beluga goes fungal: P. cubensis edition

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beluga

beluga

Oh, hi, folks!

First and foremost - I should have a general disclaimer attached to all of my posts - they might get verbose!
And this one is not going to be immune to that at all. I will, however, try to pepper in enough eye candy to break up the tedium.

I do these big posts on a desktop, so I also apologize for any formatting that may not be mobile-friendly.

I'm no master guru, but I know how to get it done with some amount of success and reproducibility and I feel like I'm pretty keen to the theory. I hope this can lend something to the community because you all have so much more than I could offer in regard to our usual subject and I've benefited from that greatly. Thank you.

I know the nature of these forums, so don't hesitate to ask questions, post your grows, etc. This shit's free-for-all. But do know that @1diesel1 has already made a general mushroom discussion thread
Here. And there's already great stuff and a healthy following there.
This will be my somewhat-personal sister thread to that.
I will head my personal informational posts with:


🍄🐳🍄🐳🍄🐳🍄🐳

Cheers!
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Introduction


So, I've recently been lured back into a passion adjacent to cannabis cultivation... mushroom cultivation!
With (arguably) fewer supplies and negligible energy and space requirements compared to cannabis cultivation, it's a very rewarding and simple hobby to get into.

That said,
I'm not going to show you what the community at large would consider the most basic introductory method - the PF Tek - because I believe it to neglect certain fundamental principles and practices to really be successful and prolific in your fungicultural endeavor.
However, I would be a total asshat to not mention
Ziran's Updated PF Tek.
He has outlined and shared an excellent bridging of the gap between the original PF Tek and those aforementioned fundamentals.
If you want to grow mushrooms fast, do that. If you want to learn to cultivate mushrooms, well...

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I hope to make this a guide to get you from point A to point A∞.
From your initial procurement of spores to the innumerable collection of subsequent spores.
Continuity. Perpetuity. The ouroboros. The three hares.
From one compatible set of spores you can have a lifetime of mushrooms.

I also believe in some fundamental principles of education.
Like nomenclature and vocabulary.
Theory before practice.
I want you to have a brief understanding of what it is you're working with before you work with it.
I don't expect people to use technical labels over common ones, but I do believe they are good to know as to assuredly identify what is being described.


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Glossary of Terms

*You may find some words missing that you think are relevant.
A. I'm not perfec. B. I may have intentionally left them out as to not introduce words that are outside of this method.

