11:43 AM.

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#4 cybele

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Posted 12 September 2019 - 11:49 AM

I like to add agar powder and other dry ingredients to cool water in a pot and then bring to a simmer. Simmer for about 10 minutes while stirring often. The agar can be clumpy and just not mixed up well if you skip the step of simmering/stirring before putting in your flask.
 
I know adding pictures is difficult and well hidden on this site. Click more reply options yellow button at bottom right and then you can attach the pics with a link at the bottom left of the post.
 
edit: you edited a lot in the few minutes that it took for me to write that post haha. Nice.


Yessir, I experimented and quickly learned with my second production run lol. From now on I’m going to mix all ingredients in room temperature water first. Then funnel it into the flask.

I was testing the pictures, and formatting right as you posted. Technically the posted above PDA was mixed and put in jars second. I did my MEA first.

This first round of pictures are out of order.

#5 cybele

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Posted 12 September 2019 - 12:01 PM

Malt Extract Agar (MEA)

A little bumpy start on the thread, figuring out the pictures. This batch of MEA was my very first attempt at mixing up some agar. Just like the PDA I used the Paul Stamet’s recipe posted above.

Tap water
Agar
Malt
Yeast
Activated charcoal* not in TMC recipe

658-EB71-F-7-AD1-4-DE7-BF3-E-2-AC9-D07-A

Here you can see the agar clumping I described above. That was an easy lesson to go ahead and get out of the way. With this hobby’s continuously found patience I shook. , and stirred for over an hour. It actually turned out well mixed. However work smart, not harder.


F3587-C13-71-B2-4461-A74-F-3265825-D33-A

Edited by cybele, 12 September 2019 - 12:02 PM.


#6 cybele

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Posted 12 September 2019 - 12:47 PM

So last night I decided to attempt laying some specimens on my freshly produced agar jars.

 

I started with a Pan Cambo spore syringe, and three agar jars. Two MEA jars, and one PDY. It did not take long for a lesson to show its face. Doing the first jar I noticed they had all sealed during the PC cycle. Even though I left the bands extremely loose. Lesson learned. I also stacked my PC full, and believe the mason jars on the second row got more condensation during the PC cycle. **Does anyone place foil around their lids like during PF jars? Note: I have ordered plastic wide mouth lids for my agar jars. I am just getting some learning, and practice in. Technique technique technique.

 

To release the pressure, and drain the condensation I placed a Clorox wipe around the jar, and released the pressure. I believe I saw catsandbats using the clorox barrier in one of his agar threads. I then sterilized the needle, and did my best to put one drop of solution in the middle of the jar. I placed the band back on the jar, and repeated that for the other two.

 

Next I started with a Pan Cyan spore print. Once again I had to learn. Holding the inoculation loop was sloppy, and unfamiliar too me. I feel like it was too large for the confined space. It also could not fit inside my iso-alcohol jar. I plan on modifying it smaller. Next go around I will also try using my scalpel to transfer some spores. I opened the jars using the same Clorox method. Sterilized the inoculation loop, and strafed it over the agar surface in a pattern.

 

Six specimens inoculated, and sixteen jars left. Now to wait for growth.

 

Up next will be some old KSSS, and GT prints. I will continue to work on my aseptic technique, and handling while I lay some more stuff on these jars. I need to use up the remaining sixteen before the new plastic lids come in. By the time they come in I should be getting the hang of this enough to lay down some exceptional specimens.

 

I would like to try transferring some mycelium from one of my jars right now to agar.


Edited by cybele, 12 September 2019 - 12:50 PM.


#7 cybele

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Posted 12 September 2019 - 01:03 PM

Here is the finished product. The blue is the PDA, the black is the MEA. This is just to show the consistency, coloring, and depth of the agar. I’m leaving these two out on the counter as a control.

9-F6360-B3-8-CCC-4-FCA-9-DF5-AE754-E6-D6

Pictured is the ball plastic lids. The quart jar has not been PC’ed and was just the extra MEA left (it is left in the fridge). Plastic lids for my quart regular mouth grain jars are here, but I am still waiting on the wide mouth plastic lids for the agar jars.

B63-FDA22-4953-4-EB5-9621-29-EFD353-C341


Edited by cybele, 12 September 2019 - 01:06 PM.

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#8 Misfit

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Posted 12 September 2019 - 11:24 PM

I seemed to have a bunch of issues when I tried using food coloring. 2 batches went worse than my first attempt. Needless to say I went back to basics.


“I’d rather have nothing than have a lie”
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#9 cybele

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Posted 13 September 2019 - 09:04 AM

I’m going to try a few different things. I feel like the condensation is going to my primary problem.

#10 Misfit

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Posted 13 September 2019 - 09:23 AM

Yea I was told that was one of the issues. Had odd discoloration.


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#11 cybele

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Posted 13 September 2019 - 09:38 AM

Yea I was told that was one of the issues. Had odd discoloration.
“I’d rather have nothing than have a lie”


The water sitting on top of the surface is going to become an interstate system for bacteria, while at the same time inhibiting the growth of fungi if I understand it correctly. I’m trying a few different methods to tackle the condensation while using jars.

What is the normal growth window if you are starting with spores?

