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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: bootster]
    #19193024 - 11/26/13 04:58 PM (10 years, 2 months ago)

Disclosure: I am going back home for Thanksgiving to spend some time with the family and therefore will be away from this thread for a bit.
But I'll probably be back on Sunday 12/1, and hopefully have some progress to report, too.



I tried to prepare some agar last night(11/25), and forgot :bigblunt: to keep the stove on low heat. Needless to say, I burnt the shit out of my agar and tried to salvage it but I woke up this morning with a clear mind and ditched that crap to start over.

This morning's agar preparation went over much smoother, and I also decided to get some snapshots of the process.

The ingredients I'm currently using:


The temperature I noticed I need to keep my stove on to keep the agar from boiling/burning:


The batch of agar waiting to be poured, plus a sleeve to keep it warm :awesome:



I was doing everything at the same time this morning... I ended up cooking my new agar, while the jars that were prepped last night were emptied and thrown into the PC for cleaning. Well, the agar was ready before the jars were finished pressure cooking. I ended up pouring the prepped agar into the large jar as a temporary means of container while I was waiting for the baby food jars to come out of the PC.

After they came out so fresh and so clean clean, I poured from the larger container into all of the small agar jars and then threw them in the PC like this:




I have to keep my jars out of the water since the lids can't withstand the pressure while in the PC. I learned this the hard way my first go-round with agar, as mentioned in the OP. This is my temporary, possibly permanent solution unless anyone has a suggestion.. like some PC rack I don't know about or something? :shrug:



Edited by Midnight Cyclone (11/26/13 05:41 PM)


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19230384 - 12/05/13 08:57 AM (10 years, 1 month ago)

It turns out pastywhyte was right, and my sterile procedures aren't up to par for agar. I thought that I could cut corners and avoid a SAB, but agar is a different beast.

These are the jars from the sterility experiment (uninoculated jars)

Tale of the tape:

3 second jars

(white growth @ 9 o'clock) -- (white milky color floating @ 8-12 o'clock)


5 second jars

(black spot @ 7 o'clock)


10 second jars

(off white mass @ 6 o'clock) -- (orange/red color floating around from 7-3 o'clock)


30 second jar

(black spot @ 6 o'clock)



Since I last posted I have been reading non-stop about doing agar work in a SAB and my view has changed entirely.

I'll be constructing something simple like this.

And I just need to re-arrange my room somewhat to accomodate for the new SAB, where I thought before I wouldn't have space. (I really don't but it's just something else that has to fit if I'm going to continue doing agar work.)

This thread will be under a little construction in the meantime, while I make the transition into the SAB.


I do have one question, should I go on using the baby food jars or just make the jump to petri dishes since I'll be in a SAB now?
I was considering just moving into petris and possibly using the baby food jars for a source of longterm storage..?

Your input is appreciated! :thumbup:


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19230428 - 12/05/13 09:19 AM (10 years, 1 month ago)

Well first let me say thank you for posting your findings honestly. It can be a hard thing to do sometimes, but the community benefits when you do. Second I'm glad to hear that you are going to continue with it and an SAB. It is extremely rewarding. Finally agar is agar, it doesn't matter if you do no pour or pour as long as your needs are met. I personally save my pour work for things like antibiotic agar which needs to be mixed while cooling as it will not survive a PC cycle. I use this for working with wild prints and clones.

To that end do what feels right. If it's easy, fits your budget and lifestyle, do it. I personally do a lot of no pour. The link to how I get it done is in my sig, have a look if you like. Good luck man, agar is awesome :super:


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Invisiblepoopy mcpooperson
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Re: Agar : My Journey [Re: Pastywhyte]
    #19230706 - 12/05/13 10:54 AM (10 years, 1 month ago)

it looks as though you used MS syringe to inoculate those jars. i had problems BIG time with MS syringes on agar. a lot of people do.

less is better for this application. just one drop will do. after that drop is placed try not to let the drop of solution move around on the agar too much.

prints are much better initially for agar but MS syringes can work.

good luck with agar and the SAB.


