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Arathu.....first agar (Sophomore Project)


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#1 Arathu

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Posted 08 August 2012 - 02:59 PM

K..Sophomore year.....The skill sets are coming together and it's time to get serious as hell. I'm a firm believer in self sufficiency and to me that means finding in the wild, returning to the lab, and cultivating eventually returning to the "not-so-wild-wilds" I'm working on the meat and potatoes of the opener to this thread in MS Word first and then I will post it here, so network time-out and such mayhem don't have me breaking glass with flying computer parts.

As the thread title says.......Arathu...first agar! Experiments are under way and I would like to solicit the attention and hopefully tutoring of agar experienced professors, grad students, seniors, and my fellows in this next level class. Building upon previous knowledge and experience, here we shall have a go from wild spores onto agar and see where it leads................. :teeth:

So I leave you with another tease of what is to come......many details to follow. Get your notebooks and supplies going, oh yeah, blowout party at Delta-Kappa-FOAF Friday night!

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Seemed a prudent way to incubate for now.
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Results of first agar ever made and first streaking ever performed by Team FOAF member X :teeth:

These are from prints, the last from wild prints, Team FOAF will attempt to maintain the same streaking pattern until death do us part...seems like a good idea.

Many details of how this was done to follow but if you go over to the archives and vaults you will find that Hipp3 and BB provided the root functions which were then digested and applied to the current experiments hopefully using experience and logic. We shall see............... :meditate: Into the darkness!

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Edited by Sidestreet, 04 September 2016 - 03:41 PM.
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#2 Il19z8rn4li1

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Posted 08 August 2012 - 03:02 PM

FIRST POST BOOYAA


Wisdom is the best teacher ;)


Nice jumping on the agar train.

Love how such "lab savy" things are becoming more and more hobbyist related rather
then actual "professional science study jive" hehe

Though simple and elegant, agar can set the stage for elite genetics/colonies


Im not to far behind you my friend hehe

Edited by Il19z8rn4li1, 08 August 2012 - 03:07 PM.


#3 wildedibles

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Posted 08 August 2012 - 04:43 PM

I need to start on this train too If I start soon it will be ready when I am ready for the next part :) Ty for the reminder to find my Agar :)

#4 psilosylum

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Posted 08 August 2012 - 08:41 PM

I'm a big fan of looking into agar...but still being in the incubation stage of my first and only grow I should slow my thoughts down :D I might start a LC from other syringes that I have for my next project - WBS :D

Looking forward to seeing your results on agar. What will the Junior and Senior projects be? :)

#5 cheetolay

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Posted 08 August 2012 - 08:58 PM

i learned from a good man, that you dont have to use pitri dishes. Tapered jars or even snapple bottles can work well with agar.

#6 psycheholic

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Posted 08 August 2012 - 09:26 PM

Well I am pulling up a chair and sit in class with you. I have been wanting to get into agar work, but time is of the essence and at a premium for me at this point in my life. Glad you started this thread, Arathu. Got Team FOAF!! LOL

#7 Arathu

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Posted 08 August 2012 - 10:23 PM

Welcome fellows...lets learn together............. :bow:

#8 ConsciousFeeder

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Posted 08 August 2012 - 11:23 PM

Arathu your just growing up so fast.... *pinches cheeks*

Hehe :hippie:

#9 Arathu

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Posted 09 August 2012 - 12:06 AM

The first installment will be the choice of the agar and the recipe's


1Why agar?

The primary purpose behind the decision to begin working with agar at this time is to allow for “cleaning up” spore prints obtained from various sources including those made from mushroom specimens gathered in the wild by attempting to germinate those spores and then isolate clean mycelium from contaminates. This is of course not the only reason one might wish to use this medium for culturing mycelium but you have to start somewhere. In the future the skills gained here will be applied to other tasks such as the cloning of tissue removed from fruiting bodies of mushrooms which possess specific desirable traits, refinement of strains of mycelium, selective breeding, and so forth.