A
Agar - an extract from a seaweed used to solidify media​
Annulus - the ringlike veil remnant on a mushroom stipe​
Axenic - free from other microorganisms; uncontaminated​
B
Bacteria- unicellular microorganisms that may cause contamination in culture work​
Basidiomycetes- a group of fungi which produce their spores externally on so-called basidia​
Basidium (plural of basidia) - A cell that gives rise to a basidiospore​
Biological Efficiency (BE) - A term for yield potential. 100% biological efficiency occurs when 1 lb of fresh mushrooms is harvested from 1 lb of dry substrate, over multiple flushes​
Brown Rice Flour (BRF) - ground brown rice​
Bruising - a bluing reaction of the mycelium from stress​
C
Cap - see pileus​
Casing - a fruiting method where a colonized substrate is covered with a non-nutritious layer​
Clone - a population of individuals all derived asexually from the same single parent​
Coir - the prepared husk of a coconut fruit, used as a substrate material​
Colonization - the stage of growth after inoculation during which the mycelium grows through a spawn substrate​
Contamination - undesired foreign material in a substrate, often other infectious microbes​
E
Expansion - the stage of growth after colonization during which the mycelium grows through a bulk substrate​
F
Field Capacity - content of water in a soil after being saturated and free drainage is negligible​
Flame/Torch - a method of sterilization in which a flame or torch is used to bring a metal tool to incandescence​
Fresh Air Exchange (FAE) - the amount of air circulation it takes to fruit a given species​
Fructification/Fruiting - the state of growth where the mycelium of a colonized substrate produces fruiting bodies, or mushrooms​
Fruiting Body - a mushroom; the part of the mushroom that grows above ground​
Fruiting Chamber - an enclosed space used to artificially create an environment that simulates fruiting conditions; vivarium​
Flush - the phasic even development of multiple fruiting bodies​
G
Germination - the spreading of hyphae from a spore​
Gills - see lamellae​
Grain-to-grain transfer (G2G) - The inoculation of grain with already-colonized grain​
H
Harvest - the gathering of crops ready for consumption or processing​
Hypha - a branching filamentous structure of a fungus, multiple hyphae are collectively called mycelium​
I
Incubation - the period of time after inoculation in which colonization occurs​
Inoculation - the process of adding spores or mycelium culture to a substrate​
ISO - 70% isopropyl alcohol; a sanitizing agent​
Isolate - an uncombined fungal strain​
L
Lamellae - the tiny segments on the underside of the pileus where spores are produced​
Light Malt Extract (LME) - a powder of malted barley typically used in brewing beer​
Liquid culture (LC) - A culture of mycelium suspended in a nutritious liquid, for use as an inoculant​
Liquid Inoculant (LI) - a suspension of mycelium in water. This is different from a LC in that the mycelium did not grow in the medium​
M
Malt Extract Agar (MEA) - a nutritious culturing medium that combines water, agar, and malt extract​
Millet - a small cereal grass grain used as a spawn substrate​
Monotub - a clear container used as fruiting chamber​
Multispore (MS) - refers to an inoculation where multiple germinations and matings occur due to the use of various spores, as in a spore solution (e.g. spore syringe) and as opposed to an isolate​
Multispore syringe (MSS) - see spore syringe​
Mycelium- the portion of the mushroom that grows underground​
Mycorrhiza - a symbiotic association between a plant root and fungal hyphae​
P
Partial Veil - a membranous covering reaching from the stipe to the pileus of a mushroom to protect spores developing in the lamellae​
Petri dish - a round glass or plastic dish with a cover to observe the growth of microscopic organisms​
Pileus - the horizontal portion of a mushroom; cap​
Pin - a term used to describe a very young mushroom​
Polyfill- a polyester fiber used for gas exchange and filtering microorganisms​
Preservation - the process of preparing food or other perishables to resist spoilage or decomposition​
Pressure Cooker (PC) - a pot with a tight-fitting lid that uses steam and high pressure to cook and preserve foods, used for sterilization​
Primordium - the initial fruiting body, sometimes referred to as a ‘hyphal knot’​
R
Relative Humidity (RH) - the amount of water vapor in the air​
Rhizomorphous (rhizo) - used to describe a rootlike characteristic of mycelium growth​
RTV Silicone - Room Temperature Vulcanizing silicone. Used as a high-temperature gasket and adhesive​
Rye - a cereal grass grain used as a spawn substrate​
S
Shoebox - a small monotub​
Spawn - a culture of mycelium on a substrate​
Spore - the reproductive unit of a fungus​
Spore Print - a collection ofspores taken from a mushroom cap, often collected on sterile card stock, aluminum foil, or some other flat surface​
Spore Syringe - a collection of spores suspended in water in a syringe​
Sporulation - when a mature mushroom releases its reproductive spores​
Stem - see: stipe​
Sterile Technique - a method of careful manipulations that prevents foreign cells from entering cultures or media​
Sterilization - the procedure of destroying all living organisms in or on a given​
Still Air Box (SAB) - a mostly-sealed transparent container designed to allow one to manipulate objects in an environment without air currents​
Stipe - the stem or stalk-like feature supporting the pileus of a mushroom​
Strain - the result of compatible spores mating; there are innumerable potential strains in a single mushroom's sporulation​
Substrate (sub) - anything used to grow mushrooms on​
Surface Conditions - the moisture content and rate of evaporation on the top of a substrate or casing​
Synthetic Filter Disc (SFD) - a woven cellulose or PTFE material used for gas exchange and filtering microorganisms​
T
Tek - shorthand for technique​
Tissue culture (TC) - a sample of tissue used to culture a genetic duplicate; clone​
Tomentose - used to describe a fluffy characteristic of mycelium growth​
Transfer (xfer) - used to describe taking a small piece of a culture from one culturing media to another​
U
Universal Veil - temporary membranous tissue that envelopes immature fruiting bodies, often leaving remnants on the pileus​
V
Variety - a distinct group of organisms with a set of unique and limited genetic traits; race (obsolete)​
Veil - see partial or universal veil​
Vermiculite - a highly absorbent substrate material made from puffed mica​
W
WBS - Wild bird seed. Millet-based grains used as a spawn substrate​

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Basic Taxonomy

This is a basidiomycetous Mushroom

basid.png

So are P. cubensis!

This is their basic life cycle

cycle.png

Neat!

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The Cultivation Process

To cultivate is to promote or improve the growth of a culture by labor and attention.
The basic needs of mushroom growth are air, water, and nutrition.

