Growing Room 1


Golden Teacher - Psilocybe Cubensis

The Golden Teacher is a Psilocybe cubensis mushroom and is a species of spiritual / psychedelic mushroom whose principal active compounds are psilocybin and psilocin. Commonly called shrooms, magic mushrooms or cubes, it belongs to the fungus family Hymenogastraceae and is a type of coprophilus fungi (a dung loving mushroom) that turns blue when cut or bruised. The non-psychedelic form of this mushroom is called Deconica and there are many species in that group. These do not turn blue when cut or bruised.

Psilocybe is a great spiritual tool as it intensifies the spiritual energy already inside you. If you consider your body and mind having a network of interconnected pipelines, be it prana or meridians or nerves, psilocybe is the cleaning agent, clearing blockages in those pipelines as effectively as possible on the planet.

The name Psilocybe is derived from the Greek roots psilos (ψιλος) and kubê (κυβη), and translates as "bare head". Cubensis means "coming from Cuba"

There are again many different strains of Psilocybe cubensis. The Golden Teacher is the most famous.

McKennaii

Some other strains:

Alacabenzi Mayiescens Psilocyne Aurumescens
Ban Hua Thanon Mazatapec Stropharia cyanescens (Mexican)
Equador McKennaii Transkei
Cambodian Naematoloma caerulescens Thai
Colombian Orissa India Nepal Chitwan
JFM Jedi (Strongest) PES Amazonian PL classic
Malabar Penis envy B+

Where to start

There are 5 ways to grow your golden teachers. The best method is listed on top:

1. Liquid culture single strain (you can buy these (sjamaan or smartific.nl), but if you did not make them yourself best is to check on agar Petri dishes - explained later)

2. Cloning using agar (basically having a good mushroom and cloning it on agar Petri dishes)

3. Spore print or spore swap (you can buy these and place them on agar plates and choose the fastest growing strain. Good but time consuming option)

4. Spore vial (Put them on a petri dish to select fastest growing contamination free mycelium.
Do not buy the spore syringe or inject the spore vials directly into a grow bag or grain jar - that is asking for trouble)

5. Grow kit (Cheap and easy but that is all.)

What you need

Still Air Box SAB Petri Dishes
Large plastic box, like a 155 liter Autoclave (pressure cooker also possible but less effective)
Phillips UVC light (such as 2x TUV PL-L 60W 4P into DUO FLUOR DELUXE FIXTURE 2 X 55W) Psilocybe Cubensis Spore Print or USA spores on Aluminium foil or liquid culture single strain
Table with aluminum surface (helps with UVC disinfection) Petri dishes
cutting tools to make holes in monotub and SAB lab bottle 500ml (Erlenmeyerkolven DURAN)
Philips 4000i air filter (acts like a laminar flow hoot for the whole room) Lab outfit - Gloves and Mask.
Oven rack food color additives
IsoPorpyl alcohol (best 70%) or Tevan Panox 300 Laser temperature sensor
  Agar-Agar
Spawn Honey or Corn syrup or Dextrose
10 jars 1 liters (Bredehalsfles DURAN) Malt extract light
ePTFE Pore size 0.2 µm DURAN jar lit (Do not use micropore tape) potato flakes
Mushroom bags XXL Powerhub @ gmx.net weight scale
For LC into mushroom bags - Beacon Hill Mushrooms adhesive self-healing injection ports bandage tape (to write on and stick on petri dishes for info)
Rye berries (Rogge) Parafilm (optional if you do not use the lock boxes below)
Millet (Gierst) (gluten free) lock box 6 liters (for the finished petri dishes to store inside a sterile environment)
Substrate Burner

Ovenbags large for the horse manure & waterproof bags

demineralized or distilled water (for in the autoclave and fog generators)
Horse manure (make sure it is dried and aged - otherwise you have a bad smell) Scalpel
Vermiculite  
Coco Coir Monotub
Agricultural Bio gypsum farming grade (so no casting stuff) Calciumsulfaat Dihydraat Sunware Q-line 120 liter monotub or similar (If you are ok with lifting 25kg otherwise go smaller)
Anova precision cooker (a sous vide device for pasteurization) 6500K daylight LED strip (to put on wooden plate on top of monotub (not inside but on top due to temperature)
Measuring cup (I use the Pyrex Prep & Store) and scale to weight things Power socket with day program (for LED lights 14 hours on / 10 hours off)
Lime (Kalk) https://geni-investments.com/ Analog waterproof temperature sensor with hydrometer (why analog because of high moisture more stable)
Extra's 5cm (2 inch) tub filters (microppose) or polyfill
Food dryer Room heater
ATCO OXYGEN ABSORBER DSP35 Temperature switch for room heater (not a plug in dimmer) (Universele temperatuurschakelaar)
Vacuum sealer with small bags Micro Mister (or reptile for generator - buy one you can put on a timer)
Vitopod propagator (or incubator) Computer fan (computer fan with "BN-link short period repeat cycle timer" and a computer power supply)
Planner  

This list will set you back around 2500 euro's.
If you follow these steps exactly it will give around 3 flushes and it will yield around 500 grams dried Golden Teacher per monotub in less then 3 month.

The normal price for the golden teacher is around 3 to 4 euro's per gram. So in the longer run this is way more economical.

The cheap option is just buying a grow kit for 25 euro's.

Liquid Culture vs. Spore Print vs. Spore vials vs. cloning

If you go for the liquid culture which is the best and easiest option, make sure it already has a living mycelium culture inside (not spores but culture) and make sure that it is of one and the same strain. Do not mix spores in the liquid culture yourself if you are not willing to transfer on petri dishes afterwards because you will have multiple strains in one vial and competition will give you slower growth and lower yield with the added problems of contamination. If you still want to do this (for legality reasons in your country), make sure you use agar petri dishes to flush out all the contaminations. Injecting spores directly into a grain jar is the worst option as you ruin it with contamination almost always.

Spore vial

Blue is the spore vial and yellow is the liquid culture.
They are from www.magic-mushrooms-shop.com

You have to transfer the mixture on petri dishes, once finished. But know that you will get mixed results and most certainly slower growing mycelium then putting the spores directly on agar plates and sector them out for the best strain.

Set a propagator or incubator at 28C for the first 3 days with the mixture inside, then 25C monitored by a temperature switch that turns the propagator heater on and off. 28C is only needed if you work with spores. If you already have a liquid culture and want them to grow, use 25C. Why use 28C for spores is because they germinate at higher temperatures. In nature these spores normally germinate inside an animals gut.

You can see that the spores have germinated and form mycelium balls. It is time to shake them up. Keep in mind that these balls are what is called tomentose mycelium and is a mixture of many different strains.

Ready to be used for agar petri dish transfer even though growth has been slow, but it will work. It is normal for spore syringes inside a liquid culture to only fill up about 5% of the LQ with mycelium.
If you use cloning or single strain liquid culture, you will see about 50% filled with mycelium when ready

Day 20 is too late as the liquid culture has stalled. Better put them in the fridge at day 15.
The black worm you see inside the mycelium is contamination. But with petri dish transfers you can filter them out.

Transfer onto petri dishes

Spores onto agar plate

Next step for the spore vials from www.magic-mushrooms-shop.com is to put the ready to use mix onto petri dishes.
What you want to see are clear white balls of mycelium. However you can expect some contamination, this is normal.

For example some of them can turn green like below.

Green is not good and indicates contamination. This is actually from the liquid culture and not from outside.

What is interesting is that these spores grow much thicker but slower then the liquid culture from sjamaan.com
Because of the contamination and multi strain and extra time needed for the spores to germinate, do not inject these spores directly into the grain spawn. Most of the time you will have contamination and have to throw away the grains.
Better first use about 6 petri dishes and put the spores or the liquid culture you made onto them. After about 10 days select the petri dish that grows the fastest without contamination. Use that one to fill 6 new petri dishes. Repeat if needed until you have a 100% contamination free fast growing mycelium strain for grain inoculation.

-- -- --

The better option is the liquid culture single strain
This is a test of the Liquid culture single strain from https://sjamaan.com
As you can see above, all healthy, clean and growing fast @ day 4.

After 1 transfer @ day 14 these are ready to be used for grain inoculation or even better, injecting the liquid culture directly into the grain jars so you can skip the agar petri dish transfers all together.

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Cloning

If you wait too long you will start to see little white balls forming on the plate that are called hyphal knots that in turn grow into micro mushrooms inside the petri dish or the dish stalls.

Enlarged photo of the same petri dish as above but left alone for 6 weeks.
It is really nice to see all the different stages of rhizomorphic growth (Rhizo is Greek for roots and Morphic is Greek for Looks like - so looks like roots) into hyphal knots (the white balls) and then primordia as seen on the large white elongated ball on the right and eventually the mushroom in the middle.

-- -- --

Spore swap

There is a whole webpage dedicated to this method: See Panaeolus page - the Universal Wind Teacher

-- -- --

Do not get the spore syringes, that should be your last option. When I tested them some never germinated, others full of contamination. Some websites offer the spore syringes and say you can directly put them into a growbag or growkit. This is officially the worst option. If you are only interested in something easy and cheap, go for the single strain liquid culture and sterilize the needle and insert them into the ready to use sterile substrate or even cheaper and easier a growkit ready to use for 28 euro's that only needs water.

Spore prints on aluminium foil is a very good option on agar petri dishes, or use the spore swaps. It takes a lot longer to get to the end result but it is also a lot of fun seeing them grow.