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Invisibleelasticaltiger
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19230912 - 12/05/13 12:02 PM (10 years, 1 month ago)

Quote:

Midnight Cyclone said:
It turns out pastywhyte was right, and my sterile procedures aren't up to par for agar. I thought that I could cut corners and avoid a SAB, but agar is a different beast.




It is the perfect growing medium for ANYTHING it comes in contact with. Wanna do a fun experiment?  Try putting a booger on a tray and watch all the different colors that grow!

Never thought of using baby food jars.  They look like the perfect little trays and shorter than those pp5 containers that I'm using now.


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Offlinetwistedty
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Re: Agar : My Journey [Re: elasticaltiger]
    #19230922 - 12/05/13 12:04 PM (10 years, 1 month ago)

what kind of GE did you use for your dishes.

also dont give up even if using a syringe you will eventually get some spore syringes to germinate properly

good luck!


Edited by twistedty (12/05/13 12:05 PM)


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: twistedty]
    #19241252 - 12/07/13 02:34 PM (10 years, 1 month ago)

Quote:

Pastywhyte said:
Well first let me say thank you for posting your findings honestly. It can be a hard thing to do sometimes, but the community benefits when you do. Second I'm glad to hear that you are going to continue with it and an SAB. It is extremely rewarding. Finally agar is agar, it doesn't matter if you do no pour or pour as long as your needs are met. I personally save my pour work for things like antibiotic agar which needs to be mixed while cooling as it will not survive a PC cycle. I use this for working with wild prints and clones.

To that end do what feels right. If it's easy, fits your budget and lifestyle, do it. I personally do a lot of no pour. The link to how I get it done is in my sig, have a look if you like. Good luck man, agar is awesome :super:




Happy to be honest, what's pride? :lol:

I would rather be corrected, than incorrect.

I'm considering buying petris and only using them once I think I have a clean dish. I'd use that as a last chance to take a good look at the "clean" culture.. as convenient as the baby food jars are, they're not very friendly as far as getting a really good look at the culture.

Quote:

poopy mcpooperson said:
it looks as though you used MS syringe to inoculate those jars. i had problems BIG time with MS syringes on agar. a lot of people do.

less is better for this application. just one drop will do. after that drop is placed try not to let the drop of solution move around on the agar too much.

prints are much better initially for agar but MS syringes can work.

good luck with agar and the SAB.




If you're referring to the jars in the above post, you're incorrect. I didn't inoculate those at all.

The water that you see in the dishes is from condensation which gradually dripped down to the agar. I can't figure out how to keep the condensation from building up, but that was before I had the SAB so I have an idea of how to correct that now.

"Less is more," that's what I've heard when it comes to agar + MS.


Quote:

elasticaltiger said:
Quote:

Midnight Cyclone said:
It turns out pastywhyte was right, and my sterile procedures aren't up to par for agar. I thought that I could cut corners and avoid a SAB, but agar is a different beast.




It is the perfect growing medium for ANYTHING it comes in contact with. Wanna do a fun experiment?  Try putting a booger on a tray and watch all the different colors that grow!

Never thought of using baby food jars.  They look like the perfect little trays and shorter than those pp5 containers that I'm using now.




I'll be sure to try the booger experiment, sounds fun  :thumbup:

Quote:

twistedty said:
what kind of GE did you use for your dishes.

also dont give up even if using a syringe you will eventually get some spore syringes to germinate properly

good luck!




I don't have any sort of GE at the moment. I was considering drilling a small hole in the lid and stuffing it with polyfill, but have yet to do anything like that.

I like to run experiments and side by sides but in order to be accurate in this community it has to be an isolate, so that is what I'm working towards.

Any idea whether the jars actually need a source of GE?
I was somewhat under the assumption that the air closed inside the jar at time of inoculation would be adequate for the culture's life duration (until I could transfer away).