Reflecting on that it seemed that in so doing little more would be accomplished than a clean multi-spore inoculation, which in itself is an accomplishment but somehow seemed a bit shallow, and so it was also decided that the trait of vigorous rhizomorphic growth would also be selectively targeted as well making the goals of this initial work:

Goals:

  • To germinate spores of targeted mushroom species, grow mycelium from those spores, and isolate the mycelium from contaminates as best as possible
  • To refine the isolated mycelium to a strain possessing vigorous rhizomorphic growth characteristic

This of course was not decided randomly but based upon reading, examples of others work, and suggestions from many sources including observations of the results of mycelium which possess this particular characteristic, specifically at the fruiting stage of its lifecycle. But, since nothing is guaranteed to wind up with desirable end results it sure can’t hurt to attempt to condition the outcome as best as possible. Simply stated we are looking for a mycelium that will vigorously produce fruiting bodies; i.e. mushrooms.

1.1Which agar?

After considerable research and study of the various kinds of agar mixtures used to grow mycelium it was decided to try and keep things as absolutely simple as possible. (Note: remembering and building on lessons learned from past experience and applying the KISS principal) So the idea is to apply previous knowledge to the new experiment while not just simply following someone else’s recipe blindly. The agar would be mixed to accomplish its mission as stated in the goals. The goals are fairly simple so we need a food source for the mycelium and a medium in which to suspend those nutrients.

Considering that many people recommend a brown rice flower PF TEK for entry level mushroom growing and having followed that advice successfully it was decided that brown rice flower would likely be an excellent nutrient component for this first agar mix.
Many recipes and techniques were read through and considered and finally the absolute bear minimum of components seemed to make the most sense for this first attempt.

Two species of mushrooms are being grown in this initial project. One of them is generally a dung loving species and the other is a wood loving species. Precedence and past experience has shown that both of these will germinate and grow on grains to develop and expand spawn, which can then be used to spawn to the targeted fruiting substrates.


1.1.1 BRFA (Brown Rice Flower Agar)

A very simple nutrient agar recipe was modified accordingly and the following recipe derived. Note this nutrient agar will be used to grow and develop the dung loving species.

40g powdered organic brown rice flour
9.5g powdered agar
500ml distilled water


1.1.2 HWBRFA (Hardwood Brown Rice Flower Agar)

The same recipe as above will serve as the medium for the wood loving species as well but slightly modified. The wood chips that will serve as the fruiting substrate were sampled and a tea created by simmering 2 cups of sun dried wood chips in 750 ml of distilled water for 90 minutes which resulted in a dark wood smelling tea. This was strained using a small funnel and coffee filters and returned to the pot for a slight reducing to the required 500ml.

40g powdered organic brown rice flower
9.5g powdered agar
500ml Hardwood tea

Next the preparation method and then pouring lava in a flow hood while remaining sterile (this will definitely be refined but I will record what was done here)
note:experience teaches like no other thing
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#10 Arathu

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Posted 11 August 2012 - 09:18 AM

Good morning Class!

First question: What the hell is growing on my agar? How will I know what I'm looking at?

My answer right now: Some of it I expect I won't but some characteristics I expect to see. From doing previous work with PF jars and spending large amounts of time looking and examining healthy mycelium and some really strange contaminants and not so healthy mycelium......I have a fairly good idea of what I want to see. (NOTE: I don't know all that much about molds, fungi, and bacterial growths, YET, but I do know enough to keep them in their jars as best as possible)

So what am I looking for?
 

  • We know the mycelium that we want (in this case) is going to be bright white in color.
  • We know that the mycelium we want (in this case) becomes rhizomorphic in its growth.
  • We deliberately use a known container and further a known pattern (streaking) of inoculation in order to further our chances of germinating, identifying, and then cultivating a specific species.

