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OfflineMidnight Cyclone
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Agar : My Journey * 1
    #19163975 - 11/20/13 08:36 AM (10 years, 2 months ago)

This is my running agar journal, and I am a beginner at the time of this posting.
I hope to get good input on how I can improve technique, how to fix anything I'm doing wrong, and of course to help out fellow shroomerites.
Please correct me if I give information that isn't spot-on, misinformation is a contagious plague.


Agar is one of the most intimidating vocabulary words to any new hobbyist.

With this "tutorial" I hope to help anyone who needs it. You'll likely get to see me fail a bunch, and hopefully succeed a time or two. So watch me progress  :awesome:


Procedure:

At this time, I am working in a standard SAB.



I currently use a no-pour tek because I am without a laminar flowhood. I don't have plans to use a pouring technique until I build my own flowhood, although it's not out of the question.

I am working with baby food jars while I use the no-pour tek.

I prepare my agar by heating 500mL water in a pan. Add agar and LME.
The agar will begin to produce flakes in the water.. just continue to heat until there is nothing solid, no chunks.
When there are no chunks, the agar is ready to be poured into your baby food jars.
Remember, you're only pouring enough agar to have a flat surface to work with.
Baby food jars are thrown in the PC for 15-30 minutess @ 15PSI.
After PC, allow to cool completely before working with the agar.


Tip for working with baby food jars:

Before PCing my jars for the first time, I was worried whether or not they could withstand the PC. I was told (but I think the question was misunderstood, I think the person who helped me was referring to mason jars being able to withstand the PC) the lids would be able to handle the pressure inside the cooker, but not 1 of my jars actually stayed closed and all lids were popped open once I opened the PC. I would suggest modifying your lids some way in order to avoid them popping off. The main problem I had happen was all my jars that were on the low shelf in my PC ended up filling up with water and ruined essentially half my jars. Don't let this happen to you!

This is my solution:

     

Also, you will create a vacuum if you twist your cap on while the jars are still hot (after the PC, without any filter). Keep this in mind because when you go to open those new jars they will equalize pressure and suck in a lot of air. This is an easy way to introduce contaminants, so just make sure you only open the jar(s) in a sterile setting.


Media:

I currently work with MEA.
My recipe is 10g agar, 10g LME, 500mL water.




Credit given where credit is due:

memberjockey does a lot of agar work and loves to post pictures!

pastywhyte pointed out a flaw in my sterile procedure, I have made changes to accomodate this.


Edited by Midnight Cyclone (12/08/13 05:32 PM)


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19163976 - 11/20/13 08:36 AM (10 years, 2 months ago)

Standard Transfer Procedure

-You need to be under proper sterile procedure, of course.
-I have forfeited proper sterile procedure here for the sake of this pictorial.

The Setup


The dishes I'll be working with


Here we go

Dish B - The little white dot in the middle is what I made an effort to transfer. It's a pan cyan on regular MEA so it might put up a little fight.


Dish D


Dish E


Final Product



-Remember that any agar work needs to be done in front of a flowhood, or in a SAB.
-This demonstration is lacking proper sterile procedure in order to get a quality pictorial.
-I DO ALL AGAR WORK IN A SAB (as of 12/8).


Edited by Midnight Cyclone (12/08/13 05:35 PM)


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19163978 - 11/20/13 08:36 AM (10 years, 2 months ago)

I've come up with a system using a letter and number so that you can know everything about that jar using the key. The letter is the strain, and the number is the number of times that one has been transferred away. If anyone has any suggestions, please chime in!

My dishes so far - updated 12/8

A - Hawaii MS (pan cyan)
B - Jamaica MS (pan cyan)
C - Amazon MS (cubensis)
D - Amazon live mycelium (cubensis)
E - Amazon live mycelium (cubensis)
F - Thailand MS (pan cambo)
G - Australia RDU MS (pan cyan)

Let the journey begin: (I know that a journal like this can get out of hand, so I'll probably keep a rolling 3 months logged here.)

11/6 - Inoculated 3 dishes. (A, B, and C)

11/14 - Inoculated 2 more dishes. (D and E)

11/18 - Live mycelium dishes have taken off (D and E)
      - Dish B shows growth, more bacteria than anything.