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InvisiblePastywhyteMDiscord
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19241293 - 12/07/13 02:41 PM (10 years, 1 month ago)

Quote:

Midnight Cyclone said:
Any idea whether the jars actually need a source of GE?
I was somewhat under the assumption that the air closed inside the jar at time of inoculation would be adequate for the culture's life duration (until I could transfer away).




This is for the most part correct, the cultures don't need a lot of GE, however with no pour teks some GE is not a bad thing as the PC cycle can cause a vacuum to build up and either make the jar hard to open or cause the agar to spatter up the sides.

I use little pp5 containers for my no pour work and the vacuum will crush the container with out some sort of GE.


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Pastywhyte]
    #19244572 - 12/08/13 11:31 AM (10 years, 1 month ago)

Okay, so here's a little update on how everything is coming along.

The SAB: (for now, considering making an update or two. If I make any changes I'll be sure to let y'all know)

The size I decided on:
-Walmart had an awful selection of clear tubs like this.. I searched for a good half hour through the mess that was the storage bin aisle but could not find a tub that was the right size for a SAB and also with a clear lid. I settled.


Marking and making holes:
-I decided to offset the holes like the instruction video suggested.
-I decided to use the lid of a mason jar to melt the holes instead of a coffee can, the coffee can is way too big IMO. The mason jar lid is a perfect fit for me.


Cleaned up and final resting place:
-If I had to do it again, I'd probably make the arm holes a little further apart considering I have long arms.
-Oh well, as you can see my arms fit just right in these holes.



My dishes as of 12/8: (All dishes pictured are X-2, the second dish-first transfer e.g. D-2,E-2)

Dish A - I can see bacteria growing but can't get the camera to focus for a picture worth posting. I plan to start a new A-1 dish with a fresh MS inoculation, since I can't even find anything here I can sector.

Dish B - I failed to transfer a clean culture from B-1 to B-2. As you can see, this yellow/orange bacteria has taken over. I even have a new white spot of growth which also supports the idea that I was not being sterile enough before I started working in the SAB. I plan to start a new B-1 dish with a fresh MS inoculation, since there is no spore germination to sector away.



Dish C - Still no activity from the spores placed on agar, don't expect anything at this point. You can see bacterial growth.



Dish D - I either picked up some bacteria during the transfer, or D-1 is hiding this as well. Close examination of the third picture (bottom view) shows this off-white/yellow bacteria pictured is actually growing under the mycelium along the agar.



Dish E - The only dish which I can't seem to identify a contamination, in other words a "clean" dish. Of course, I can't be sure it's clean and in all likelyhood it's actually probably contaminated. But it looks nice so far :smile:




Some things in the GH:

This one has a couple cool things..
-First veil pop of the recent set of trays.
-Big dark pumpkin head on the guy front and right/center.
-A little up-turned gill action in bottom leftish.


A couple of them have turned themselves inside out..
-The first two pictures are yesterday, the third picture is from this morning 12/8.
 

And another patch of noms..



Projects and Possibilities to Come

- Dishes D and E need transferring, first time using the SAB. :awesomenod:
- Dishes A, B and C need to start over from dish 1 (inoculation).
  - I think I'll inoculate Dish C with MS instead of that old print I was using.
- I have a few more panaeolus varieties that I'd like to start as well.
  - I thought this would be faster and require more attention, but most of it is just waiting! Therefore I feel I'll add a few more dishes to the mix, add some excitement!
- If I get bored, I'll probably try the booger (thanks ElasticTiger) experiment. I imagine growing rainbows.
- I'd also like to start some dishes with an LC inoculation from a couple old LC's I have. Who knows what they will show!


I just built the SAB this morning, so I'll likely post later with some updates about what I've done in the SAB!