For my technique I will try and use a specific streaking pattern always,
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So I'm just training myself to use this shape with my inoculation loops and that is my first indication that what I'm trying to culture is actually something I'm targeting. In the photos we can see that in fact there is growth that has begun within the streaking pattern I have chosen.....this MIGHT BE what I'm after....... I think in this game it is best to reduce the guessing to absolute minimum.



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So what are these? It's white, it looks like mycelium, maybe we can save it, or see what it turns out to be? My answer......HELL NO......I won't, it's failing my first test and I'm going to be picky as hell.



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Strong bleach solution flooding and soak....I don't care what was on those plates and don't have the facility or the time to try and figure it out either....bleach solution strength 1/4 cup to a gallon of water. This way I will immediately rule out anything not growing in my known streaking pattern (note this is not Arathu running around naked, that is outside of the scope of this thread, see above for definition of this Arathu's streaking pattern) :teeth:



So we have some candidate growth that looks likes the snaking pattern made by my inoculation loop. Sweet! Funny they are all from the locals......hmmmmmm......I like symbiosis!

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This one serves as a decent example although I'm sure as I practice more I'll get better and better at this with hopefully higher germination rates. But I can clearly see my pattern in the growth.....fucking bonus!

Now what? OK from the snaking pattern there is obviously what appears to be white mycelium growing....now I from past observation I know that I want rhizomorphic growth.

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And here, although not anything like the killer photography others have provided of it, at the edges of this growth is what definitely appears to be sectioned rhizomorphic growth of mycelium. I have found the places where I will take my first wedges for transfer of mycelium to new plates for continuing the expansion of the cultures.

So what about all that other stuff.....it's just wasted Arathu, all that time and those precious spores! NOPE......it's called learning and EXPERIMENTS, amateur science, REALLY amateur. Some of the stuff I see folks doing is kinda scary actually, but I'm opinionated and I digress. IMHO that is what experimentation is all about and those other cultures, they will be done again, and again, and again until this kind of results are obtained. Besides there are literally millions of spores, even on a light print, crunch a bunch, they'll make more!

Still, we have early results that are at least conforming to a set of boundaries that should be very strict IMO (I think this follows along with and supports attaining goal number 1, germinate and isolate)
Next I will grab some of that rhizomorphic growth, what I think is the most vigorous and transfer it to another set of plates.

SIDE NOTE: I'm going to cook up a batch of PDA per HPP3's recipe and pour that into some flat jelly jars and PP5 jars for additional work with a recommended starting medium (see I'm always trying to do shit like run before crawling BAD IDEA)

My prep method for the above, minus pouring by syringe, came from [Buckaroo Bonzai]'s great write up

Much more to follow as we GROW!

Class dismissed!

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Edited by Sidestreet, 04 September 2016 - 03:39 PM.

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#11 psycheholic

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Posted 11 August 2012 - 10:45 AM

Excellent thread my friend. I really enjoy the way you conduct them. Looks like I will have to spread some rep around, but you definitely deserve it. Very informative and detail oriented and organized extremely well. Do we have to use a laminar flow hood for agar work?

#12 Cindysid

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Posted 11 August 2012 - 02:27 PM

I am a newbie and I have been wanting to learn about agar. I have a stupid question. Is the agar in your recipe agar agar, or is it a mix? If it isn't, then I have another question, also stupid...Do you not have to have a sugar source in agar? I thought you had to put in dextrose or some sort of sugar. Is the BRF taking it's place? I want to learn to make agar and I'm looking for the simplest and most effective recipe. I will be watching this thread. Thanks!

#13 Myc

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Posted 11 August 2012 - 02:36 PM

Arathu,
Flip your plates over - up-side, down and the condensation will go away.
Another way to say it - incubate them while they rest on the lids. Looks to me like you have good mycelium there and I'm personally curious if you don't mind trying.

Ooopsie,
I just noticed you're not using Parafilm. I love the stuff.