Inoculation

This is the process of germinating spores or transferring mycelium to a sterile and highly-nutritious spawn substrate.
This spawn substrate is most typically a whole or ground cereal grain such as rye, oat, millet, wheat, or rice.
Colonization
After inoculating, the germinated spores or the transferred mycelium start the vegetative process of growing on the spawn substrate known as colonization.
We sterilize this substrate before inoculating to minimize as much as possible, the opportunity for competing microorganisms, or contamination.
Expansion
Once the spawn substrate is 100% colonized, it is then known simply as spawn. We then expand this spawn to a mildly-nutritious bulk substrate in a process known as spawning.
Properly prepared bulk substrates provide a medium for the mycelium to thrive in which helps maintain adequate hydration (water) and respiration (air). Bulk substrates prepared properly are said to be at field capacity.
Fructification
Or, simply, fruiting is the stage of growth after the mycelium has expanded as far as it can go and is now developing fruiting bodies, or mushrooms. Indoors, this is carried out in a fruiting chamber that simulates natural fruiting conditions - hydration, respiration, and evaporation of the top layer of the colonized bulk substrate, collectively known as surface conditions. Fruiting starts with mycelium joining together in hyphal knots (primordium). Those hyphal knots then go on to develop the first recognizable fruiting bodies, or pins.
Harvest
Once the mushrooms have developed fully, they are preparing themselves for the end of their reproductive cycle - sporulation. Before they open up to spread their spores, their gills are protected by a membranous layer called a veil. Harvest typically happens at or near the time of the veil just starting to tear, before the mushrooms have dropped their spores from their gills.
The other crucial aspect of harvesting is collecting those spores so that we may perpetuate our cultivation. This is done by taking a cap that has not yet dropped its spores to a controlled environment and setting it on a piece of foil or paper to take a spore print.
Preservation
After the mushrooms have been harvested, they must either be used fresh within their short shelf-life or be thoroughly dried and stored in order to preserve them for later use.
The main factors in spoiling of dried fruits are moisture and sunlight, so it is best to store them in an air-tight and opaque container.

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This Technique

The technique that I'll be using will be, essentially, making clean spawn to expand to a properly hydrated inert bulk substrate in a self-maintaining environment, or monotub.
It's a common one. People have variations on this theme. It is not the end-all-be-all of mushroom cultivation by any means. I did not create it nor do I take credit for any of its processes. I'm just sharing it with you.

Cell Culturing on Agar
We'll start with bringing spores - either a (multi)spore print or a (multi)spore syringe (MSS) - to a sterilized nutritious agar media on petri dishes or no-pour plates.
Through a series of transfers, we'll learn how to take our desired culture away from any contaminating culture to be left with an axenic culture.
Colonizing Grain Spawn
Once we obtain an axenic culture on agar, we'll next learn to properly hydrate and sterilize grain spawn as to provide optimal conditions for colonization.
Expanding to Bulk Substrates
Once the grain spawn is 100% colonized, we will then learn how to hydrate coir to field capacity. This can be tricky, so I will also suggest the use of vermiculite to help us obtain that field capacity if we happen to oversaturate the coir. After we have our hydrated bulk substrate at field capacity, we will then spawn our 100% colonized spawn to the buk substrate at a ratio ranging from 1:2 - 1:4. For example, 1 quart of spawn to 2 quarts of hydrated bulk substrate.
Maintaining Surface Conditions
Once spawned and mixed with our bulk substrate, we will then simply need to insure that our envrionment is optimal for growing out our mycelium and not encouraging contamination.
This is achieved via adequate fresh air exchange, relative humidity, and temperature. P. cubensis is a very tolerant species and can grow in many ranges. So there is wiggle room, but some extremes can promote contamination.
Harvest, Spore Collection, and Cloning
Once the mushrooms have developed fully, we'll then harvest them (!!!). But we want to make sure we can keep on growing, so we will learn to collect spores with minimal contamination and also learn to clone selected specimens to get the best mushrooms with the most dense canopies.
Preservation and Recipes
After the mushrooms have been harvested, then we eat them!!! But you'll have too many to eat them all within normal parameters of maintaining your sanity. So, we'll go through proper drying, making teas, ice cube cubes, etc. as to store and them keep them from spoiling. Also some added information on MAOI's and things to help make your experience a smoother ride.

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Supplies

I'll do my best to make a supply list relevant to each section, but I'll also preemptively add everything I can think of here for reference.
Along with these lists, I'll do my best to give you places to get them. I don't know international suppliers at all, so, please bear with me and maybe spark up conversation with your fellow countrymen.
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Supply Key

Follow these symbols to see generally where you could find things.
I would mention online supply/Amazon, but that's kind of a given and, for me at least, generally where I get all my stuff except heavy items like 50lb bags of grain.

