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InvisibleStipe-n CapMDiscord
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Why Agar? * 18
    #28188696 - 02/15/23 01:21 PM (11 months, 6 days ago)

Why do we employ agar for mushroom cultivation?





At first glance it may appear perfectly reasonable to grow mushrooms straight from spore; plants grow from seeds planted in soil, after all, so why not reduce the complexity of mushroom cultivation by doing essentially the same with spores? Let’s look at what agar is first before we move on to the why and how.

Robert Koch initially utilized potato slices to culture bacteria during his investigations into the germ theory of disease, Koch postulated that:

1. The microorganism must be abundant in all organisms suffering from the disease but should not be found in healthy organisms.

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

To prove these postulates, Koch would require media to both grow and then isolate these organisms for identification. Gelatin was widely available but failed to produce a growth medium that could reliably culture bacteria due to the protein structure of gelatin. Bacteria would often utilize those proteins as an energy source, gelatin failed to remain stiffened at higher incubation temperatures and would often melt when bacteria produced protease enzymes that break down proteins.

Agar, on the other hand, is made up of carbohydrates extracted from seaweed. These carbohydrates have a much higher melting temperature than animal proteins which bacteria do not readily catabolize for energy. Koch would eventually graduate from potato slices to agar-stiffened liquid media poured into jars; Julius Richard Petri would further develop Koch’s culture dishes into the Petri dishes we recognize today.

How is this germane?

When streaking microbes for identification there may be a wide variety of bacterial species present including yeasts and molds; sound familiar?

To I.D. the species of microorganisms responsible for disease, there must be a method for isolating a pure culture, the same is true for the cultivation of mushrooms; this is achieved by streaking to agar.

Agar-filled Petri dishes provide a sterile environment to germinate and grow all present microorganisms allowimg you to isolate a pure culture of your target organism. Mushroom fruit bodies grown in an open environment like tubs or tents will be exposed to environmental contaminants like molds and bacteria.

The removal of caps for printing, swabbing, etc, risks further contamination via handling. Spores are to be considered septic/contaminated by default.



When spore syringes, spore prints and swabs are viewed from this perspective it becomes obvious why agar is necessary: to produce a pure mushroom culture free of contaminant organisms. Failing to produce a pure culture prior to inoculation will result in a very high risk of failure.

Pouring /wrapping blank plates





Streaking

There are three common methods for streaking:

1. Quadrant method
2. T method
3. Cell spreader

The quadrant method of streaking produces 4 zones of growth. The first contains the initial inoculum at full concentration, each subsequent quadrant aims to pull material from the previous by thinning the cell concentration utilizing a zig-zag pattern while either replacing your disposable loop or flaming in-between:





This process thins the concentration of cells allowing for separation of growth, separation facilitates isolation. Without proper separation the cultivator risks growing mushroom mycelium on top of bacteria, yeasts, and molds, making it difficult to isolate a pure culture free from contamination.

T method is essentially the same except with 3 zones of growth as opposed to 4:



If using spore prints, just add a drop or two of sterile water to the edge of your plate to facilitate streaking/spreading of dry spore material:




Cell spreaders are used to evenly distribute cells across the surface of the agar, cell spreading is the easiest of the three and produces excellent results:



Add a single drop of spore solution to the center of the plate, then gently smear the solution evenly across the entire surface of the agar with the L shaped spreader.

The result:




Notice the amount of growth produced from a single drop of spore solution? This is a great example of spores' septic nature and the sheer number of both target species spores and bacteria. Each milky dot in these pictures are bacterial colonies, had I not used the cell spreader all of the bacteria and mycelium would have grown together, infecting the culture. Should I have chosen grains or any other substrate other than agar, well, I think you get the point. Bacterial contaminations may persist even after multiple transfers. Isolating clean mycelium early on will prevent any downstream frustration.