I've heard of masking tape, glad press-n-seal, glad clingwrap, and similar materials being used in a jam.

#14 Myc

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Posted 11 August 2012 - 02:46 PM

I am a newbie and I have been wanting to learn about agar. Is the agar in your recipe agar agar, or is it a mix?

"Agar agar". The food-grade variety available at Oriental markets and such.

Do you not have to have a sugar source in agar? I thought you had to put in dextrose or some sort of sugar. Is the BRF taking it's place?

Yes, the Brown Rice Flour doesn't need additional sugar. Same with Malt Extract recipes.
Very cool stuff here.

#15 Arathu

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Posted 11 August 2012 - 02:56 PM

I am a newbie and I have been wanting to learn about agar. I have a stupid question. Is the agar in your recipe agar agar, or is it a mix? If it isn't, then I have another question, also stupid...Do you not have to have a sugar source in agar? I thought you had to put in dextrose or some sort of sugar. Is the BRF taking it's place? I want to learn to make agar and I'm looking for the simplest and most effective recipe. I will be watching this thread. Thanks!


First....as the thread says......I'm noob 2......1st agar work...... nuff said there. :teeth: Second, stupid questions don't exist unless you ask the same one over and over and over....... nuff said there too.......:teeth:

The agar I am using is labeled 100% pure Agar Powder from NOW Healthy Foods, because the local health food store carries it. Since this was the first time I ever made the stuff I figured a very simple recipe was in order. So, looking at brown rice nutrition information and the fact that it is used as the sole food source in a basic PF TEK the answer is yes, it is the only nutritional source in the mix, it has it's own carbohydrates and sugars. I seem to remember in my readings of the various threads and articles that "less was better" in agar. That is the reasoning behind this mix, and I didn't have a potato on hand. :teeth:

Now I do.

Immediately following I am preparing new "plates" which will actually be small jelly jars, with PDA (Potato Dextrose Agar) from Hippy3 for continued work and comparison. I can recommend anything to you yet and I am seeing growth on my mix but the recommendations from the local gods is definitely PDA and BB recommends purchasing a premixed batch. That is up to you of course. I'm hard headed as hell and learn best by failing a few times, personally.


Happy reading and agar explorations......looking forward to seeing some mycelium............... :teeth:

Edited by Sidestreet, 04 September 2016 - 03:40 PM.


#16 Arathu

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Posted 11 August 2012 - 02:59 PM

Arathu,
Flip your plates over - up-side, down and the condensation will go away.
Another way to say it - incubate them while they rest on the lids. Looks to me like you have good mycelium there and I'm personally curious if you don't mind trying.

Ooopsie,
I just noticed you're not using Parafilm. I love the stuff.

I've heard of masking tape, glad press-n-seal, glad clingwrap, and similar materials being used in a jam.


Thanks Myc.....I've noticed the blunder there and will seal them on the next go........Like I said......I have to learn by screwing things up first.......... but the Ovoid mycelium has me all silly. Transfer to new plates coming shortly or do you think I should wait just a little longer?

Flip your plates over - up-side, down and the condensation will go away.
Another way to say it - incubate them while they rest on the lids. Looks to me like you have good mycelium there and I'm personally curious if you don't mind trying.


I have had them in a vented PP5 storage container in the incubator upside down as suggested and some of the condensation has gone away but as you can see, not all of them. I really need to seal the plates too. Surely I've invaded and contaminated my work...........blundering my way to higher learning............... :teeth:

I'm loving this stuff............... :teeth:

Edited by Arathu, 11 August 2012 - 03:08 PM.


#17 Arathu

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Posted 12 August 2012 - 12:29 PM

Hi all, a little more rhizomorphic development here.