Farm & Garden supply - 🚜
Laboratory supply - 🧪
Medical supply - 💊
Hardware supply - 🛠
Kitchen & Grocery supply - 🍳
Big Box/General supply - 🛍
Brewery supply - 🍺
Pet supply - 🐶

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Agar and Culturing Supplies

Still Air Box - 🛍🛠 Pressure Cooker - 🛍 🍳 Petri Dishes / Bulk - 🧪 Media Bottle - 🧪🍳🛍 Agar Agar - 🧪🍳🛍 Malt Extract - 🧪🍺 Torch - 🛠🚜🧪 Scalpel / Blades - 🧪💊 Nitrile Gloves - 🧪🛍🛠💊 70% Isopropyl - 🧪🛍🍳💊 Cling Wrap - 🍳🛍 Inoculation Loop - 🧪 Synthetic Swabs - 🧪🛍🍳💊

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Spawn
Quart Jars - 🍳🛍🛠 PP5 Lids - 🛍🍳 Synthetic Filter Discs (SFDs) - 🧪
Polyfil - 🛍 RTV Silicone - 🛠 Rye -🚜🐶🍳🛍 Millet - 🚜🐶🛍 Wild Bird Seed (WBS) - 🐶🚜🛍

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Bulk Substrate/Habitat
Monotub / Shoebox - 🛍🛠 Coconut Coir - 🐶🚜 Spray Mister - 🛍🐶🚜 • Vermiculite - 🚜


If you aren't able to find something here - let me know! There can be substitues or different places to procure these things.
For instance, if you can't find SFD's, you can use Polyfil instead. If you have trouble finding Polyfil, well, most pillows with synthetic stuffing are made with it!
There are, however, some things I find absolutely necessary - like a pressure cooker. Most any model will work in some way or another.

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Alright folks! I think that's about it for my introduction. I sure hope so... 😅

Now, if you want to join along - find some spores! And get everything I have listed under Agar and Culturing Supplies.
I'll give a bit of lagtime to prepare myself and so others who want to follow can prepare themselves. But don't feel like you need to keep up pace... do it at your leisure for your own satisfaction.
Anyone ahead of me, feel free to ask questions, but, again, remember there is The Mushroom Thread. I keep tabs on that and love to advise any way I can there, too.
 
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beluga

beluga

🍄🐳🍄🐳🍄🐳🍄🐳

Alright, I'm gonna get into another couple bits of crucial information before we go into the process.
I know. I'm not keeping up with my eye candy promise. And that's why I inserted the 'try' caveat - immediate dismissal of commitment.
I don't make promises. I do stuff or I don't and the result is what it is. Imagine how my wife feels. 😉

Oh, it's been brought to my attention that some of the links may be broken in the original post.
I'll eventually poke at a mod to see if I can't edit that for continuity.
Sorry 'bout that. Feel free to inquire about anything... it'll help make this a more solid thread for everyone.


In this segment, we're going to discuss....

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Sterile Technique, Pressure Cooker Use, and SAB Use.


All of these touch on the same fundamental: sterility and avoidance of contamination.
It's important to always have the idea in mind of how the microbial world works and how the laws of physics can help us to manipulate that in our favor.

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Sterile Technique

Present in the air, on every surface, and on you are innumerable amounts of living microorganisms.
When working with a cell culture such as fungal mycelium, it is necessary to ensure that culture is axenic - free from other microorganisms.
We call any organism aside from our intended culture a contamination - bacteria, yeast, other fungi, insects, and even debris can all be contaminants.
To ensure that a culture is axenic, we use what is referred to as Sterile Technique.

Sterile Technique is used to avoid contamination of sterile media and equipment during cell culturing.
In this practice, our sterile technique will be executed primarily by means of heat, filtering, and environmental control.
We will subject our media to very high temperatures in a pressurized atmosphere.
We will flame sterilize our equipment by using flame to bring them to incandescence.
We will filter contaminating microbes out of the air that contacts our media while our cultures are growing on them.

Sanitization vs. Sterilization
Very simply put: Sanitization removes some microbial life from something. Sterilization removes all microbial life from something.

Chemicals such as isopropyl alcohol and chlorine bleach are effective for cleaning surfaces and reducing the amount of microbes on your work surfaces or your self.
It should be assumed and expected that something sanitized will contaminate your sterile media.
YOU DO NOT NEED HYDROGEN PEROXIDE IN THIS PRACTICE

If something has not been effectively treated with high temperatures, it has not been effectively sterilized.

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Using a Pressure Cooker

First and foremost:
DO NOT OPERATE UNTIL YOU HAVE READ INSTRUCTIONS
This is printed on the gauge of almost every make and model of pressure cooker that exists, but it is the most ignored aspect of cultivating. I cannot stress enough,
DO NOT OPERATE UNTIL YOU HAVE READ INSTRUCTIONS.

The basics of using a pressure cooker:
1. - DO NOT OPERATE UNTIL YOU HAVE READ INSTRUCTIONS.
2. - Pressure cookers should be cleaned, maintained, and each component should be inspected before and after operating.
3. - Fill your pressure cooker with the amount of water specified by the manufacturer for canning.
4. - Load your contents
5. - Bring water to a boil
6. - Secure your lid with the regulator/weight/rocker OFF
7. - Vent for 10 minutes
8. - Put the regulator/weight/rocker in place
9. - Bring to desired PSI
10. - Cook at desired PSI for desired duration

But don't just take my word for it,

Here are the canning instructions from the Presto 23 Quart Pressure Canner and Cooker
presto23-lrg.png

This is NOT an exemption from READING THE INSTRUCTION MANUAL in full.

