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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
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Agar : My Journey 1
#19163975 - 11/20/13 08:36 AM (10 years, 2 months ago) |
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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 
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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Midnight Cyclone
StrangerDanger
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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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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
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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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TheApprentice
back at it



Registered: 09/25/11
Posts: 4,727
Last seen: 2 years, 1 month
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Do you have any idea how excited I was to see pictures...
and u have none.

--------------------
RR Videos -Best $9 Ever Spent * No Pour AGAR Tek * Easy COIR Trays! * Pink Oysters on Newspaper TEK "Yeah? Well, DRACULA called... and he said he's coming over tonight, and I said OK!"
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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I only made the thread 15 minutes ago, give me a chance 

edit: Thanks for being patient with me.
Edited by Midnight Cyclone (11/23/13 08:40 AM)
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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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.
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.
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tripdawg420
low life with no life



Registered: 02/02/09
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good work
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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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 
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.
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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Quote:
tripdawg420 said: good work 

Quote:
Pastywhyte said:

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 
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.

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! 
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. 
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.
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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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 
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
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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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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blindingleaf
blue collar underworld



Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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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
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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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.
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
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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. 
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.

Thanks bud.
I'll be sure to let you know how the un-inoculated jar experiment goes!
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Midnight Cyclone
StrangerDanger
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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.
Happy Shrooming
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krikkrew
Manifestation



Registered: 05/25/09
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Last seen: 10 years, 2 months
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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.

I concur, he's a helpful dude. Good luck with agar. That'll be a project for me soon.
-------------------- Welcome to Nirvana
Have a seat, we have much to discuss.
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Midnight Cyclone
StrangerDanger
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Re: Agar : My Journey [Re: krikkrew]
#19183052 - 11/24/13 03:04 PM (10 years, 2 months ago) |
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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.
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magickspore



Registered: 12/11/12
Posts: 798
Loc: Center of the universe.
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Nothing to add, just wanna watch.
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d0urd3n
Just call me "D"


Registered: 09/15/10
Posts: 5,237
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Soon as I get a PC I want to start agar. Shall be following.
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bootster

Registered: 02/22/11
Posts: 1,531
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Re: Agar : My Journey [Re: d0urd3n]
#19183279 - 11/24/13 03:52 PM (10 years, 2 months ago) |
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
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Re: Agar : My Journey [Re: bootster]
#19193024 - 11/26/13 04:58 PM (10 years, 2 months ago) |
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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 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 

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? 
Edited by Midnight Cyclone (11/26/13 05:41 PM)
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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
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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!
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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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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
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poopy mcpooperson
Pogonophobia



Registered: 10/17/13
Posts: 124
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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.
-------------------- love and laughter
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elasticaltiger
Like Tigers in Coitus




Registered: 06/24/13
Posts: 8,059
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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.
-------------------- First time growing cakes? DON'T make a Shotgun Fruiting Chamber The Shmuvbox. - The Old TC's Like it Afraid to Start Growing From Your Own Prints? Drop it Like a Tiger! No Pouring. No Syringes. No Cutting. No flaming. No Contamination. No Bullshit. "The best thing to do while your waiting is to start more stuff. I usually got so much happening that I have tossed projects simply because I didn't have time for them. -Pastywhite QFT Pastywhite's Easy Agar Tek (PastyPlates) Tiger Drop Video Demos By munchauzen Van Gogh would’ve sold more than one painting if he’d put tigers in them.―Bill Watterson EZEKIEL 23:20
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twistedty
Forcefully Retired



Registered: 07/01/12
Posts: 5,487
Loc: Middle
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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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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
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Re: Agar : My Journey [Re: twistedty]
#19241252 - 12/07/13 02:34 PM (10 years, 1 month ago) |
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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 
Happy to be honest, what's pride?
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 
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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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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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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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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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 

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.  - 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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Pastywhyte
Say hello to my little friend



Registered: 09/15/12
Posts: 37,810
Loc: Canada
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Good looking SAB 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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Midnight Cyclone
StrangerDanger
Registered: 05/29/13
Posts: 399
Loc: oo ess aye oo ess aye
Last seen: 10 years, 11 days
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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 )
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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twistedty
Forcefully Retired



Registered: 07/01/12
Posts: 5,487
Loc: Middle
Last seen: 3 years, 6 months
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rahahhah, srry i seen your bed and laffed because i knew what happend before i read it.
nice SAB though
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blindingleaf
blue collar underworld



Registered: 07/19/13
Posts: 22,008
Loc: sub-surface unseen
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Re: Agar : My Journey [Re: twistedty]
#19251259 - 12/09/13 05:11 PM (10 years, 1 month ago) |
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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
-------------------- A few thoughts on cultivation MICROBIAL HUSBANDRY!!!! The whole is greater than the sum of its parts
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Trustybadmash
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Registered: 02/24/17
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Nice work mate.
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