Agar Petri dishes

Now that we know the different options, let look at one of the best methods, the agar plates (petri dishes).

This process can take up to 60 Days (for the 4th transfer to be ready for inoculation into grain jars)

Obviously you need all the items listed in the beginning of this webpage before you can begin. Best are the larger (10cm x 2cm) glass petri dishes, explained later.

Growing room

Label the mycelium strain that will match the agar color on a box where you will store the dishes for growth.

Isopropyl alcohol clean the workplace. Best table surface area is metal or aluminium if you are working with UVC.

Still Air Box (SAB) with UVC does not need to be cleaned.

Place SAB on the table (on an aluminum or metal surface)

The still air box that I use has a DUO FLUOR DELUXE FIXTURE 2 X 55W and 2 TUV PL-L 60W 4P UVC light tubes. These are in total 120W UVC so be careful with your eyes.
Also my SAB is connected to a philips 4000i air filter that filters 99.9% of all bacteria and even viruses and creates positive pressure inside. This helps to cool down the dishes faster and avoids condensation on the walls of the SAB and UVC light tubes. The other 0.1% is killed by the 120W UVC. It is surrounded by aluminum plates so the UVC can reach everywhere. In the picture below you see how I cool down my freshly poured agar dishes. With this setup you can even leave the dishes open as there is no contamination, but better is to close them as the moisture content remains exact. I poor them at around 90C. Also if you close them you can stack them on top of each other. This way you can do more dishes. Stacking them also reduces the condensation inside the dishes.

I use a rack for faster cooling with the air filter on. You can also leave the UVC on, but I found this was not needed.

This setup only works for creating and cooling agar plates. Do not transfer dishes with the air filter on as this blows the contamination all around the inside of the SAB if you have contamination.

A word of caution. Because these UVC lights are very powerful and the philips 4000i air filter creates positive pressure, you will breathe in ozone if you work in front of it. So time it right and better use the UVC before and after working in the SAB.

That said, these steps were all needed for a 100% contamination free workspace as I had to go through many trial and errors to end up here.

120W UVC Still Air Box with a philips 4000i air purifier connected to it (acts like a flow hoot near the entrance of the SAB or acts like a cooler inside the SAB and also cleans the air outside the SAB) on a stainless steel metal table with UVC protection glasses. There is also a working LED light installed inside the SAB so you can choose only that light for working inside the SAB or with UVC together or only UVC. The aluminum plate on the lower left can be used to seal the 2 entrance holes in the SAB. It is aluminum because it reflects UVC the best. There are also 3 other aluminum plates installed on the side walls of the SAB, all to reflect UVC everywhere inside. The SAB itself blocks UVC from exiting so you do not go blind by looking at the SAB with the UVC on and the cover plate over the holes.

Prepare the glass petri dishes - clean with isopropyl alcohol and place them inside SAB. What I do is put them all in an ovenbag and put them inside the autoclave. When finished I tie wrap the bag and bring it inside the SAB. Leave room for a 500ml lab bottle filled with hot agar. Turn the air filter on and turn the UVC on for a couple of minutes and all is ready and sterile. After this you can open the ovenbag with a sterile pair of scissors.

If you leave them open for cooling, clearly separate the cover and base of the petri dish so you know which one to fill. In the case above, the large top covers are below and the smaller dishes that need to be filled are on top of the rack. This can only be done if you are 100% sterile. If not, leave the dishes closed. Once you fill them at a lower temperature of around 70C, you close them again and you will get condensation but stacking them one on top of the other will reduce the condensation .

UV-C does not go through the glass or plastic, so be careful for shadow. Because I autoclave them, there is no need to UVC them both sides though.

UVC test inside and outside a petri dish. Like the SAB, the petri dish will also block all the UVC.

— — —

Kitchen

High Potency Agar (HPA)
-
this is not an official name, but makes sense:

1 liter destilled water or chlorine free tap water
(for 25 petridishes 10cm x 2 cm)

20 grams organic agar-agar
15 grams potato flakes
10 grams Light Malt extract
5 grams honey
5 grams yeast
Optional for hard to grow mycelium - 5 grams peptone

total sugars / carbohydrates <4%

Potato flake Dextrose Agar (PDA)

(CLASSICAL FORMULA):

500ml water (chlorine free, so be careful with tap water)

10 grams potato flakes
7.5 grams organic agar-agar
5 grams honey (or dextrose or corn syrup)

Malt Extract Agar (MEA)

(CLASSICAL FORMULA):

500ml water (chlorine free, so be careful with tap water)

15 grams Malt extract

7.5 grams organic agar-agar

500ml is for 10 large petri dishes. You want the large dishes (10cm that are 2cm thick) as less moisture buildup inside and more fresh air.

As a general note, do not go beyond 4% sugars / carbohydrates, be it honey or glucose or similar.

Light Malt Extract, total sugars 90%

Malt is germinated cereal grain that has been dried in a process known as "malting". The grain is made to germinate by soaking in water and is then halted from germinating further by drying with hot air.

Malting grain develops the enzymes (α-amylase, β-amylase) required for modifying the grains' starches into various types of sugar, including monosaccharide glucose, disaccharide maltose, trisaccharide maltotriose, and higher sugars called maltodextrines. Malt also contains small amounts of other sugars, such as sucrose and fructose, which are not products of starch modification, but which are already in the grain.

Peptone ingredients and total sugars 20%

Peptone is rich in amino acids, peptides, vitamins, carbohydrates, nucleosides, minerals, and other components, so they are a good mix for the LQ.

 

Potato flakes 85% sugar

Potatos contain 20% carbohydrates but dried potato flakes loose the 77% water which means dried potato flakes are 85% carbohydrates.

Bring to a soft boil and pour into lab bottles and add a food color (best is brown and dark blue - this to make it easier to identify contamination). Leave the screw cap a little open so it is not air tight. Best is to put them inside a ovenbag in case they overflow plus contamination proof when moving the bottle out of the pressure cooker.

Potato flakes provide a nutritive base for luxuriant growth of fungi. Potato Flakes also enhances the production of morphological structures necessary for the identification of many pathogenic and opportunistic molds. Dextrose is added to the medium as a source of energy. Agar is a solidifying agent. Light Malt extract is to let the mycelium make the required enzymes so it will be succesful when transferring them into grain. Yeast provides nitrogen compounds and vitamin B complex and other nutrients for growth. Peptone is used for detecting bacteria inside your mycelium, so it is faster at cleaning plate transfers.

-- -- --

So what is the best growing medium for mycelium?

HPA (High potency agar) - factor 1.8

1. MEA (Malt extract agar) -factor 1.5

2. PDA (Potato Dextrose Agar) - factor 1.3

3. YMA (yeast Malt Agar) - factor 1.3

4. GPA (Glucose Peptone Agar) - factor 1.1

5. SDA (Saboraud's Dextrose Agar) factor 1.0

However a combi of them all, like above, is the best.

Researched by Nagalakshmi Muthu and Krishnakumari Shanmugasundaram (factor means with MEA the mycelium grows 1.5 times faster then SDA)

Clearly HPA is the fastest and best but sometimes the HPA creates weaping - small yellow droplets on top the mycelium.

Peptone

Due to the peptone, you can see a sector on the left bottum that is most likely contaminated. It is thinner and a little yellow. Without peptone, you will not detect this. The droplets on top of the mycelium is called weaping mycelium - moisture from the HPA that is sucked up by the mycelium.

Put in autoclave for 90 minutes at minimum 121C (use distilled water in the autoclave as the autoclave will survive longer). Also put the glass petri dishes inside (I put them in a oven bag together for easy transport into the SAB).

There are stories going around that over 30 minutes in a pressure cooker the agar mix will caramelize and give less than optimal results. I tried this many times, almost always with contamination. So this is an incorrect statement. Also Caramelization happens at 185°C and not at 135°C. So just do 90 minutes and use MEA or PDA or both and you are fine.
With a pressure cooker do at least 120 minutes as those systems normally only reach 110C. The 15psi you find on internet is a standard number for most of the old pressure cookers. It is better to go higher as the agar looks better and is more sterile.

With the VOS autoclave it keeps the temperature at around 128C

The petri dishes that you will use, you put in the autoclave together with the agar lab bottles. Best is to place them inside an ovenbag and slightly tie wrap the whole.
The moment you open the autoclave, you tighten the tie wraps and bring them inside the SAB - when they are still very hot. The bag will vaccume seal itself a bit.

Next step is take them out of the ovenbag (inside the SAB) and place them on the rack.

Back in Growing room

Put on your protective gear (gloves cleaned with isopropyl alcohol, and mask)

At around 100C fill the petri dishes half full (You need an air purifier with positive pressure otherwise be prepared for condensation on the SAB walls and low visibility. But low visibility also means the UVC lights might short circuit so again use positive pressure)

Fill petri dishes and directly close them and stack them on top of each other.

Put the dishes in an air tight box and keep them empty for at least 3 days. This way you are sure the dishes are 100% clean and no moisture buildup before placing anything inside.

If you have liquid left in the bottle, because you made too much, do not throw that into the sink - It will block your sink ! (yes I have done it...)

-- -- --

Time to inocculate the petri dishes with spores or use clone samples or transfers or liquid cultures.