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Using a Still Air Box

The function of an SAB is to create an environment in which the air is still and will not carry microbes on currents and into your sterile media or equipment.

Here's a Still Air Box
(and some weirdo doing some weird shit in it.)
sab-stuff.jpg


Let's dissect this picture a little bit.
- It's a clear tote... clearly, a pretty large one.​
- There are a couple access holes... big enough in circumference and wide enough apart for free movement of that guy's arms... small enough to not invite room air currents.​
- That weird guy's got some cool hats.​
- To the right (dominant hand) there is a lit propane torch laying down (adhered with duct tape to the table.) - He'll use this to flame his knife.​
- To the left, a spray bottle - I bet it's 70% isopropyl alcohol.​
- There are working surfaces in the middle of the table - his hands and arms aren't passing above them.​
- He holds his knife at the end of the handle - to avoid touching some of the shaft that may pass over sterile media​
- Just from a couple still frames, you can make out the delicate and deliberate movements that he makes. His arms never touch the sidewalls of the access holes. His hands never pass above a working surface. They have to, of course, pass over the top of a petri dish from time to time, but it is lidded and the fuction of petri dishes is such that this is okay. Microbes will not crawl under, through, or over an inert surface without external physical manipulation.​
So, that was a pretty good rundown to give you an idea of SAB use.
We move deliberately and delicately - as if moving through water and trying not to splash or cause a wake.
We never want to abruptly bump our table, our SAB, or anything else. This can cause any microbes on the ceiling of the SAB to fall right into our work. It can also cause the air to be disturbed or kicked-up... generating turbulent currents that can spread microbes. We also don't want anything that touches sterile media to touch the floor - the floor is lava! (Actually, it's just teeming with microbes.)
What you can't see from this picture is to have everything planned out before you start your work. This takes practice. All of this takes practice. But, being prepared and having everything easily accessible keeps you from making accidents.


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Short but crucial lesson.
I'll go over more specifics later that are pertinent to each stage of the process.
Remember... microbes are endemic to everything. There are even ones that survive those sterilizing temperatures - extremo/thermophiles. These don't tend to be an issue for us (as far as I know) because they don't really thrive in normal conditions (as far as I understand).
The point is... think of everything - every surface, your body, the air - as teeming and crawling with life. And do your best to keep that life away from the one life that you're trying to grow.
 
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beluga

beluga

🍄🐳🍄🐳🍄🐳🍄🐳


Alright, folks! Now to the exciting parts where you actually get to do shit.

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The Still Air Box (SAB)

stillairbox.jpg

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Supplies:

• Still Air Box - 🛍🛠​
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Seeing as I already have a SAB, I'm not going to show you the process because once you have one, you have it forever unless you do some dumb shit and break it burn it or something. In all my terrible drunk "clean work" and moving houses and storing my mycology supplies in it, I have never damaged mine.

It's very simple and you can do it however your ingenuity leads you to do it.

Just take your large clear tote without its lid, flip it upside-down, and you're halfway there!
Now, cut two ~6" (15cm) holes in it, roughly shoulder-width apart, roughly in the middle.. but a little closer to the bottom.
Just think about how you would most comfortably be able to move with your arms relaxed at your shoulders.

Some ways you could do it:
- A sharp blade​
- A soldering iron (wear a mask in a ventilated area)​
- (Very Clever) A large metal coffee can placed on an element burner (wear oven mitts and a mask in a ventilated area)​
- A pin in the center of the hole with a string attached to it and another pointy object attached at the other end of the string (like a compass)​
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Making & Pouring Agar in Petri Dishes
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Supplies:
Pressure Cooker - 🛍🍳
Petri Dishes / Bulk -🧪
Media Bottle - 🧪🍳🛍
Agar Agar - 🧪🍳🛍
Malt Extract - 🧪🍺
Nitrile Gloves - 🧪🛍🛠💊
70% Isopropyl - 🧪🛍🍳💊
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Agar or MEA (Malt Extract Agar) is the base ingredient for cell culturing. It gives your cultures a place to germinate (spores) and replicate (mycelium).
You don't have to use LME (Light Malt Extract) but it is the standard ingredient. It also withstands overcooking without caramelizing.
Agar agar can be found in a couple different forms - powder and a kind of stick form. They both work fine, but you will need to powder the sticks if those are what are available to you.
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A Basic MEA Agar Recipe