When first starting out I advise you resist the urge to buy or use liquid culture for the following reasons. Don't use LC until you have all of the lead up techniques including the ability to produce reliably clean spawn nailed down. Stay as far from LC as possible until then, otherwise you'll be chasing variables and failures forever.

Each skill builds upon the next.

1. Spores to agar to produce culture; cleaning to produce pure culture, making transfers, and agar to grain.

This step will familiarize you with the fungal life cycle and introduce you to the basics of identification for both contamination and healthy mycelium. During this phase you'll learn how to reliably produce clean cultures and sterilize both grains and media.

Once these skills are mastered, move to skillset #2

2. Grain to grain expansion.

This step expands upon the previous production phase by introducing how to expand your pure culture exponentially. This is a more advanced stage dealing with inoculation techniques and aseptic techniques which will prepare you for the next step:

3. Liquid inoculant/liquid culture.

Liquids will not work for you if you haven't learned to identify pure culture, contamination, or prepare sterile media which was learned way back in step one. Liquids won't work for you if you don't understand/master the aseptic techniques required for expansion.

Walk before you run, otherwise there's too many variables to troubleshoot. Each phase carries it's own set of criteria for troubleshooting problems, if you skip them, you're going to be fucked.



Making Agar







Agar is quite simple when dealing with non-selective media. Standard 2% gelling and nutrient strength are all that's required to produce healthy fungal colonies. Amending with peptones, yeasts, etc, is not necessary but if that's your thing, to each their own; for most of us this will be all you'll ever need:

for a sleeve of 25 @ 2% concentration:

600ml distilled h20
12g agar
12g Dry malt extract

for a sleeve of 20 @ 2% concentration:

400ml distilled h20
8g agar
8g Dry malt extract


A note regarding yeast extract and peptone:

Quote:

Yeast extract and peptone provide nitrogen compounds, vitamin B complex and other nutrients for growth. Yeast extract agar allows separate counting of aerobic mesophilic organisms that form visible colonies in this medium after 24 hours incubation at 37°C and those that form colonies after three days incubation at 22°C. The two methods give different results.




https://www.biotrend.com/en/buy/cat-yeast-extract-agar-non-selective-5494.html

Peptones are specifically used for culturing bacteria, particularly bacteria associated with fermentation, or food spoilage. When used for culturing fungi, it's used to isolate and diagnose pathogenic and non-pathogenic fungi. Peptones were originally made from anamal products, beef in particular. Peptones are partially digested proteins.

Protein = Nitrogen.

When considering gourmet/active culturing techniques, ask yourself whether a nutrient dense media is beneficial. Agar is not only a growth medium, it's an inoculum. When used as growth medium we utilize for isolation from contamination (cleaning), spore germination, pure culture expansion and storage. When germinating spores, spores are often comingled with aggressive contaminant organisms; a nutrient dense, nitrogen rich medium may not be beneficial.

When utilized as inoculum there may be an argument made for rapid expansion, this would likely be the case with large volume liquid culture broth production. Use of peptones with agar, on the other hand, seems like a waste when considering its wide application for germination, cleaning, culturing, and storage. Nitrogen rich media would be counterproductive when used for cleaning or germination due to the probable presence of aggressive contaminant organisms that would benefit from the excess nitrogen.

Malt extract provides ample nutritional resources, further ammendments are not necessary:






https://consteril.com/steam-sterilization-cycles-part-2-liquids/

Adjust sterilization time to the volume of water. I always cycle for 45 minutes, vent the pressure cooker for 15. The longer cycle will ensure that solids have completely dissolved. Most people are running their cycles for 15-20 mins, this is a mistake. Less is not more with regards to sterility assurance.  The standard 15- 20 minute cycle falls well below the minimum threshold for the standard volume people are using for agar and LC. Most people are mixing 500-1000ml when prepping agar.

There's no requirement whatsoever to fill the PC/autoclave with water. Place your agar vessel(s) on an elevated trivet, fill water for the cycle up to the trivet:



Allow the pressure cooker guage to reach zero before removing, any attempt to evacuate steam/hasten cool down will result in boil over.