Get the spores or liquid culture or mycelium or clone inside the SAB. (No more UVC and no more fan if you have one at this point, as otherwise you might kill the spores or mycelium)

Fire sterilize scalpel or needle outside the SAB (Be careful for a fire risk inside SAB if you cleaned anything with isopropyl alcohol)

Inoculate petri dishes

Close and parafilm seal the petri dishes. It is much better to still parafilm seal the dishes even though you have them inside an air tight box. First, you keep the moisture contend in a better shape and second, cross contamination with other petridishes are almost impossible.
(If you place them inside an air tight box, you have a UVC the box or use an air filter to make sure the air is clean or parafilm seal the dishes making sure the air is germ and spore free.)

Then place the air tight box with the parafilm sealed dishes inside a heated propagator or heated incubator or warm location.

If you use a heated propagator make sure you place them on top of an oven rack or small table so the base of the petri dishes are lifted at least 2cm above the base of the propagator.
This will prevent unequal heating of the propagator and too much moisture buildup in the petri dishes.

Set the temperature to 2*C for spores and 24C for mycelium (27C they grow best but mycelium warms the petri dish from the inside so 24C is the best temperature inside a propagator keeping in mind the moisture buildup as well)
Make sure the sensor does not touch the base of the propagator but is on the same level as the petri dishes.

A test to see what happens to a plate when it is placed at an angle.
Condensation will drip onto one side and increases the chances for contamination .

A test to see what happens if you place the petri dish upside down.

Petri dish (plate) with mycelium first 2 days straight up and then upside down. As you can see the moment when it got turned upside down the mycelium started to grow very thin and strange. You can still use this but better have the thick white mycelium.

So, do not put them upside down or sideways.

Best solution is to get bigger petri dishes that hold more air and to lower the temperature to 23C. Also once you filled the dishes, leave them inside an air tight box for 4 days. This way you do not get condensation anymore and you are sure the dishes are contamination free before usage. After 4 days you have no problem using the dishes and heating them up to 28C as the condensation in minimal.

I use 28C for the panaeolus agar dishes and they aee doing fine as longn as I wait 4 days before using the dishes. The mycelium is actually sweating so moisture buildup is normal. Stacking the dishes also helps with excessive moisture buildup in the beginning.

Heated propagator (incubator) with large 12cm petri dishes inside a sterile still air box and also parafilm sealed. Stacking helps with too much moisture buildup.

10 cm x 2 cm plates is the sweet spot. If you put them in a sterile box there is really no need to parafilm them unless you suspect contamination in one of the dishes. You do not want to spread that over the other dishes.

Mold contamination sample 50X enlarged on a petri dish that I did not seal in the open air to show what can happen. These types of contaminations are often formed near the edge of the petri dish because air with contamination enter this way. So if the mycelium is not white, starts from the edge of the petri dish or behaves differently (that will only be recognized with experience), make sure you transfer the good mycelium until you have a clean dish.

Placing the petri dish inside an air tight plastic container avoids the agar edge contamination, just like parafilm does, but is more breathable.

Below a test to see what happens to green mold in pasteurization:

Green / blue mold test.

This plate is being pasteurized at 60C on the right and tested for growth afterwards... see below

RESULTS:

After 5 days - no growth, so pasteurization works

After 10 days - everything is dead and sterile
- click image to enlarge

No more growth of both mycelium and mold. Even after a month no further growth. So 60C pasteurization works.
I also checked if the spores of the mold were still alive on a new petri dish. Nothing was growing.

Below some other examples of plates:

This is a plate with the first transfer after spore swap on agar. There is some yellow contamination and a whole area that looks weak or contaminated. It is important to turn the dish around and look for the healthy sectors. See image to your right.

The same dish as on your left. On the left bottom you see some contamination that blocks the growth that cannot be seen in the top view. The yellow and green areas are healthy growth. The mycelium actually changes the agar color if you use food colors. On the lower right side you can count about 8 healthy sectors meaning 8 different strains. The best one is at the 4 o 'clock position as it growth the fastest. Use that for transfer.

This is a clone looking at the back side of the dish and you see black contamination on the left. The 2 ear looking areas are actually healthy. When you use clones, it tends to grow into one direction. This is normal and is called rhizomorphic growth. Only transfer if you have contamination. No need to sector them out.

This is from a liquid culture single strain sample, all looking healthy. With single strain liquid cultures there is no need to transfer unless you have contamination.

Rings (second transfer)

Rings will show up like the plate above if the temperature changes from day to night. Mycelium grows faster during the day when it is warmer.
Almost like a tree where you can count the years instead of days. In this case it was 10 days. The first 3 days is the solid white in the middle and 7 rings.

Isolate cultures in spore prints or spore swaps

One of the components that play into the complexity of mushrooms is the fact that each spore print, has the capability to grow millions of individual cultures. When grown out on agar, however, sectoring out that many would be a bit of a hassle. Usually, you’ll be able to isolate up to five individual cultures. These cultures can then be grown out on their own plates, put in grain, and ultimately fruited.

Once collected, the spores are caught with an inoculation loop which is used to create streaks across the agar plate or in the case of a spore swap you can draw a S on the agar plate directly, taking care to space them out to help make the sectoring process easier. They will then need time to grow enough to see the individual cultures (see below good sectors). As they grow, the cultures will develop different looks and grow out mostly from the streaks, so isolating them shouldn’t be too difficult.

Sectors (3rd transfer)

You want to find the fastest growing mycelium and the thickest and take that onto a new agar plate. Do not only use one good sector but use multiple good sectors.

What works really well is to use an oven rack with a LED light (like a phone or small LED strip, but make sure it is sterile) under it to find the sectors. Use the thick sectors and pick them from the edge of the growth.

Backside - Use the thick sectors for transfer.
What looks like dust inside the plate is actually potato flake remains, as this is PDA.

Once the fluffy mycelium (Tomentose) turns into the more rooted form (Rhizomorphic) after multiple transfers, you have got yourself and isolate. This you can use to inoculate grains.

Fourth transfer - one more transfer needed as you can see the rhizomorphic growth and you want the whole plate like that.

-- -- --

In real life the spores incubate inside an animals gut like a cow or horse at temperatures of 38C.
After 3 days the young mycelium starts to grow inside the manure (shit) at temperatures around 26C - so yes warm and moist...
Happily enough we do not have to emulate this strategy. That being said, horse manure is the best medium. Also when using spores, the initial 3 days temperature must be over 28C to be successful. This means dripping petri dishes or you have to use a liquid culture.

It takes around 3 days for bacteria to show up if any and about 5 days for the spores to germinate and show the white mycelium growth and about 2 to 3 days for a liquid culture to show the same.
Between 7 to 10 days about 80% is filled and you can cut the healthy white mycelium and transfer that into a new agar plate. The fastest growing mycelium is the best. After transfer it normally takes 14 days for a whole plate to fill up. Do not go beyond the 14 days as mushrooms might start to form.

 

You can slow down the growth rate by decreasing it's temperature.
After about 3 transfers you should have clear white Petri dishes that are ready for inoculation. If using spores it might take 5 or 6 transfers to get to the Rhizomorphic stage.

Grain Jars

If you use the 120 liter monotub, you can fill it up with about 30 liters of growing material. That would mean it weighs about 18 kg.

6 liters of grain (it will expand to 10 liters), 10 liters of horse manure and 10 liters mix of coco coir with vermiculite and some gypsum.

-- -- --

6 liters Rye berries or a mix of rye berries and millet (volkoren bio rogge en gierst)

Millet is gluten free and it seems better for the mushrooms as well as for you consuming them. However you cannot boil Millet. So soak millet for 24 hours instead.

1. Rinse the rye berries with water until clear water

2. Soak 24 hours in room temperature with 1 tablespoon gypsum per 3kg rye berries (so they become less sticky and better PH). Add 10cm extra water as the grain will expand.
Why soak - because spores will germinate, specifically Bacillus spores that can survive sterilization, but not if germinated.

3. Warm it up until you see steam and bubbles, then take it off the fire. You can also check if you find any burst grains. When you spot the first one, stop the heat.

- Without soaking, boil for 20 Min.

Drain the grain in a beerfilter (these are the large filters to get most of the water out.) Then put the grain on a drying rack (make one yourself as the big racks are almost impossible to find on internet) and after dripping is finished shake and spread over the rack. Shake every 10 minutes until 30 minutes or let them dry for 3 hour on the rack.

Beer drain for rye berries

Drying grain on a self made sieve

Fills jars to 80% and close but do not seal the lid (air has to be able to pass a little). Best is to use the ePTFE membranes.

Duran bottles of 1 or 2 liters I like the best as they can be closed air tight (but with just a quarter twist, air can flow without too much risk of contamination with the normal caps) and you do not need to use aluminum foil or other extra's as the lid is very big. Also the bottles do not shatter if broken and it is up to 140C autoclavable. In the cause above these bottles are fitted with a ePTFE membrane, so you can just leave them closed also inside the autoclave.
No need to drill holes inside them either if you use the still air box to inoculate the jars.

If you want to mix Rye berreis with millet, soak them both for 24 hours in a separate container. Boil the rye berries for 5 minutes but leave the millet alone. Dry them both on the rack for 3 hours and put them in the glass jars or mushroom bags.

Depending on what type of jars you have, cover jars with Aluminium foil, shiny side out (not needed for the DURAN). This is to avoid water entering the jars.

Pressure cook / autoclave for 4 hours. Why 4 hours, because it takes a long time to get the inner grains above 100C. Wet spot contamination occurs often below 2 hours pressure cooking.

The grains will shrink in size a bit during pressure cooking and when you fill the jars 80% you will end up having 70% filled after pressure cooking. But you want the extra space, not only for shaking later but also for air exchange.