11/20 - Planning to do some transferring here in the next day or two.
      - Dish B looks to have some transferrable mycelium. (if that's mycelium, too hard to tell.)
      - Dishes A and C show no activity
      - Dishes D and E ready to begin sectoring

11/23 - Cut and transferred from dishes B, D and E.
            - I expect these dishes to be "not the cleanest" because of the detailed pictorial.
      - Dish B looked to have some pretty fast growth, isolated the very tip of the best growth. Interested to see what I get out of my first transfers.
      - Dishes A and C still show no activity, about to ditch them considering it's day 17 with pretty much no signs of anything.
        - I'll probably just set them to the side and give them a bit more time. If nothing happens by 12/6 - one month from inoculation - then they will be dumped.

11/24 - Sterility experiment started. Pastywhyte questions my sterile procedure.
        - Experiment concluded 12/5, he was right... I'm not being sterile enough without a proper SAB.

12/7 - Complete overhaul on sterile procedure.
            - Building a proper SAB.
        - There's no use in throwing out the dishes, the nice thing about agar is you can always transfer away, so we don't have to lose the data thus far.

12/9 - New dishes inoculated - first time using the new SAB.
        - Restarted on dishes A, B, and C. Dish C inoculation changed from a print to MS.
        - Transfer from D-2 and E-2 to make D-3 and E-3.
        - Inoculate dishes F and G.


Edited by Midnight Cyclone (12/08/13 05:39 PM)


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OfflineTheApprentice
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19163998 - 11/20/13 08:45 AM (10 years, 2 months ago)

Do you have any idea how excited
I was to see pictures...

and u have none.

:picdidnthappen:


:tongue:


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: TheApprentice]
    #19164006 - 11/20/13 08:52 AM (10 years, 2 months ago)

I only made the thread 15 minutes ago, give me a chance :rolleyes:
:waitingpatiently:


edit: Thanks for being patient with me. :heart:


Edited by Midnight Cyclone (11/23/13 08:40 AM)


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19178059 - 11/23/13 08:40 AM (10 years, 2 months ago)

Woke up this morning hung over as fuck... ($1 top shelf all night) but I've been putting this off for a couple days now and it just needed to get done.

Oh, my head.  :mypoorbrain:


The Setup


The dishes I'll be working with


Here we go

Dish B - The little white dot in the middle is what I made an effort to transfer. It's a pan cyan on regular MEA so it might put up a little fight.


Dish D


Dish E


Final Product


I'm not exactly expecting spotless dishes this time considering the effort I put into the pictorial; but I shouldn't have to do that again so the dishes after these should be cleaner!

Time to wait.  :justcantwait:


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19178067 - 11/23/13 08:43 AM (10 years, 2 months ago)

good work :thumbup:


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19178101 - 11/23/13 09:00 AM (10 years, 2 months ago)

:smbfacepalm:

Dude get an SAB. Its not expensive or hard to make, but it will help. Without it you are just going to go through an endless cycle of contamed transfers. Shit even a cardboard box with armholes and a saran wrap window will give you a better chance than what I see here.

I don't think you quite realize how much contamination there is in the air, or on your body for that matter. A cubic foot of air has somewhere around 500,000 - 1,000,000 contams floating in it at anytime. The number of contams on your belly that I see in your second last pic probably number in the billions. You have open agar right next to it? Could just do your agar on the toilet then.

I don't want to be a dick, but this is terrible. I will be utterly shocked if you a) get a truly clean culture, and b) manage to colonize and fruit any jars inoculated in the same manner :shrug:

Quote:

Midnight Cyclone said:
I'm not exactly expecting spotless dishes this time considering the effort I put into the pictorial; but I shouldn't have to do that again so the dishes after these should be cleaner!




They will not be clean. I would bet a fully colonized monotub on it. Think about it for a second. When we use a torch to flame a scalpel red hot, why do we need it red hot? To kill any contams that might land on it in the one second it takes us to bring it back into the SAB (or in front of the flowhood). Sorry man, it looks like your trying, and I applaud that, but this whole project reeks of fail.

:goodluck:


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Pastywhyte]
    #19178179 - 11/23/13 09:35 AM (10 years, 2 months ago)

Quote:

tripdawg420 said:
good work :thumbup:




:awesome:

Quote:

Pastywhyte said:
:smbfacepalm:

Dude get an SAB. Its not expensive or hard to make, but it will help. Without it you are just going to go through an endless cycle of contamed transfers. Shit even a cardboard box with armholes and a saran wrap window will give you a better chance than what I see here.