Before and after pc cycle:



No boil over; no caramelization; no maillard effect; no crashing; no sediments; no problems.
Your light, dry malt extract will produce varying levels of clarity depending upon the Lovibond rating of your malt:



Clarity does not affect growth, simply aesthetically pleasing. 2 Lovibond will produce the greatest clarity, the same is true for distilled h20: a greater concentration of dissolved solids = more yellowing of the end product.

These are the products I use:




Wrap plates with Parafilm or cling wrap.





The following plates were poured using the above 2% agar and dry malt extract powder recipe listed above.




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InvisibleShrimps
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Re: Why Agar? [Re: Stipe-n Cap] * 3
    #28188697 - 02/15/23 01:22 PM (11 months, 6 days ago)

:leocheers:

Great writeup p9!


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Invisibledowodenum
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Re: Why Agar? [Re: Shrimps] * 1
    #28188721 - 02/15/23 01:31 PM (11 months, 6 days ago)

Posting in a future oft-referenced thread.

:rockon:


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Offlinehazyhorse
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Re: Why Agar? [Re: dowodenum] * 2
    #28188724 - 02/15/23 01:34 PM (11 months, 6 days ago)

hell yeah stipe. excellent resource to point people to when explaining agar. thanks for all the banger write ups


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InvisibleLotKid
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Re: Why Agar? [Re: hazyhorse] * 1
    #28188856 - 02/15/23 03:10 PM (11 months, 6 days ago)

props for taking this kind of time and effort.


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InvisibleBaba Yaga
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Re: Why Agar? [Re: LotKid] * 3
    #28188888 - 02/15/23 03:31 PM (11 months, 5 days ago)

Always good to keep things tight and fresh. Sometimes it feels contradictory when telling someone they are using old information and then referring to a 6 year old post explaining the basics although these are still valid posts.

This will be the agar thread I'm referring to from now on.


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InvisibleStipe-n CapMDiscord
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Re: Why Agar? [Re: LotKid]
    #28188895 - 02/15/23 03:34 PM (11 months, 5 days ago)

Quote:

LotKid said:
props for taking this kind of time and effort.




Keeps me busy.


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Offlinejohnukguy
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Re: Why Agar? [Re: Stipe-n Cap] * 1
    #28289904 - 04/22/23 01:58 PM (9 months, 2 days ago)

:takingnotes:


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OfflineSikjakass
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Re: Why Agar? [Re: Stipe-n Cap]
    #28534947 - 11/08/23 10:52 PM (2 months, 18 days ago)

Commenting so I can find again lol


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OfflineAdvil_Liquigels
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Re: Why Agar? [Re: Sikjakass] * 1
    #28534956 - 11/08/23 11:12 PM (2 months, 18 days ago)

Thank you so much for this.

"Don't shoot spores into grain" should be the number one biggest advice to new people. I wasn't aware that spore syringes had contaminants in them, I thought they could be manufactured to be "clean" but now I know and that's honestly been the biggest thing steering me toward agar work.


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Invisiblemeta_mmxxii

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Re: Why Agar? [Re: Advil_Liquigels]
    #28534982 - 11/08/23 11:49 PM (2 months, 18 days ago)

Quote:

Advil_Liquigels said:
Thank you so much for this.

"Don't shoot spores into grain" should be the number one biggest advice to new people. I wasn't aware that spore syringes had contaminants in them, I thought they could be manufactured to be "clean" but now I know and that's honestly been the biggest thing steering me toward agar work.



Solid advice. :thumbup:


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InvisibleStipe-n CapMDiscord
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Re: Why Agar? [Re: meta_mmxxii]
    #28535160 - 11/09/23 06:49 AM (2 months, 18 days ago)

Clean syringes can be made, however, they're the exception and not the rule. Many headaches can be avoided by assuming all spores are dirty, doing so will save you in the long run. Even with proven clean spore solution, agar is still the weapon of choice for the reasons mentioned in the OP.