 

Here a video with another type of grain, but the idea is the same.

It is best to open the autoclave when the grains are still over 100C steaming hot. Use heat resistant gloves and put the bottles inside an air tight wooden or metal box (plastic will melt) or leave them inside an oven bag tiewrapped (this is my preferred method). When the jars cool down, they suck in air, so be careful with contamination. 99% of the contamination in the air will be killed above 60C so use 60C or above for transport if there is any chance of air entering the jars.

For bulk growth use mushroom bags. Easiest is to put the mushroom bag filled with grain inside an ovenbag and tiewrap the ovenbag but with a little air exchange. Once the pressure cooking is finished and the temp is still above 100C, open up and tiewrap the ovenbag air tight. This way the mushroom bag is contamination proof.

If you use the DURAN® Membrane Venting Screw Cap, GLS 80® you still want to put them inside a heat resistant container, as you do not want to screw the lid too tight as when the bottle cools down you cannot untighten the lid.

0.2-µm ePTFE (Polytetrafluoroethylene) membrane - Membrane for Pressure Equalisation during Autoclaving

The microporous structure of the ePTFE membrane enables sterile gas exchange in two directions, while blocking the penetration of liquids or solids and the bottle contents remains sterile.

The PTFE membrane and its polypropylene mesh support are ultrasonically welded in place, so you do not have to worry it will break during large pressure changes.

Another method is to fill mushroom bags prior to sterilizing. After sterilization, you seal the mushroom bags. Use a Liquid Culture and a self healing port on the bag to get the mycelium in.

If you seal yourself make sure you use high grade thick PP plastic and not PE plastic as that will melt in the autoclave. PP plastic normally melt around 160C and PE around 70C.

Best way for contamination proof operation is to put the mushroom bag inside a oven bag, tiewrap it but not air tight and then autoclave. After 4 hours open the autoclave and tiewrap the ovenbag air tight. This way the mushroom bag remains sterile while cooling. Bring the cooled tiewrapped bag inside a still air box, open it and inocculate the bag and seal the bag with a good quality sealer. The cheap sealers will not seal PP plastic. I had to look for different sealers and found the audion working the best.

The big advantage of mushroom bags are that you can mix the mycelium inside the grains very easily. This is much more tricky with the glass jars. Also mixing it inside substrate is much easier as you just cut the bag open.

Inoculate (Spawn)

After 10 hours cooling, UVC the SAB for 5 minutes and seal jars air tight again (all inside still air box)

I add one extra step and that is I first make the whole room sterile using the Phillips 4000i air purifier for 20 minutes prior to starting the transfer inside the SAB.
Just make sure you wear a mask when you transfer and do the steps below or use a positive pressure SAB like described below.

INOCULATE

 

Place the petri dishes that are 100% mycelium filled inside the SAB and put the jars inside as well.

Inoculate jars with agar samples by cutting them in small pieces. Flame sterilize outside still air box to avoid combustion inside due to the alcohol if you use alcohol to clean the SAB.

First I turn on my philips 4000i air purifier for 20 minutes so the room is clean. Second I use Panox to clean the inside of the SAB and the metal table and metal rack. Then I UVC the work area inside the SAB for a sterile workplace.
The aluminum plates inside the SAB reflect the UVC all around the box. I also sterilize the gloves that I am working with and I use gloves that are very long so nothing non sterile enters the SAB.

The air filter, if connected to the SAB, CANNOT be on when you start to work inside the SAB (only use it in the beginning to clean the SAB and the surroundings). The simple reason for this is that anything you want to transfer inside, if it is contaminated, the air will blow the contamination all around inside SAB and contaminate the petri dishes. So this setup does not work like a laminar flow hoot unless you transfer from the outside where the air exits to the inside into the petri dish.

That being said, you can use the air filter with UVC to cool the petri dishes that are just filled with sterile agar. This works very effective.

So once everything is clean and ready, you can place the grain jars and mycelium inside the SAB with the air filter still on.

Once everything sterile is inside, turn off the air filter and flame sterilised scalpel, cool it a bit and start cutting the mycelium in small squares.
Open up the grain jar and put the squares inside. Close the lid and repeat for all the other grain jars.

Once everything is filled and the caps are on you can open the SAB and shake the jars so the mycelium is spread everywhere.

Inoculation

Make a monotub specifically aimed for growing mycelium in grain jars. Make the monotub air tight so nothing can go in or out and drill 4 large (3 inch) holes on the lower 4 sides of the box.
Place microppose filters or polyfill in / over the openings and you have a sterile grain jar monotub. This is only needed if you unscrew the grain jars for fresh air exchange. If you use the Duran ePTFE membrane this grain jar monotub is not needed.

Once shaken place the inoculated grain jars inside the grain jar monotub and put a UVC on top before unscrewing the caps and closing the monotub cover. Or use the air filter with positive pressure and close all the 4 openings with polyfill.

Ozone is not good for the mycelium but it will not interfere as the ozone sinks to the bottom in a SAB (ozone is heavier than air). This will leak out of the microppose filters.

Ozone will not be created inside the jars as the UVC in unable to penetrate the glass. (only quarts allows UVC to pass - hence the light tubes are made of quartz)

The with UVC on, cover the lid of the box and store them. Leave jars slightly open, UVC the inside and set temperature to 24 C if you can.

Now if you have a air filter you can skip the UVC and just clean with panox and seal the grain jar box.

Another yet easier method is buying the 0.2-µm ePTFE Duran caps and you can skip the whole grain jar monotub. See image below.

UV-C test card inside a glass jar and outside. As you can see there is no UVC entering the DURAN glass jar.

Grain to Grain transfer

Grain to grain transfer is the most simple and effective way to rapidly increase your mycelium volume.

Just place the best looking 100% white grain jar inside the SAB and fill as many new grain jars as you like with this healthy mycelium.

In other words you can transfer up to 50 grain jars if you want with one healthy grain jar.

This is better then multiplying liquid cultures as you cannot see the contamination as well as in grain jars. If the grain jar is 100% white without discoloration and the smell is mushroomy, you are almost 100% good for transfers.

The 0.2-µm ePTFE Duran caps surely makes life a lot easier and within a month you can grow as much as you can dream up :)

Another great benefit in grain jar transfers is that you can keep the cycle of growing grain and mixing into substrate almost indefinately without the need to germinate new spores and start all over.

Sealing

Using the ePTFE membrane has multiple advantages over storing them inside a SAB with the lid slightly open. The ePTFE keeps the humidity correct, leaves contamination outside and is easy to transport. Just know that the caps last only 5 to 6 times of usage, so it is a bit more expensive. But 10 times should be workable as well.

Storing

8 days after inoculation, seal jars air tight again if you are not using the ePTFE filter and check for contamination and mycelium growth (should be around 30% white. Shake jars (using a bicycle tire or knee protection pat) and UVC SAB again before unscrewing the jars.

Another 4 days and the whole jar should be white and you are ready for the next step.

Contamination

"Wet spot" - Bacillus contamination (will stall mycelium)

In grain spawn jars and bags, one commonly encounters Bacillus, which sometimes survives the sterilization process as heat resistant endospores. A dull gray to mucus-like brownish slime characterized by a strong but foul odor variously described as smelling like rotting apples, dirty socks or burnt bacon makes uncolonized grain appear excessively wet, hence the name "Wet Spot". Pallid to whitish ridges along the margins of individual grain kernels characterize this contaminant. Bacillus primarily reproduces through simple cell division. In times of adverse environmental conditions, especially heat, a single hardened spore forms within each parent cell body - bacterial endospores.

Bacterial endospores are simplified forms of the bacteria, consisting of the DNA genome, some small amount of cytoplasm, and a specialized coating that confers resistance to heat, radiation, and other harsh external conditions. Endospores are virtually immortal, and can be re-activated, under favorable growth conditions, after lying dormant for hundreds or perhaps millions of years.

So how to deal with bacterial endospores - soaking. Soak your grains at least 24 hours so the endospores are re-activated and are easier to kill in the autoclave. But make sure you autoclave at least 3 hours, because the grain takes at least 1 hour to get above 100C.

Wet spot grain test on agar
- as you can see, part of the grain has no mycelium because the Bacillus has taken hold of it, but it does not kill the mycelium, it just inhibits its growth.

- Click to enlarge

The most practical method for eliminating bacterial endospores involves soaking the grain at room temperature 24 hours prior to sterilization. Endospores, if viable, will germinate within that time frame and then be susceptible to standard sterilization procedures. And, new endospores won't form in the moist environment of the resting jar of grain

Liquid culture

If you like to use mushroom bags with filter, or want to inoculate a lot of grain jars, get yourself familiar with LC (liquid cultures).

The problem with mushroom bags is that you often run into trouble with grain to grain transfers. I thought this was a nice idea and ended up with a lot of contamination. First because in my setup you cannot open a bag of grain filled with mycelium as the air filter will blow and spread all the mycelium and contamination around the inside of the SAB. Without the air filter you cannot use the UVC because your hands are inside and get burned. So you need to be extra careful and still you get in trouble.

To solve this, use LC and leave the grain bags sealed outside the autoclave.

Simply put the self-healing injection port on the outside of the bag and inject the LC in it. Or inject it directly through the plastic and seal it off with a waterproof bandage. Off course you use a flame to sterilize the needle before use.