I don't think you quite realize how much contamination there is in the air, or on your body for that matter. A cubic foot of air has somewhere around 500,000 - 1,000,000 contams floating in it at anytime. The number of contams on your belly that I see in your second last pic probably number in the billions. You have open agar right next to it? Could just do your agar on the toilet then.

I don't want to be a dick, but this is terrible. I will be utterly shocked if you a) get a truly clean culture, and b) manage to colonize and fruit any jars inoculated in the same manner :shrug:

Quote:

Midnight Cyclone said:
I'm not exactly expecting spotless dishes this time considering the effort I put into the pictorial; but I shouldn't have to do that again so the dishes after these should be cleaner!




They will not be clean. I would bet a fully colonized monotub on it. Think about it for a second. When we use a torch to flame a scalpel red hot, why do we need it red hot? To kill any contams that might land on it in the one second it takes us to bring it back into the SAB (or in front of the flowhood). Sorry man, it looks like your trying, and I applaud that, but this whole project reeks of fail.

:goodluck:




Thank you for your harsh criticism, I need things like this.

I guess I should explain my "sterile" technique, and you can tell me whether this is satisfactory or not. I'm extremely new to this area of mycology so I am a blank canvas, looking to learn.

1. I am always freshly showered.
2. I am always naked when I do my work, fresh out of the shower I am as clean as the human gets pretty much. - I knew I would accidently get some body in these pictures so I threw on some boxers.
3. I wear a surgical mask covering mouth and nose so that I'm not breathing contaminants on my work.
4. On top of the mask, any time an agar jar is open I hold my breath for the duration.
5. The area I'm working in (my 2'x2' closet) is completely set up meaning all tools are in place ready for use, the closet is then sanitized with a metric shit-ton of Lysol, then I shower, and when I get back in the closet, and after I seal the door behind me, I spray Lysol again up over my head and all around - choking on it if I wasn't wearing a mask.

Only at this time do I begin any work. By the way, I just flat out don't have extra space for a SAB. I don't have room for what I already have, nonetheless an extra empty box just laying around! :lol:

You're shocked that I can colonize a healthy jar under these conditions? I've been under these conditions from day 1 and nothing has changed.. I guess it's not that bad. :shrug:

When I say "I'm not exactly expecting.." that means I know they're not going to be clean. Usually I am a lot quicker, lid off a jar for less than a second, but I was taking pictures. I'm sacrificing clean cultures this go-round to help people who learn best from a pictorial instead of just words of how-to.

Quote:

Sorry man, it looks like your trying, and I applaud that, but this whole project reeks of fail.




Harsh, but thanks.

Hopefully I can prove you wrong, I love doing that.  :douchewink:  :hehehe:


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InvisiblePastywhyteMDiscord
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19178233 - 11/23/13 09:51 AM (10 years, 2 months ago)

You may have done G2G in open air and gotten away with it (still is a horrible practice), but agar is a different beast. Maybe your house is just that much cleaner than mine, but one time I opened a plate in open air for a second, and a couple days later had all kinds of nasty shit growing inside. Fact is that agar is a super hospitable environment for all kinds of microorganisms. Stuff will germinate on agar, that might never have a shot on brf or grains.

You may have showered etc, but all that means is that you cut the number of contams on your body down to a billion rather than 4 billion. It helps if you think of yourself as a contam.

I really don't want to come off as harsh but what we often forget is that for every person posting on these boards, there are 1000's of noobs lurking around, reading your posts. If you do have success with this that's great, but I hate to think of all the lurkers who will read this, think its a good idea, and take a trip on the fail boat.

Here's what you should do for a control of your setup. Take a fresh jar, open it up for 5 seconds, then close it and see what happens. If nothing grows in it, then its one of three things, either there are no nutes in it (doubtful), its too dry for anything to germinate (also doubtful), or you sir are the cleanest man who ever lived and your house is also unbelievably clean.

If you do get contams (which I suspect you will) you can be certain that this will never work, and start thinking of ways to get enough space for an SAB.

Again sorry about being a dick but we gotta think of the children! I mean noobs and lurkers :lol:

Edit: you do have room for an SAB. Put it on the stool where you do your work. Even a smaller size SAB is better than none at all :shrug:


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Pastywhyte]
    #19179503 - 11/23/13 04:10 PM (10 years, 2 months ago)

Quote:

I really don't want to come off as harsh but what we often forget is that for every person posting on these boards, there are 1000's of noobs lurking around, reading your posts. If you do have success with this that's great, but I hate to think of all the lurkers who will read this, think its a good idea, and take a trip on the fail boat.