Ingredients

• 500mL tap cold Water (H2O)​
• 10g Agar powder​
• 10g Light Malt Extract (LME)​

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Instructions

• Add manufacturer's specified amount of water to Pressure Cooker and start to bring to a boil​
• Add LME to media bottle​
lme.jpg
• Add tap cold water to media bottle and dissolve LME​
• Add Agar powder and shake to incorporate (you won't get it dissolved)​
agaw.jpg
• Place media bottle in heating Pressure Cooker​
mix-heat.jpg
• Once boiling, wait five minutes, and then vigorously swirl your media bottle to mix Agar in​
mix-swirl.jpg
• Put foil over media bottle lid; loosen slightly; return to PC​
mix-boil.jpg
• PC for 30 minutes at 15-18PSI​
psi.jpg
timeryes.jpg
• Remove PC from heat and allow to come to atmospheric pressure naturally​
• Now, we have one of two options:​
1. Let it cool and solidify for later use​
2. Pour some fucking dishes!!!​

Since I don't have a microwave and it takes a long time to re-liquefy them otherwise, let's pour!​
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Pouring Agar
We need to bring the agar to 117ºF to pour it properly.​
This gives it a good consistency for pouring, makes it easier to handle, and also helps to prevent condensation buildup in your petri dishes.​
The two common ways of doing this are to either let it cool naturally or put it in a hot water bath (cool, in this scenario).​
Agar starts to solidify at ~107ºF, so you also need to pour it before it reaches that temperature.​
A note
- 500mL of media will fill 20 100mm x 15mm petri dishes to the appropriate level.​
- You'll want to learn to pour stacks of 10 - they're manageable and pouring like this also helps minimize condensation on all but the top plates. It also helps reduce clutter and the chance of passing anything over your sterile media area.​
Nervous? Don't be! You got this!​
Instructions
• Prepare a hot water bath by adding ~120º water to a pot large enough to cover the MEA in your media bottle.​
The media bottle will spike the temperature, so keep a thermometer in there to monitor when it comes to 120ºF​
water-bath.jpg
• Prepare you SAB while you wait:​
- Get your isopropyl handy​
- Give a cursory wipe-down of your SAB​
- Put on a pair of gloves​
- Spray down your hands​
- Put your dishes in there in stacks of 10​
poursup.jpg
• Once cooled to ~117 - 120ºF, spray your gloved hands with ISO once more and put the media bottles in the SAB​
tempgood.jpg
(Take a breath and plan out your execution:)​
*The following will be covered visually in the video following these steps
• Remove foil, discard, and remove lid​
• Grab your media bottle with your dominant hand. Grasp with your fingers and thumb like you would a teacup, the lid of the bottom-most petri dish​
(Pinkies Up!)​
• In a tilting motion, lift the lid and the rest of the stack up at a slight angle, giving yourself just enough area to be able to pour a stream of MEA into each dish​
• Pour enough to coat the entire petri dish ~1/8-1/4" thick​
• Let the stack gently fall back down, putting the lid back in place​
• Move on like this through the rest of the stack.​
Pouring Agar


Fuck yeah! You just poured agar! Feel like a scientist?​
So, from here, you can do a few things:​
- Allow them to solidify and cool for a couple of hours before inoculating with spores or cell tissue​
- Let them sit in your SAB if your SAB is going to remain stationary - I'll usually cover up the arm holes with something if I do this.​
- Or, allow them to solidify and cool, then wrap them with a few passes of cling wrap cut to 1.5" - 2" rolls, then put them back in the sleeve they came in for later use.​

•••••••••••
•••••••••••
Cell culturing ended up being my absolute favorite part of this hobby.
You get to see the results of your actions within days and there's something about growing a thing on a two-dimensional surface that satisfies your curiosity and thirst of observation like nothing else. It's all there in front of you for you to see however closely you would like to look.
Even catching contamination is enthralling and prods your fascination.
I'd like to introduce a mindset that you shouldn't really reserve too much space in your brain for, but is apt to fungiculture as a whole. And that's that..
We see from the life cycle of mushrooms that, aside from spores, every tissue is damn near the exact same makeup. And, furthermore, the demands for those tissues throughout almost all of their life cycle is the same.​
Culturing on agar is no different from growing on a substrate. Media/substrate can be thought of as one in the same.​
MEA : Spawn/Bulk Substrate - inoculate, colonize, fruit.​
Petri Dish : Monotub - habitat.​
It's so cathartically simple. I love fungiculture for that reason. It has real sense of eliminating complication in that, the more you complicate it; the harder it gets : the more you simplify it; the easier it gets. It's Zen. It's Wu wei. It's 1 + 1 = 2.​
Of course there are parameters and concentrations and levels and specifications... but, I find if you take a step back and just sympathize and accord yourself with the nature of the species, you'll find that the solutions are childlike in nature... they're very watery, so they must want humidity. They like to breathe in oxygen and exhale carbon dioxide like we do, so it's probably best to give them circulating air. They respond to light like we do, so we don't need to bombard them with specified levels of PPFD, lux, spectrum, etc.; they just want a little light to get some pigment and not grow funny.​
 
Dirtbag

Dirtbag

Supporter
Cool cool. I can already see where i fucked up a couple times lol.