To make a LC is not difficult. Use distilled (demineralized) water and add:

LQ ingredients

1 liter chlorine free water:

15 grams Light Malt extract (14 grams carbs/sugars)

15 grams honey (14 grams carbs/sugars)

Corn syrup is clear and that makes it easy to spot contamination inside. That is also the reason why you filter the potato flakes. Light malt extract same story, it is clearer then the dark one.
Boil it and put in container with ePTFE filter or regular lid.

35 minutes in autoclave. (Do not go over the 40 minutes as the sugars will caramelise)

I like to put the LC bottles inside an ovenbag and put that into the autoclave. Once sterile, I seal the ovenbag with a tie wrap and bring it into the SAB.
I turn on my air filter so the cooling process will be quicker. Once cooled down I stop the air filter and turn on the UVC for disinfection. Then I proceed to transfer my petri dish into the LC bottle. Best way to do that is to use some of the liquid inside the bottle and poor it into the plate that you are going to use. Mix the liquid and mycelium by scraping the top layer with the syringe or better an inocculation loop. Then I use the inocculation loop to collect some mycelium and put it into the bottle and then seal the bottle. I tried using the syringe but those get clocked up so you cannot transfer the mycelium that way.

Agar transfer to Liquid Culture

If you fill the bottles only 50%, it is easier to shake and mix the mycelium. You should mix once or twice a day. Also best is to use the special duran breathable caps, so no CO2 buildup that can kill the mycelium eventually and to make sure it is 100% contamination proof, also parafilm the lower cap.

LQ made of 4% light malt extract in chlorine free water - 30% filled with mycelium after 8 days

It is better to use 1.5% LME and 1.5% honey as it is clearer and easier to detect contamination. Only use 4% LME if you are sure it is 100% contamination free.

After 2 or 3 weeks it is ready for inocculation.

Use the LQ to inoculate grain bags or jars directly. This is the best solution if you grow in big quantities. Just drain the top liquid so you are left with mostly mycelium and poor the mycelium directly into the grain bags or jars. If the grain is very dry, then just shake the mycelium inside the liquid and do not drain but pour directly over the grains. Also the LQ you can easily put in the fridge for long term storage.

Substrate

The best substrate for the psilocybe mycelium is horse manure (cow manure is not so good, that is better for the panaeolus mushroom) as the mycelium live in symbiosis with the living bacteria in the horse manure, just like the amanita muscaria live in symbiosis with the birch tree. But it is possible to use other material or combine the horse manure with other material.

 

CVG recipe

CVG recipe (as in film above) for 40Q or 40 liter monotub

430 gram dry coco coir (that is about 6 liters when expended)

1,2 liters Vermiculite

0,2 liter bio gypsum

6 cups (2,9 lit) water

Total of 8 liters

CVG recipe (as in film above) for 60Q or 60 liter monotub

650 gram dry coco coir (that is about 9 liters when expended)

1.9 liters Vermiculite

0,3 liter bio gypsum

9 cups (4.3 lit) water

Total of 12 liters

CVG recipe (as in film above) for 80Q or 80 liter monotub

850 gram dry coco coir (that is about 12 liters when expended)

2,5 liters Vermiculite

0,4 liter bio gypsum

9 cups (5,7 lit) water

Total of 16 liters

CVG recipe (as in film above) for 100Q or 100 liter monotub

1100 gram dry coco coir (that is about 15 liters when expended)

3 liters Vermiculite

0,5 liter bio gypsum

9 cups (7,2 lit) water

Total of 20 liters

When you use CVG and sterilise it, make sure you pasteurize the casing layer later on as you want the beneficial bacteria. More info about this later.

First put all the dry ingredients together and mix them inside a monotub. Then add water and check for field capacity. When well mixed you can put it in an oven bag and tie wrap it. Make sure the tie wrap allows for airflow if you sterilize it, otherwise the bag will blow up. Also I find using a double bag better as sometimes there is a leak.

Autoclave for 90 minutes and directly take it out and seal with the tie wrap.

HM-CVG recipe

75% dried horse manure HM
(Make sure it is leeched - sun dried - otherwise you can have an ammonia smell)

10% Coco coir

10% Vermiculite

5 % gypsum

Easiest is to measure the length and width, multiply the 2 and divide by 100
(L x W) / 100 -- That is the volume in liters for a 10 cm thick substrate

I found around 25% water in volume seems to bring it to field capacity

Do not add coffee or stevia or other things to the HM-CVG. Coffee grounds from shops where you collect them can contain decaf and that means there can be chlorine in it. Stevia creates contamination.
What you can do is use a bit more Vermiculite as the horse manure tends to keep the moisture not equal everywhere and on the bottom it is more moist. With Vermiculite you can be safer.

-- -- --

Phase 1:

The HM-CVG needs to be at field capacity in the open air (around room temperature) for 24 hours - this to aid in the growth of beneficial aerobic microbes (So make sure the substrate has access to O2 and is able to remove CO2. This will actually happen if you use a polyethylene plastic bag or simply place it inside a monotub. Manure will breathe oxygen, and PE plastic actually lets oxygen through a little - and that is also why you still smell stuff through plastic, even though it does not leak, but better is to leave the bag open as there is more exchange.

Aerobic microbes will convert ammonia into protein which is food for mycelium before pasteurization, as long as they have the proper temperature and oxygen they require that is.

A good indication is that the ammonia smell in the HM-CVG is much less or is almost gone after 24 hours.

Incorrect mixing
If you first put everything in an oven bag and hope to mix it well inside, you get what you see above, a bad mixture where certain areas have too much gypsum and some areas without vermiculite.

Correct mixing - dry with pre-labelled tub. So mix it when it is dry

After your first try you know exactly how much water you need to add. Just write it on the box.
In this case 6.5 liters

Phase 2:

Easiest method to attain this phase is with a sous vide and a double ovenbag filled with HM-CVG.
Keep in mind plastic containers or bags normally can only handle max 70C and start to leak or break near that temperature. So use oven bags instead.

The problem I encountered in the beginning was too much air inside the bag. Well that s easily solved by submerging the bag underwater and tie wrap them in the water. This way it sucks in vacuum before you seal the bag.

(start timing for 12 hours the moment the water reaches 140F. I found that I need to put the sous vide to 142F to reach 140F water temperature average.)
You also do not want to wait too long as you need oxygen for the good bacteria to proliferate.
What you can also do is find the core temperature and wait until that reaches 140F and start timing for 6 hours.
But I find 12 hours in a double ovenbag easier. The sous vide does the work quite well.

Plastic bags gave me trouble almost 50% of the time as they tend to leak over time. So you can also use glass containers.

Here some failures:

Failure because the HM-CVG never reaches 140F evenly because the top is always cooler then the bottom. After 6 hours I measured a core temperature of 120F while the water was 140F.

Failure as the temperature was too hot (160F).

But... give it a try though as some have great success at temperatures of 162F like Jakeoncid419 from shroomery - and he is a master.

Failure because the bag leaked.

So use 2 oven bags. I sometimes even use 3 bags just to be on the safe side.

Bath pasteurize glass bottles is not possible if fully submerged. They start to leak and suck in water. However you can leave them open, like the cider bath pasteurization.
Problem here is that the amound of substrate you can do in 12 hours is way less plus you need to find ways to keep the bottles 95% submerged and still leave room for the sous-vide.

Cider pasteurization.

The best way I found to bath pasteurize :

Fill it up in plastic bags. (In this case waterproof fish bags)

Seal them but not air tight. Air should be able to exit out of the bag.

They will leak after many hours of pasteurization if you seal them air tight.

One extra safety layer. Best is to keep this bag open on top and sticking out of the water so it is breathable

A stainless steel fruit basket with lifting weights tie wrapped to the side to keep the substrate bags submerged.

3rd layer all open at the top. Pasteurizing for 12 hours

The substrate bags can touch the metal base as the base is also 140F, so the whole bag is surrounded by 140F. If you heat the pan with fire you have to watch out for the base temperature. The bag might melt. (Normal PE plastic melts aroud 70C). Also be careful with the underground. If the surface is made of plastic it might also melt. Large cooking pans like the one above need 2 sous-vide as one cannot maintain temperature.

An effective mushroom substrate pasteurization will eradicate harmful bacteria, nematodes, insects and fungi and leave the good microbes alive (the thermophilic bacteria). It is the best substrate for your psilocybe mycelium. Food pasteurization needs higher temperatures because you want to kill more stuff.

Cow manure from India have for example the following beneficial bacteria that live together with the mycelium of Psilocybe Orissa:

Actinobacillus lingieresii Brahnamella Pasturella haemolytica Streptococcus cremoris
Agrobacterium Erwinia herbicola Pasteurella ureae Streptococcus Haemolytica
Bordetella Microcossus    

All are thermophilic and to be more specific all belonging to the Actinobacillus. (bacteria occurring as parasites or pathogens in mammals, birds, and reptiles that are nonmotile and non-spore-forming)

These Actinobacillus will survive sun drying (hence you want sun dried manure) and survive pasteurization at 60C (140F).

If the HM-CVG never rises much above 140° F there is minimum effect on the Actinobacillus and these will convert ammonia in the manure, so you will have no bad smell !!!

In other words, do not use the suggestion of 160 or 170F that you find on some forums as it kills the ammonia converting bacteria.

The professional mushroom farmers slowly decrease the temperature after the 6 hours, and slowly I mean 5F per day.
The process of going through this temperature range will produce the most protein or the maximum amount of food for the mushroom. This is a bit of an overkill if you ask me.