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OfflineSupaThaRipper
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Re: Why Agar? [Re: Stipe-n Cap] * 1
    #28535178 - 11/09/23 07:11 AM (2 months, 18 days ago)

Really appreciate this thread Stipe. I’ve been filling my PC up with water to the level of the agar to prevent boil over. Same thing with LCs. It’s really nice to know it’s unnecessary. Do you have a write up going in depth, like this one, about transferring and isolating. I’ve read about dropping nutrients with each transfer but that seems to be unnecessary. Not really sure and would like clarification.

Also, “caramelization” is something that’s definitely heavily circulated. Realizing such a thing isn’t something to worry about. That’s why people are doing 20 minute cycles


Edited by SupaThaRipper (11/09/23 07:15 AM)


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OfflinePnin
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Re: Why Agar? [Re: SupaThaRipper]
    #28535182 - 11/09/23 07:17 AM (2 months, 18 days ago)

This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look.
https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1


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InvisibleStipe-n CapMDiscord
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Re: Why Agar? [Re: SupaThaRipper]
    #28535186 - 11/09/23 07:19 AM (2 months, 18 days ago)

There's plenty of bad or incomplete info in circulation, I do my best to present reliable information; I'm glad you've found it helpful. I don't have any posts that cover culturing, unfortunately.


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OfflineSupaThaRipper
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Re: Why Agar? [Re: Pnin]
    #28535189 - 11/09/23 07:22 AM (2 months, 18 days ago)

Quote:

Pnin said:
This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look.
https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1




Thanks pnin! I’m going to check this out. Pretty old but hopefully reliable lol


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Offlinetryptkaloids
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Re: Why Agar? [Re: SupaThaRipper] * 1
    #28535386 - 11/09/23 09:55 AM (2 months, 18 days ago)

That link is 100% reliable and the one I link to peeps.


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Offlinec01h
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Re: Why Agar? [Re: Stipe-n Cap]
    #28583695 - 12/15/23 09:42 AM (1 month, 13 days ago)

:threadmonitor:

Stipe at it again :bowdown:


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InvisibleStipe-n CapMDiscord
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Re: Why Agar? [Re: Pnin]
    #28583740 - 12/15/23 10:10 AM (1 month, 13 days ago)

Quote:

Pnin said:
This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look.
https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1




I think Stro meant well but this must be the source material for some of the ideas circulation about sectoring, strain isolation, and the use of the term "monoculture" to refer to a single strain isolate. None of that information is correct.

Single strains cannot be isolated on agar from clone or multistore uaing a scalpel, "sectors" are not individual strains, "monoculture" isn't a synonym for "isolate" in the sense that it'sa "single strain isolate".

A monoculture is a single propagated species, all plates cultures free of contaminants are a monoculture (pure culture), which should not be mistaken for a monosporic, or single strain isolate.

Quote:

As you can see the cultures are looking healthy and the strains ar showing their individual characteristics in A and B and as suspected C is a true monoculture.

As a learning tool, I will go over one or two things here. When I first posted the photo that were taken seven days after transfer someone was asking me a few questions and I said;

Quote:

Stropharis said:
C is now true monoculture, yes. As for A I believe there are two strains but it is hard to tell





Outdated and definitely incorrect.


Quote:

SupaThaRipper said:
Quote:

Pnin said:
This one is ten years old, but it is the one I return to for pics and to read. Might be worth a look.
https://www.shroomery.org/forums/showflat.php/Number/18430998/fpart/all/vc/1




Thanks pnin! I’m going to check this out. Pretty old but hopefully reliable lol




Not entirely.


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OfflineSupaThaRipper
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Re: Why Agar? [Re: Stipe-n Cap] * 1
    #28583827 - 12/15/23 11:00 AM (1 month, 13 days ago)

Yeah it’s circulated, that’s for sure


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