I was SUPER careful to make sure my hands didnt go above any sterile grain, but a couple times my gloved hand was above the cultured agar while i was cutting it into slices. It was sprayed with alcohol.. but still, sounds like thats a deal breaker.

Guess Ill need a longer knife. And ill need to get more cultured agar as ive likely infected all of mine... Boourns.
 
beluga

beluga

🤣🤣

Yeah, you'll quickly see how a scalpel or craft knife really makes a difference.
Something I didn't mention about the scalpel that I thought of last night was how they don't transfer heat far into the handle at all. Which... fuck. When I used a craft knife, it was hell... ended up throwing a few passes of electrical tape over the butt and that still didn't quite keep it from nearly blistering my fingers.

As far as colonized dishes go, it's a good practice to set their lid aside, pick them up, and work on them at a decent angle... like so..
20201122-211429-1.gif


(sorry it's sideways...)
 
beluga

beluga

🍄🐳🍄🐳🍄🐳🍄🐳


Shut up, beluga! Let's start growing!!!

•••••••••••
•••••••••••


Inoculating Agar with Spores (germinating)
•••••••••••
Supplies:

Torch - 🛠🚜🧪
Scalpel / Blades - 🧪💊
Nitrile Gloves - 🧪🛍🛠💊
70% Isopropyl - 🧪🛍🍳💊
Cling Wrap - 🍳🛍
Synthetic Swabs - 🧪🛍🍳💊
•••••••••••
A couple notes before you do this:
- You'll want to cut your cling wrap into 1.5" - 2" sections so we can neatly wrap the plates that have cultures on them.​
- If you have a spore syringe - shake the fuck out of it. There are (probably) millions of spores in there, so you wanna disperse them well and make get your use out of it. Each 10cc syringe can probably inoculate hundreds of plates - you only need one drop for each inoculation.​
- Plastic inoculation and synthetic/cotton swabs are one-time use.​
- Nichrome inoculation loops need to be sterilized and can be used quite a few times, but will lose their efficacy and become brittle.​
- You'll be using a gas torch. Be smart about it. It's very hot and is a cylinder filled with a flammable material under pressure. Don't knock the valve. Don't let it run unlit. And absolutely - whatever the fuck you do - DO NOT OPERATE THAT SHIT IN THE SAB.​
- On that note, you also are using 70% isopropyl alcohol. That's also a flammable material and when you have it inside your SAB, you are creating a pressurized container. Whatever the fuck you do - DO NOT USE OPEN FLAME INSIDE YOUR SAB. Not only are you asking for a plume of fire blasting out of the holes (I've seen the aftermath of this happening), you're also destroying your still air. Be smart.​
- It's good to have some kind of stand in your SAB for your tools, the lid to petri dishes you're working on, etc. Anything works.. petri dishes, a wire rack, lids.. whatever, so long as you can spray it with ISO and it keeps your stuff from touching the floor.​
•••••••••••
•••••••••••


Cut your Cling Wrap


It's as simple as it sounds. Cling wrap comes in a long roll. Make it into smaller rolls of ~1.5" to 2".
I use a sharp utility knife - hold the knife steady and roll the cling wrap as I slowly depress the knife gradually into the roll.
This is the type of shit people hurt themselves on. Be smart.
I've also seen people use a vise and serrated knife. Whatever. As long as it cuts and doesn't mar your cling wrap so badly that you have burrs in it that will easily tear when using it.

Mount your Torch

Also as simple as it sounds. It's a torch. Having a lit torch be able to roll around or be knocked over is seriously dangerous. Use duct tape. A vice. A sack of sand. Whatever. Just so long as it keeps it stationary and gives you easy access to be able to sterilize your tools. Be smart.

•••••••••••

Inoculating with a Spore Syringe

Instructions

*The following will be covered visually in the video following these steps

• Prepare your SAB and workspace​
- Cursory SAB wipe-down​
- Load SAB with pre-poured petri dishes, tool stands, cling wrap, and (if applicable) disposable loops/swabs (in their individual packaging)​
- Ready your work station with ISO spray bottle, paper towels, scalpel/knife, torch (safely mounted to easily sterilize your instruments)​
• Take your spore syringe (assemble if needed, leaving the needle cap on) and shake it until any clumps are broken up​
• ISO your hands, the spore syringe, your knife and inoculation loop handle (if applicable)​
• Light your torch​
• Get a petri dish ready in the middle of your SAB workspace​
• Start to remove disposable tools from their packaging​
OR sterilize your inoculation loop and bring it into your SAB, setting it on your stand with the tip far away from the stand surface (not in contact with anything)​
• Remove cap from spore syringe, sterilize ~1/2 - 2/3 of the needle (don't get too close to the plastic), and bring it into the SAB​
• Give it a light squirt - it will spurt out a bit of steam (don't point it in the direction of your sterilized instruments)​
• Grab your inoculation loop or swab in your free hand and carefully drop ONE drop of spore solution on it. Set your spore syringe on your stand​
• Carefully remove your petri dish lid - you can either set it on another dedicated stand, or you can just carefully hold it away from the base​
- this is the most critical part in not letting your unsterilized tools or self pass over the media - take care to make deliberate movement and manipulations
• With your incoulation loop, streak your plate in a zig-zag pattern, as such:​
zig-streak.gif