Best is to bring the manure at field capacity, leave it in the open air for 24 hours and then put in a double ovenbag and pasteurize under water for 6 hours in 140F water. Make sure the manure is not compressed inside the ovenbag but is airy so the good bacteria that use oxygen will proliferate. After pasteurization put it in a monotub and let it slowly cool down.

The longer the microbes in the HM-CVG remain in the range of 115 - 140F with all the critical growth requirements available the faster the ammonia will be converted.

Another advantage of the thermophilic bacteria is that they keep the manure warm, which in turn keep the surface air moist and increase the humidity inside the monotub.

-- -- --

Make sure the HM-CVG never rises much above 140° F and is not too wet so there is minimum effect on the good microbes that convert ammonia. And trust me the smell can be horrific if you kill the ammonia converting bacteria !

Also make sure from the beginning of mixing the substrate with spawn, that the upper air holes are open for oxygen. This again for the thermophilic actinobacillus that convert ammonia and produce food for the mycelium. If you use the CVG you need less air exchange. But with the HM-CVG do not open the lower air exchange holes as that will reduce the CO2 in the monotub and that will slow down or stall the monotub.

I have tried everything to make the HM-CVG workable after sterilization, but all with minimal success. So save yourself the trouble and only sterilize the CVG and pasteurize the Horse manure.

Below an example of sterilised HM-CVG versus pasteurized.

HM-CVG sterilised, 7 days after inoculation (without plastic bag so you can see the growth). As you can see, not good.
The manure is compressed, which makes it more difficult for the mycelium to spread. The manure does not produce heat so less humidity and everything is super sensitive to contamination.

14 days later same monotub (mycelium did start to grow but much later and green mold (Trichoderma harzianum, T. viride, T. koningii) mixed with the mycelium, eating the mycelium plus lipstick mold on the bottom which is a type of candida).
Yes I could have sealed the monotub air tight and use the microppose patches for fresh air exchange but as you will see below, pasteurizing is a lot easier.

HM-CVG Pasteurized @ 140F / 60C (without plastic bag so you can see the growth). Same conditions, same time, same substrate, be it a bit more, same single strain fully colonized grain jar like the other sterilised one above 7 days after inoculation. Really fast growth healthy looking mycelium and no smell - well actually smells mushroomy :) Also humidity very good and the substrate was totally immune to contaminations - the box was left 10 % open in a living room full of potential danger. Even after 2 month no contamination !

In other words Psilocybe cubensis not only loves horse manure, it actually grows in symbiosis with the bacteria inside of it, helping and protecting each other.

Mycelium also has the ability to create enzymes to fight off harmful bacteria and viruses just like us humans and need the good bacteria to stay healthy.

This is officially called Fungal-bacterial endosymbiosis and is a very big subject. Some examples of the endosymbiosis below:

Fungal-bacterial endosymbiosis (this is a generalization, not specific to cubensis)

Bacillus subtilis, which is another example, is one of the best bacteria for Fungal-bacterial endosymbiosis as it survives pasteurization and works together with the mycelium, giving the mushrooms vitamin B1 Thiamine that in turn helps the mycelium go into hyphal knot stage.

--

If you do not like to go the extra mile and want to go really easy, just use coir. Basic formula is the weight of the coir x 5 and that will be how much water you need. Easy with good smell and good results because coco coir is already sterile and works anti-bacterial. Yes the horse manure is better and mycelium growth faster but the idea that you consume mushrooms that grow on poo, well...

That being said, the best spiritual mushroom in this category, the Panaeolus Cyanescens, will not even grow on coco coir and need grass and manure that is properly pasteurized as it needs the beneficial microbes for symbiosis

MIX

10 Days

Ratio 2:1 (2 parts substrate, 1 part spawn)

Jars or grain bags should be 100% white and check the smell of the jars before using (should be shroomy and not sweet or sour)

Agar plate transfer in grains - DURAN 2 liter bottles with GLS80 lit.

Liquid culture transfer into grains - DURAN 2 liter bottle with ePTFE membrane.

The 2 liter bottles filled 50% are the best as more air inside and shaking is a lot easier.

Check the smell of the grain - should smell like mushrooms (not sweet, not sour, not plastic not like ammonia or anything else)

Mix substrate (HM-CCVG or CVG) with Spawn. Fill up to 10 cm thickness. 2 liters substrate in measuring cup for every 1 liter spawn jar.

This is a 1 by 1.5 fill, meaning 1 part grain spawn, 1.5 part substrate. Normally 1:2 is better.

After mixing, top it off with a substrate layer so it looks black on top and try to make it as level as possible.

Same monotub as above but with a top layer of pure sterilised coco coir. Why a top layer, well because it protects the mycelium against harmful bacteria from the air.

Spray top with water. Best is to use sterile water (just boil water for 15 minutes and let it cool down). This to imitate a recent rainfall.

At this stage the contamination risk is much lower and you do not have to be as careful as the steps prior to this, unless you use sterilized the horse manure, then be really careful.

Leave the top air holes open (filled with polyfill or other breathable material that blocks water). If you do not have vent holes just make sure you do not seal the monotub

 

Below a sample of stacking 120 liter monotubs with a LED strip with timer on top and humidity and temperature sensor inside. Here you see the microppose covers for fresh air. The top ones are on the left and right side. The big middle one will be sealed for CO2 buildup (CO2 is heavier then air so remains low). The black line denotes 10cm thickness for the fill. Do not use Micropore tape to cover the holes.

You want more then 5000 PPM CO2 buildup near the surface. Max 40.000 PPM CO2.

Stacking

Keep in mind that the monotub temperature is always 1 to 2 degrees warmer then the outside air temperature.
If you compacted the substrate mixture or used less or not fully colonized grains or use a lower temperature it might take over a month to fully colonize.
there are stories of people waiting 3 month until full colonization.

Climate

In nature manure is warmer than the surrounding air and is very moist. These 2 factors create a humidity of over 90% near the surface - hence you see steam coming from the manure in the morning.
This is the climate you want to reproduce.

Why is manure warmer then the surroundings?

This is because the Thermophilic Actinobacillus (the good bacteria) are composting the manure into heat. This is also why you do not want to pasteurize hotter then 60C or 140F, so you keep them alive.
If you use CVG you might want to add some coffee grind to it as this adds nitrogen that helps these bacteria to grow - unless you really want to go 100% sterile.

Here you see that inside the monotub the Thermophilic bacteria AND the mycelium are heating up the substrate by 2 degrees C compared to the outside temperature - in turn create higher humidity inside the monotub (between 70 and 80% humidify)

To create higher humidity you can do 4 things, easiest first:

1. make the vents (air holes) smaller

2. Use a reptile fog generator (the ultrasonic ones)

3. make the substrate warmer (like using a heated propagator)

4. let cool air in while maintaining constant warm substrate temperature

The best method I have tested is a combination of all 4, heated propagator with a fog generator and letting in more fresh air but holding the moisture in longer by using computer fans to suck air out so fresh air comes in via filters and the fog generator brings the humidity back up to 90% and above.

Airflow inside a monotub without fan

You want about 600PPM CO2 level near the surface. Basically outside air is 400PPM so if you use a room for your mushrooms it already has a higher level of CO2, near the 500PPM. So a fan with a fog generator will work nicely.

If you do not fan the monotub twice a day or use a computer fan to suck out the air every 2 hours or so, the airflow will go from the lower to the higher openings, see image above. This is due to warm temperatures rising and sucking fresh air in from below. So even though CO2 is heavier then air and will normally sink, due to the mycelium warming up the substrate, the CO2 actually exits from the top openings as long as the bottom openings are open. If the bottom holes are closed, the CO2 will increase and you will slow or block the fruiting condition, but high CO2 is needed for colonization. So during colonization only leave to top air holes open and when all is ready for fruiting conditions, open the bottom holes.

Another effect if you do not fan the monotub is that the monotub will form pins mostly in the direction of the airflow. If you look at the picture above you see most pins parallel to the airflow and none in the middle. This will not be the case if you fan.

Colonization 2.5% of the lid surface area should be open. In other words the 2 top openings

In fruiting condition 5% (all 4 open)

Diameter of each air opening in cm or inches (you will need 4 of these in total)

L x W = Length times Width of the monotub cover (Lid)

For the 120 liter monotub (80cm x 50cm lid) it comes to 7.5cm diameter openings. If you live in a desert (really dry) the formula is not 2 times but 1 and if you live in the tropics the 2 should be 3 times (just use 4 holes in the top instead of 2).

If you use a fan you should also mist. Make sure you use a micro mister or a reptile ultrasonic mister. You do not want to spray directly onto the mycelium but it should mist down.

ONLY MIST WHEN DRY AND NO PINS VISIBLE !

When pins form, stop misting.

The cover should preferably look like the roof of a house or dome, because this way the condensation droplets will not fall onto the substrate but actually leak to the sides. Here the plastic bag comes in useful as the droplets will not form pools of water inside the monotub. Drops onto the hyphal knots will actually destroy the knots in the early phase and in turn give less harvest. But this is not practical if you stack but if you can, this is optimal.

Dome setup error test

For smaller to medium size monotubs you can use a dome setup to increase the space for mushrooms to grow and use polyfill to filter the air.
A dome setup is easy as you just use the same monotup on top of another. Just make 2 vents on the long side in one tub and on the short side in the other tub.

Be careful for a couple of common mistakes though. Like above, the right one has too much polyfill and the vents are on top of each other. You want one tub the vents on the side and on the other tub the vents on the front and back side. Also in between the 2 tubs it needs to be sealed (like using a tape), otherwise contamination can easily enter through the slit.
The left setup is also wrong because the mushrooms will dry out as condensation will not return plus bad fresh air exchange.