Questions for the experienced: Any particular optimum timing for the choice of mycelium to transfer? I'm sure there must be and that experience plays a big role in understanding each on an individual basis. So I took a virtual stab (pun intended) with MS PAINT before I go doing it with a scalpel, of mycelium that looks like it wants to be transferred to its own plate. I post it up for some critique, if anyone will volunteer up some grading and advice (what specifically to target, how much material to transfer, when, and etc)

Please feel free to grab some of these pictures and E-Chop wedges with paint and etc.....if you would be so kind.............. humming along we are............:meditate:


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LK with some nice looking growth going on..what would you transfer, if anything at all, to the next plate(s) and why?



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It's clear that this mycelium development is as a result of my streaking the agar with an inoculation loop. :thumbup:
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Which have been created from SSS dental picks (note: after the photographs and some additional reading the loops were fully closed)


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Possibles? Wait longer and see if anything stands out as the over achiever? Decisions decisions! :eusa_thin

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This looks to me like a good candidate of vigorous rhizomorphic growth, would one transfer a wedge like in the photograph or just out at that leading edge of growth?

POV2_selection_001.jpg
I'm surprised I don't see a bunch of contaminates here, of course that doesn't mean I'm not just blatantly ignorant as hell but this looks like healthy mycelium to me.

What do you all think? Get ready to transfer? Wait a few days and exercise that patience thing again? Quit and get into crochet?

Myc, I'm incubating these on the lids.....I don't understand why the condensation hasn't gone...... perhaps the agar already saturated with water???????? It's not runny or loose so......IDK. The plates that I transfer these too will be taped. I figured it was better to leave well enough alone, and mostly hands off, at this point and get it when I do the transfers especially since they seem fairly clean already. I must of done a decent job taking the prints. Of course this says very little about bacteria, or does it?

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#18 Myc

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Posted 12 August 2012 - 05:17 PM

The condensation has dramatically reduced due to your efforts. Photos are clear and easy to analyze.
Nice inclusion of the smaller ziploc baggie - an new one for me and a great improvisation.

When you use tape, look at Buckaroo Banzai's thread again - where he gets to the Parafilm application. The film is applied to the entire circumference of the dish. You seem to use tape to hold the lid on. Parafilm seals the entire seam between the lid and the plate and acts as a breathable membrane which allows gas exchange but excludes particles.
Membranes which do not breathe - plastics like Glad products, ziploc bags - have a finite air supply and are not desirable for long-term use. The mycelium will exhaust its oxygen supply - CO2 will build up - competitors will emerge (trust me, they're on your "sterile" plate).
Posted Image
For more clarification, the above plate has been wrapped with Parafilm.

Now for your transfers.
Select growth from the leading edge of the areas you've chosen - good choices
You'll want to transfer the tiniest amount possible from the leading tip of each of those advancing "fingers" - Located at the radial edges of the "wedges" you've drawn for consideration. Grab a tiny spec of the agar just in front of the advancing mycelium (where tissue is near invisible).
Label and monitor these.
From these subcultures, you can fruit and develop isolates.

#19 DirtyViperMan

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Posted 12 August 2012 - 05:52 PM

If you are trying to isolate a sub strain it is best to take the smallest possible transfer. The larger the wedge the more likely you will be doing another transfer in a weeks time, not to mention you want to keep the mycelium as close to its genetic start as you can. I generally do 50 to 75 petri dishes per pour around 2,250ml of MYPA always take ncopies of each transfer. Good lick and have fun

#20 Arathu

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Posted 13 August 2012 - 01:35 PM

First transfers done and incubating.....taped everything I touched with micro-pore tape. I also created some PDA in jars two days ago and did some transfers to them as well. We'll see what works and what doesn't.

So both are being tested on the BRFA, HWBRFA, and PDA.

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Little freaking tiny wedge of mycelium from the leading edge of rhizomorphic growth. I tried to repeat this with each transfer as best as possible.

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All work done in the HEPA airstream with a flamed and then cooled (in the target agar) scalpel.

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All plates that were touched were taped with micro-pore tape and then sealed into new ziplocks with air trapped inside.

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Incubation time.........:meditate:

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