I never let this leave my mind, that's why the third sentence in this thread relates directly to that.

Quote:

Here's what you should do for a control of your setup. Take a fresh jar, open it up for 5 seconds, then close it and see what happens. If nothing grows in it, then its one of three things, either there are no nutes in it (doubtful), its too dry for anything to germinate (also doubtful), or you sir are the cleanest man who ever lived and your house is also unbelievably clean.




I'll start this experiment in the morning.

I'll run normal sterile practices and do just this, with all the jars I have remaining that have not been inoculated. (I won't need any more jars for at least a week so I'll be fine, not to mention I have other baby food jars that are empty and ready to be poured.) I think I have 6 or so remaining, but I will add this experiment to this thread in order to gain a conclusion based on evidence instead of opinion.

Quote:

Edit: you do have room for an SAB. Put it on the stool where you do your work. Even a smaller size SAB is better than none at all




The closet doubles as a microphone booth when not used for mycology, so the stool is usually stacked with recording equipment.


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Invisibleblindingleaf
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19179643 - 11/23/13 04:49 PM (10 years, 2 months ago)

haha u work naked? thats funny.  My roommate thinks its weird when I'm doing agar stuff in my SAB when he comes home, but if I was naked??? haha i might just have to try that with a hidden camera to post his reaction.  Naked with arms in a laundry tote holding a petri dish and a flaming hot scalpel hahaha


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Re: Agar : My Journey [Re: blindingleaf]
    #19179704 - 11/23/13 05:05 PM (10 years, 2 months ago)

Glad your gonna run the test I suggested. I hope all goes well. I personally love agar so much that it pains me to suggest anything that might turn a person off of it. I got one more suggestion for you. Try to look around for a collapsible cardboard box that could have a saran wrap window cut in along with armholes. Should not be too hard to find on. Then when you don't need it, just fold it up and stash it away.

Regardless, you seem to have a good attitude and no matter your methods, I hope you can make it work, agar is such an excellent tool to have.

:kenthumbup:


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Pastywhyte]
    #19180416 - 11/23/13 08:28 PM (10 years, 2 months ago)

Quote:

blindingleaf said:
haha u work naked? thats funny.  My roommate thinks its weird when I'm doing agar stuff in my SAB when he comes home, but if I was naked??? haha i might just have to try that with a hidden camera to post his reaction.  Naked with arms in a laundry tote holding a petri dish and a flaming hot scalpel hahaha




Only if you manage a simultaneous black hole. :lmafo:

Quote:

Pastywhyte said:
Regardless, you seem to have a good attitude and no matter your methods, I hope you can make it work, agar is such an excellent tool to have.

:kenthumbup:




Thanks bud.

I'll be sure to let you know how the un-inoculated jar experiment goes!


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19182977 - 11/24/13 02:50 PM (10 years, 2 months ago)

Updated OP (3rd post) with the transfer pictorial.

Also, I added the experiment that Pastywhyte suggested to see whether my workspace is clean enough for agar work.

:mushroom2:  Happy Shrooming  :mushroom2:


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Offlinekrikkrew
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Re: Agar : My Journey [Re: Pastywhyte]
    #19182991 - 11/24/13 02:52 PM (10 years, 2 months ago)

Quote:

Pastywhyte said:


Regardless, you seem to have a good attitude and no matter your methods, I hope you can make it work, agar is such an excellent tool to have.

:kenthumbup:




I concur, he's a helpful dude.
Good luck with agar. That'll be a project for me soon.


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OfflineMidnight Cyclone
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Re: Agar : My Journey [Re: krikkrew]
    #19183052 - 11/24/13 03:04 PM (10 years, 2 months ago)

Quote:

Good luck with agar. That'll be a project for me soon.




Thanks bud, you guys will get to see every step along the way.

Just make sure to stay tuned and this thread should explode with good discussion/information before too long.

:awesomenod:


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Re: Agar : My Journey [Re: Midnight Cyclone]
    #19183094 - 11/24/13 03:13 PM (10 years, 2 months ago)

Nothing to add, just wanna watch.


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Re: Agar : My Journey [Re: magickspore]
    #19183206 - 11/24/13 03:36 PM (10 years, 2 months ago)

Soon as I get a PC I want to start agar. Shall be following. :crazy2:


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Re: Agar : My Journey [Re: d0urd3n]
    #19183279 - 11/24/13 03:52 PM (10 years, 2 months ago)

:popcorn:


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