Set outside temperature around 23 C and 90%+ humidity inside the monotub. This means you cover the lower air holes with a beer coaster or similar so limited airflow out of the monotub. The mycelium will actually create a highly humid environment by itself at a certain moment, so no need for humidifiers, but these extra steps might help.

When the top is white (can be around day 6 or later) you can turn on the 6000K LED lights on top of the monotub (not inside, it will get too warm plus led lights do not like all the humidity). Put the lights on a timer, 14 hours on and 10 hours off. For the cubensis, red in the beginning and blue when the pins are there seem to be the best but the daylight LED are even better. 12 hours on and 12 hours off is not optimum test have shown. Somewhere around 14 hours on is the best.

8 days inside monotub, check for white hyphal knots and check for dryness. If too dry, micro mist or turn on the humidifier.

The monotub will grow much slower if you compacted the substrate. The more air inside the substrate the faster the mycelium will grow.

Guttation

Guttation is the exudation of drops of xylem sap (water that is sucked up from the mycelial network and going up) on the surface of a monotub or on the mushrooms themselves. Guttation is not to be confused with dew, which condenses from the atmosphere onto the mushroom / mycelium surface. Guttation generally happens during the night time and is a perfectly healthy phenomena.

Contamination in Monotub

Lipstick Mold - Sporendonema purpurescens (Geotrichum candidium)

This fungus looks like Guttation but is not and colonizes compost or casing. As spores mature, the color of the mold changes from white to pink, to cherry red, and finally to dull orange. It is slow growing. Spores spread in air, during watering, and on pickers. The lipstick mold utilizes certain fats in the compost. It is an uncommon problem. Do not fruit any monotub that has color as color means mold spores and will create havoc in your growing room. Also some molds are not healthy and you do not want to be a test pilot.

Cobweb mold or Dactylium Mildew - (Hypomyces sp.)

A cotton like structure that sometimes looks like a spider web normally starting from one place on top of the substrate before the mycelium is visible and is thinner, bit more grey and growing much faster then mycelium. If this happens there is no problem. It normally indicates it is a bit too moist and not enough fresh air. Mycelium will eventually take over, so let it be.

There are many other types of mold that are not ok and means you should start over, generally growing faster then mycelium and having different colors: Green Mold, cinnamon mold, black whisker mold, pink mold, red mold, Olive Green Mold, pin molds, Plaster Molds and Flour Molds.

There are 2 main reasons why things go wrong if your work has been done correctly and sterile:

Main reason is not enough FAE (Fresh Air Exchange) - so you need to leave the top air holes open during colonization and the other main reason it too wet. You cannot have pools of water on top of your casing layer.

Fungus gnats and other insects...
These are small flies that infest the substrate and their larvae feed and destroy the mycelium. They are slow moving and easy to kill (just put a bucket with water and lemon juice next to it, but the larvae are difficult to get rid of. Best is to leave the monotub closed with polyfill so these buggers do not enter the monotub.

Casing Layer

In basic terms, a casing layer is a layer of moist material – either organic, inorganic, or a combination of the two – placed on top of a colonized substrate prior to fruiting. The main function of the casing layer is to aid with moisture retention in the substrate and inrease the relative humidity near the surface. Hence you use material that can hold a lot of moisture but will not be consumed. This will create more flushes and more sturdy mushrooms.

So when the top substrate is mostly covered with mycelium, it is time to add a casing layer.

Use 50% Peat moss and 50% vermiculite as you do not want mycelium to consume the layer, but rather use it to get extra moisture from its surrounding during fruiting. Pasteurise the casing so the good bacteria remain alive. This will help pinning and protect the mycelium from green mold.

Mold will grow on almost any organic source, if the humidity is sufficiently high.. The molds which grow on peat moss however are saprophytic (Alternaria spp. Aspergillus spp. Penicillium spp.), meaning that they feed on dead plant material and are not pathogenic or harmful to plants or people. But you also pasteurized the casing so no mold should be present.

A great helpful bug present in peat moss is Bacillus subtilis that is working together with the mycelium giving it Vtamin B1 that in turn signals the mycelium to start pinning.

Also at this stage with the living bacteria, even sweat inside a monotub will not do any harm.

Pure peat with vermiculite (50/50) is very acid. PH 5

Casing layer must be PH 7.2 (+/- 0.5)

In this case 5 liters casing (field capacity) needed about 20 full table spoons of Lime (Kalk) to reach PH 7.2 (Normally that is about 2%)

Psilocybe Cubensis will also fruit without a casing layer but you get less flushes and less optimal fruit.
Panaeolus species need a casing, otherwise no fruiting.

Fruiting

Wait 24 hours after cassing and start fruiting condition.

Turn room temperature down to 22 C and open up the lower air holes. It has been found that if you turn down the temperature at this point and give it light, the mushrooms will have a higher quality, denser fruit bodies. Also turn on the LED lights on a timer (14 hours on and 10 hours off) as this helps pinning and will give direction to the fruit bodies. If you leave the CO2 in there the mushrooms will grow taller but get fuzzy feet and might stall due to suffocation.

Mist the substrate 2 times a day only if the surface is dry (with casing this is more difficult to judge), but do not spray directly on top of the mycelium but rather above it or use a micro mister so that the small droplets will form on top of the mycelium.
Even better is using a ultrasonic mister on a timer with a computer fan. Mist and fan for 1 minute and let the fan continue for another 30 seconds to pull in fresh air.

You fan for fresh air exchange that will help evaporate the droplets and create hyphae knots but make sure some of the droplets remain. Easy method is to fan twice a day by using the cover of the monotub and wave over the substrate a couple of times.

The white hyphal knots should show up within a couple of days after FC. This only happens if the whole substrate is almost 100% colonized. Pins form when there is no more room for expansion.

See video below.

 

Another method is using the microppose 3 inch adhesive Tub Filters For Mushroom Cultivation, stick one on the inside of the tub and place a 80mm computer fan on the outside.
Or like the image below put a computer fan inside a tube. Make sure the fan is as close to the casing layer as possible as you want the fog to be sucked over the substrate, into the tube and out of the monotub.

Fan 1 minute on then

Fan + Fogger 1min on then
(so use CMFR-66 to delay 1 minute fogger and put fan on a 2 minute total)

Fogger 1 minute on then

Everything 15 minutes off.

You need a "CMFR-66 time relay" for this.

The idea is to evaporate the small water droplets on the surface first by using the fan for 1 minute. Evaporation helkps create premordia (pins)
Then remoisturise the monotub.
Best is to stop everything before the mushrooms start to release spores , otherwise your filters will clog up with spores and the mushroom caps get too moist.

This is how to keep the power supply always on. Just use a paperclip.

This way if you put the power supply on a timer it will work.

 

CMFR-66

This is connected to the BN-LINK and the output is connected to the computer power supply. It is set for a 2 minute delay to be turned on.

So what happens is when the BN-LINK is turned on, the CMFR-66 delays it by 2 minutes and than turns on for 3 minutes (because the BN-LINK is set for 5 minutes on)

BN-Link

The LED lights are on a 14 hour on and 10 hours off cycle.

The "BN-LINK Short Period Repeat Cycle Intermittent Timer" is for the computer powersupply and fogger. It is set for 5 minutes on and 10 minutes off.

Microppose filter on the inside of monotub with computer fan and mister outside

The fogger is connected to another BN-Link that is set for 3 minutes on, 60 minutes off.

What happens is when the first BN-LINK turns on for 5 minutes, the second BN-LINK turns on for 3 minutes. The 60 minutes off only means it will not turn on again within the first BN-LINK 10 minute timer resets.

The end result is 2 minutes fog on, 1 minute fog with fan on and then 2 minutes only fan and then 5 minutes all off.

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The 3 different stages

Colonization stage

Mycelium in CVG growing out of the top substrate. This is the right time to add the casing layer.
Water droplets evaporating are needed to form hyphal knots on top of the casing layer (hyphal knot = a concentration of mycelium commonly seen as white balls that form in a network of mycelia)

Click on the image to enlarge to see the water droplets... that turn into hyphal knots over time...

This is the stage where you turn on your LED light on a timer (14 hours on and 10 hours off) or use daylight and open up the CO2 holes at the bottom of the monotub and fill it with polyfill or microppose. This is about 24 hours after casing.

When the mycelium cannot find any new areas to grow into for finding food it will start to form Hyphae. Actually the same counts for the ivy plant and many others. When the food source becomes scarce it will flower and produce berries.

So pinning is triggered by:

- Fresh air

- Evaporation

- Bacteria (optional - better with)

- Light (optional - better with)

Hyphal knot stage

Mycelium in horse manure growing out of the casing layer. Here the hyphal knots are visible as small white balls that later turn into primordia (baby pins that grow into mushrooms)

- Click on image to enlarge. -

Primordia stage

Pre-Pins (otherwise called primordia) are elongated hyphal knots that have a brown dot on them that eventually grow into pins and mushroom.

- Click on image to enlarge. -

Pinning or pin stage

Pins are baby mushrooms and they grow fast. Make sure there is no CO2 near the surface by using FAE (fresh air exchange) using microppose (polyfill) holes or fanning.

- Click on image to enlarge. -

Ready for harvest.