Using a Loop to Streak a MSS (multispore syringe)


















There you have it!

•••••••••••
A common question is, "how much spore solution do I need to germinate?"
The answer - one drop.
There are millions of spores in one 10cc syringe. One drop will see you with probably hundreds of spores. Those dark clouds of collected spores you see? Hundreds of thousands of spores.
Be frugal and you can have one syringe last you hundreds of plates.
Be frugal yet and you can have one drop last you a lifetime of mushrooms.
•••••••••••
Let's go over why we streak instead of just dropping spore solution directly to a plate.
Well, it's fairly simple.
By streaking it out in a long line, you're depositing the solution over a greater area so to separate individual colonies.
If your syringe or whatever you're using to germinate/inoculate has contaminating cultures in it, streaking will do a much better job of isolating those from your intended culture.
Via the same mechanism of action, it will also separate your spores from each other, getting you closer to an isolate - isolates give us more unified traits and are generally more favorable to work with.
If you blop a drop right in the middle of your plate, you have all that mayhem just raging out together in one small pool.
Streaking also gives us a better means to have a clear working area on the agar media. Because you have these uniform lines, you can more easily distinguish which culture you'd like to choose from and which ones you'd like to stay away from. And hopefully have some open, uninoculated space in-between.

Good stuff!
•••••••••••
•••••••••••
 
Dirtbag

Dirtbag

Supporter
Awesome man! So far im learning a lot. And you know, i usually learn from mistakes lol. Sitting over here waiting to see "all that mayhem just raging out together in one small pool."..
 
beluga

beluga

Awesome man! So far im learning a lot. And you know, i usually learn from mistakes lol. Sitting over here waiting to see "all that mayhem just raging out together in one small pool."..
I did it that way at first, too. It's not like it doesn't work, it's just more prone to issues.
Honestly, I like all the mistakes. If I didn't make them my first go or so around, I probably wouldn't have the articulation of the process. Also teaches you how to work with the less-than-ideal... which happens even if you're following everything the way you're supposed to.
 
Dirtbag

Dirtbag

Supporter
I did it that way at first, too. It's not like it doesn't work, it's just more prone to issues.
Honestly, I like all the mistakes. If I didn't make them my first go or so around, I probably wouldn't have the articulation of the process. Also teaches you how to work with the less-than-ideal... which happens even if you're following everything the way you're supposed to.

Agree. I personally welcome the mistakes. Each one is a lesson and a valuble part of the process of learning man..
 
Lynch_Ironside

Lynch_Ironside

Hey bro, awesome thread and info. I wanted to get your opinion on this pressure cooker. I tried to find the specs on it to no avail.
To sterilize inoculation jars its reccomended to run 15 psi, do you think I'd be ok with running this cooker for 90 min or so since I have no idea what the pressure would be?
 
beluga

beluga

Hey bro, awesome thread and info. I wanted to get your opinion on this pressure cooker. I tried to find the specs on it to no avail.
To sterilize inoculation jars its reccomended to run 15 psi, do you think I'd be ok with running this cooker for 90 min or so since I have no idea what the pressure would be?
It should absolutely work... just have to figure out what PSI it gets to to know how long you need to run it to adequately sterilize...


Found those two manuals and both seem to suggest 7-8PSI. I believe that's gonna be what the manufacturer uses for all of their pressure cookers.
So, you'll have to up those cooking times by quite a bit... lemme find a chart...


So, that would suggest about 5 hours to properly sterilize at 7-8PSI.
 
Lynch_Ironside

Lynch_Ironside

Wow, I did not think it would take that long! I think I would run out of water in the cooker if it steamed that long... I'll give it a try and see what happens 👍
 
beluga

beluga

Wow, I did not think it would take that long! I think I would run out of water in the cooker if it steamed that long... I'll give it a try and see what happens 👍
Yeah... be careful, of course.
If you have the heat set right, it shouldn't allow very much steam to escape at all, so you should likely be okay.


Man... I really need to update/reboot this thread. Sorry to anyone following... more on my plate than my lazy ass can keep up with.
I'll get around to it 😎🍄
 
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