Edited by Midnight Cyclone (12/08/13 11:40 AM)


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InvisiblePastywhyteMDiscord
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19244585 - 12/08/13 11:36 AM (10 years, 1 month ago)

Good looking SAB :thumbup: Also I like how you made the right hole higher, should make G2G easier. Is the one thing I wish I did when I made mine. Gonna make another one soon and will be sure to do it on that one. Anyway looks like you are on your way now, hope to see some excellent stuff from ya soon.


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Pastywhyte]
    #19250351 - 12/09/13 02:12 PM (10 years, 1 month ago)

Alright everyone so I made a few updates to the OPs; added some pictures and organized them a little more.
The posts seemed out of order and extraneous so I twisted them around and I trimmed them up so hopefully it's less daunting to read!


12/8

In a rush to use the new SAB, I was beating a colonized quart against the tire when I missed and hit the metal bed railing, "SMASH!" "...damnit.."



After cleaning up that mess, I got another quart and managed to shake it loose without any more damage.
I had 6 uninoculated quarts ready for G2G.
I got 3 spawn bags, 6 uninoculated quarts, the colonized quart and went to the SAB.
I consolidated the jars into bags, two quarts per bag.
After, I took the colonized quart and did my best to split a 1/3 to each bag.

It worked out alright: (now time will tell if I stayed sterile my first time in the SAB)





12/9

Getting a start on working in the new SAB:




About midway through: (figuring out a system of order, so I can stay as organized as possible in the small workspace)

- New syringes yet to be used: left
- New dishes yet to be inoculated: top left
- Used syringes: top center
- Dishes to be transferred away from: top right
- Tools: right/center
- Dishes already inoculated: bottom right
- Sharpie/Tape: bottom center




The new dishes: (my first completed in the SAB :awesome:)

A-1 = Hawaii MS (pan cyan) x 2
B-1 = Jamaica MS (pan cyan) x 2
C-1 = Amazon MS (cubensis)
D-3 = Colonized grain transfer (AMZ cubensis)
E-3 = Colonized grain transfer (AMZ cubensis)
F-1 = Thailand MS (pan cambo) x 2
G-1 = Australia RDU MS (pan cyan) x 2




I'm most interested in agar to help with my panaeolus mushroom adventure. That is the reason I created two of each of the A,B,F,G dishes because I want to give those the best chance at working. In a week or two I'll most likely pick the best of each letter and toss the other. I'm just trying to give myself a better chance of successful inoculation since these are proving trouble so far.


I've got a couple questions for those who have experience with agar..

1. When transferring a wedge from dish to dish, do you put the mycelium facing up or down?

2. When working in a SAB, how do you also apply heat sterilization to scalpels and syringes?
  - Is this done before you close the box?
  - Does this have to be done between each cut and transfer?


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19250362 - 12/09/13 02:14 PM (10 years, 1 month ago)

rahahhah, srry i seen your bed and laffed because i knew what happend before i read it.

nice SAB though


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Re: Agar : My Journey [Re: twistedty]
    #19251259 - 12/09/13 05:11 PM (10 years, 1 month ago)

for the heat on the syringe/scalpel, don't do it in the box or before closing the box.  Have the flame sitting close to the box and once it gets red hot, slowly bring it in, fast enough that metal is still hot, slow enough to not disturb the still air.  For super good sterile procedure (i.e. by the books) yea you wanna flame scalpel every transfer.  Im sure there will be a variety of opinions on it.  I flame between each petri, so if I'm taking 5 wedges from one petri, the blade makes 5 cuts, then I slowly pull the one arm out with the blade, get it red hot, then put it back in slowly.

Ive had wedges face up and down, and not noticed anything significant in difference.  for some reason I think RR suggests face up (myc facing air).  I plop it in and where it lands is good for me as long as its in the middle.

that sucks about ur grain jar!  brings back memories because my grain jar cracked being hit against my bike tire that was inside next to my coffee table....literally like ur pic haha


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #24385281 - 06/07/17 01:17 PM (6 years, 7 months ago)

Nice work mate.


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