#12 Misfit

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Posted 13 September 2019 - 10:05 AM

It takes longer with spores usually a week plus.
Also I started inverting my jars so that helped.


“I’d rather have nothing than have a lie”

#13 cybele

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Posted 15 September 2019 - 10:42 AM

It’s always fun playing the waiting game.

Six of the agar jars were inoculated on 9/11. Three of them are already showing growth three days later. So far here are the results.

I had a little MEA left over from pouring into the small jars. So I left that in a quart jar on the counter top with a plastic lid. I did not PC it because I wanted to use it as a control. It should prove if the PC run was successful, and it didn’t disappoint. I also left an unopened agar jar next to it that was sent through the PC run.

Two days after pouring, and the inoculation of a few jars I saw growth on the counter top control. I wanted to wait seventy two hours before checking the agar jars so I gave it another night. Today it’s very easy to distinguish the growth in the control jar. This small experiment proved fruitful. One, the PC run was successful because the control showed contaminated growth after 2 days. While no agar jar shows contaminated growth, so far, including the six I inoculated. Also the unopened agar jar that was counter top control is also showing no growth.

969-FBCC3-F4-CE-4019-BAA5-304-A39-AD7016
[some type of green mold, and a yellow blob. Not sent through PC left on counter]

So if any contamination shows itself in the agar jars it’s my handiwork.

Out of the six the Pan Cyan are all three showing growth. The two on the MEA have very wispy growth the size of a pen dot, and its aerial. The one showing growth on the PDA looks weird. I suspect it’s the condensation, it may be the food coloring. Two early to tell.

7-C1-CD4-D8-07-FD-4-AE0-8-A39-BE6-CE96-D
[waiting to see if this turns into Pan Cyan mycelium]

The Pan Cambo’s are showing no growth yet.

#14 bezevo

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Posted 15 September 2019 - 12:05 PM

great post  thanks for sharing keep it coming ...learning 


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#15 cybele

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Posted 16 September 2019 - 01:07 PM

Four days in, and the growth is incredible.

The pan cyan is taking off inside the MEA. It went from one germinated pin dot to almost germinating the entire symbol I strafed with the inoculation loop. It’s only germinated one spot on the PDY, and the growth is minimal. To reiterate a spore print was used.

A spore syringe was used for the pan cam, and no growth is visible on any three of the agar jars.

#16 cybele

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Posted 17 September 2019 - 12:17 PM

Tomorrow will be one week since inoculation, and we have no contamination. That is quite the confidence builder since I thought I was doomed. Between the condensation, the lids being pressurized and my sloppy handling I was sure at least one would go bad. The Pan Cyan’s are still shooting off, and that is what I have pictured.

DB25-DEF8-4-AFB-4-DFC-8712-C1758427-A1-E
[here you can see the mycelium growing up. The growth on this MEA is whispy so far]

9-DD8-C9-CE-21-DA-4251-9-D3-F-FBC9-C7-CA
[the growth on the PDY looks different]

Although the appearance in the mycelium differs. I believe they are both Pan Cyan mycelium, but what do I know? Lol. You’re watching me learn this as I go!

The Pan Cam is showing growth in one jar along the very side. I believe the spore water, Along with the condensation caused some trouble with these.

#17 cybele

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Posted 20 September 2019 - 11:13 AM

Just watching the growth now. The Pan Cyan's are all germinated, and growing steadily. It should not be that much longer before I need to start isolating these. The Pan Cam's finally decided to grace us with an appearance. The pan cam sample pictured below germinated four days after the cyan's, and it caught up to size within a day.

 

 

F3-EA296-E-106-A-470-C-B9-BB-904-D25-DA4

[Pan Cyan on PDA]

91-F01-F58-26-DD-44-C5-82-A9-CEA3-C1-F64

[Pan Cyan on MEA]

7-E1-DCED7-3-D83-46-CA-958-D-A9-F22-A224

[Pan Cam on MEA]


Edited by cybele, 20 September 2019 - 11:19 AM.


#18 Asura

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Posted 21 September 2019 - 10:43 AM

Looking good!


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#19 cybele

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Posted 23 September 2019 - 11:15 PM

Looking good!


I appreciate it bud! Is isolating wispy aggressive cultures specific to cyan’s or a general rule of thumb for all pan’s? All my pan cambo cultures are extremely tomentose. Picture the cotton on the end of a q-tip, but pristine, and glowing. It’s growing nearly a half-inch above the agar.

Edited by cybele, 23 September 2019 - 11:16 PM.


#20 cybele

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Posted 23 September 2019 - 11:34 PM

The process of agar has two major benefits.

One being something we all love. Being contaminate free!
Two being able to isolate and test strains to find a keeper.

We’re always practicing clean work because, well, waste not want not.
So that leaves us with room to experiment.

I have successfully kept these cultures fed, and happy. Now here comes the fun right? Isolating, and fruiting.
Which leads to a bonus major of agar work. Who needs bulk inoculate? Not you if you work with agar.

It also leads to questions. When do you start transferring to isolate? I see two options. Let them colonize the entire plate, and watch it to see how they act, and react to each other. Then transfer based on observations. Or take the smallest transfer from the smallest growth of each germination point from the original dish that was inoculated with spore/syringe. Then grow them out on individual plates, and watch them side by side.

Any input/advice is welcomed.




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