This is a monotub with casing and very good fresh air exchange (be it on the dry side as you can see on the casing color).
You can also tell if a monotub has a lot of fresh air as the mushrooms are shorter like this one. The dark caps in the middle are spores.
What happens is that the mushrooms are actually warmer than the outside air, so when the spores are released they actually climb up (called convection) and the cap radiates heat as well creating a local low pressure above the cap sucking the spores on top of the cap.

Problems

Side pins

In this setup it also becomes obvious why you should use a plastic bag. The liner or plastic bag not only collects condensation but also prevents side pins from forming everywhere and if you take a look at the bottom right and left there is a whole group of side pins growing there.

Poor fresh air exchange

Pin stall

Pins will stall and even shrink a little and their caps will turn darker and become dry and leathery when there is poor fresh air exchange.

bacterial blotch - Psudomonas

Basically any discoloration of the caps other then spores. This is mostly caused when you spray directly on top of the caps.

CO2 suffocation (Bad FAE - Fresh Air Exchange). Stem produces hairs, pins stall (turn dark and do not grow anymore).

Mushroom stems will turn hairy and hollow if too much CO2 and not enough fresh air. Mushrooms want to grow out and above the CO2 level.

Dry Bubble - verticillium infection

Harvest

On average it will take around 2 month from starting point to finish if you follow this method exactly.

So when is the best time to harvest. Best is if the veil is still on or just broken - they are more potent this way and cleaner as the spores have not been released.

Good time to harvest (the black area on the stem are spores)

Many proclaim that twisting the mushrooms and pulling them out is the best, sometimes even taking some of the substrate along with it, but I tend to say just use a sterile pair of scissors and cut them near the base. Potency wise there is no difference if you dry them immediately and you dry them without horse manure ! Also cutting them you have better flushes afterwards and more protection against contamination in the substrate, especially when using a casing layer..

A day too late with harvest - spores turning mushroom caps black

The black caps are actually spores sitting on top of the caps. You can easily wipe them off but that is not needed.
The main problem with overgrowth is FAE. Often the mushrooms grown near the air vents and when things get crowded, these vents will block. Problem with blocked vents are over-humidity seen as pools of water on the substrate and CO2 suffocation - seen on the stem of the mushrooms - they get hairy.

Fresh mushrooms and truffles can be safely stored in the fridge at 2-4 degrees Celsius for up to a month.

Drying

In many countries, including the Netherlands, Jamaica, Costa Rica, mushrooms are legal but drying psylocybe mushrooms is not allowed.

Dry the mushrooms in cold or warm air ?

There are many conflicting stories on the internet. Let me make this clear.

Some of the arguments you find say go cold. (this is from Fastfred from shroomery.org)

"Actives decompose fairly rapidly until the drying is nearly complete. Any water left allows the enzymatic dephosphorylation of psilocybin to psilocin. The psilocin then oxidizes fairly easily. Any time you raise the temp by 10C (~18f) you are doubling the rate of reaction. (See the Arrhenius equation) If you use even 90f to dehydrate you are doubling the rate of decomposition compared to drying at room temp. So if you think that raising the temp 18F is going to dry them twice as fast then go for it. Otherwise you're wasting the goodies. I don't personally believe this is the case, so I don't think heat drying is a good idea unless you have no other option due to high humidity. Why use heat in the first place? Well, heating the air lowers the RH, allowing this relatively drier air to more effectively absorb moisture from the material. But the main reason is that dehydrator makers are usually too cheap to put a fan in their units, so they need the rising hot air for airflow. Unless you live in a high humidity area, fan drying is a far better way to go. It dries them faster, especially at first when they are wettest and most vulnerable. If you are going to use heat, then use it at the end to get them fully dry. It's pretty easy, and a lot cheaper to build a proper fan drying setup. You'll get better fruits in less time than using heat. I'm in a fairly low humidity area so it works excellent to fan dry, then store in a container with desiccant. The fan drying gets them 98% cracker dry (very slight bending before snapping), then the desiccant storage gets them ultra dry (break apart if you look at them wrong). There is no magic number for heat damage of actives. Psilocin can be vaporized in a gas chromatograph and survive. (Psilocybin gets dephosphorylated in the process and shows up as psilocin) The actives are not stable at any temp and will decompose and oxidize steadily at a temperature related rate. ANY heat speeds this up and any cooling will slow this down. IMHO it's dumb to use any heat unless you absolutely had to. If I had a bunch to dry and plenty of fridge space I'd make a fan drying setup in the fridge."

It is true as you see this reaction happening really fast if you cut or slice the mushroom open. They turn blue. But it is the enzyme PsiP and PsiL that do this.

So knowing this you do not want the PsiP and PsiL enzymes to stay alive when drying. Enzymes are proteins and they start to break down above 104F or 40C. So when drying at high temperatures there are no more PsiP left so the psilocybin stays intact. The enzymes in your body will do the magic to create psilocin from the dried psilocybin.
Also you do not want oxygen to enter the inside of the mushroom when drying so keep the mushroom whole.

This means it is best to use 50C - 60C air dryer for 24 hours to disable the enzymes but leave the mushroom intact so the air does not oxidize the inside. Above 70C it is said you will loose potency.

Do this for 24 hours so you can easily break the stem (in other words the stem does not bend but actually snaps)

Stöckli Ovens Dehydrator

If you did the twist and pull method, once they are dry, clean the mushrooms from any substrate left on them. It is much easier to clean when they are dry then to try and clean them before drying. Also you prevent oxygen from entering the mushroom in the drying process.

But better is to cut them because the moment they are dry but still warm, you put them in a vaccume sealed bag with desiccants and seal the bag. When the shrooms cool, they do not suck in extra air / moisture, so the potency stays higher this way.

After all the PsiP enzymes are gone and everything is dry, you can grind them into powder. But better keep them whole in the bag and when ready for ceremonial use, grind them.

If you grind them into powder after drying and planning on vacuum sealing the package, the best desiccants are the ones that also absorb oxygen like the ATCO OXYGEN ABSORBER DSP35

Powdered dried shrooms have problems with oxygen-centered radicals (OH and HO2 - moisture) and UV radiation. Putting it in a plastic bag protects them from UV radiation as plastic blocks UV and vacuum sealing with a ATCO Oxygen absorber means you prevent oxygen-centered radicals from decreasing potency.

When an oxygen absorber is removed from its protective packaging, the moisture in the surrounding atmosphere begins to permeate into the iron particles inside of the absorber sachet. Moisture activates the iron, and it oxidizes to form iron oxide. Typically, there must be at least 65% relative humidity in the surrounding atmosphere before the rusting process can begin. To assist in the process of oxidation, sodium chloride is added to the mixture, acting as a catalyst or activator, causing the iron powder to be able to oxidize even with relatively low humidity. As oxygen is consumed to form iron oxide the level of oxygen in the surrounding atmosphere is reduced. Absorber technology of this type may reduce the oxygen level in the surrounding atmosphere to below 0.01%.

Storing them this way the shrooms will last you at least 10 years with minimal loss in potency if any.

Potency

The psilocin / psilocybin contents in Psilocybe cubensis is 0.14-0.42% / 0.37-1.30% in the whole mushroom.

Fresh and dry psilocybe mushrooms are generally considered equivalent using the 10% dry to wet ratio. If you dry a mushroom you will loose 90% of its weight (mostly water).
The psilocin contained in the fresh mushrooms will also disappear in the drying process. About 66% is psilocybe and 33% psilocin, so 33% is lost in potency by drying process and another 10 to 20% psilocybin that turns into psilocin during drying and also disappears. So the equivalent would be more accurate 7% of fresh weight.

Also keep in mind that Psilocybin content varies from mushroom to mushroom, from flush to flush, and between strains.

Psilocybin content changes in the different stages of development of a mushroom also. Miniature pinheads that just didn’t grow bigger are more potent than larger mushrooms per gram of weight.

Caps and stems contain different levels of psilocybin.The psilocybin contents in the golden teachert are in the range of 0.37–1.30% in the whole mushroom, with 0.44–1.35% in the cap and 0.05–1.27% in the stem. This means that the caps are, on average, slightly more potent than the stems, and that there are tiny bits of stems that contain almost no psilocybin. In order to equalize, it is best to grind your mushrooms to a fine powder - see below.

Dosage

To microdose mushrooms, you need approximately one tenth of a standard dose. This means you will use somewhere between 0.1 – 0.3 grams of dried powdered psilocybin mushrooms per dose.

Standard dose is 3.5 grams dried

A heroic dose is 5 grams or more.

Health and Safety - psilocybe

Golden teacher Retreat

MycoMeditation

Multiple Flushes

First flush harvest (the green blue color is not green mold but an enzyme effect on psilocin - basically indication the potency of the mushroom)

The mushrooms have an enzyme called PsiP that when a mushroom is cut or bruised, the phosphorus portion of the psilocybin molecule is cut off and turns into psilocin, the psychactive molecule.
Another enzyme produced by the mushroom is called PsiL that makes the psilocin stick to other psilocin molecules and some loose their hydrogen molecule during the process and that makes it turn blue.

In the human body we make a similar enzyme to PsiP, converting psilocybin into the psycoactive psilocin without the blueing effect.

Second flush forming after 1 week (as you can see, the cut mushrooms are overgrown with fresh mycelium)

Third flush
(1 day late for harvesting as the black spores are all over the place, turning the caps black. This is due to the mushrooms and substrate being warmer then the air and that creates convection (vertical air movement) and the spores go up and fall back onto the caps. Interesting is that the mushrooms in the third flush are bigger)

So there are 2 techniques for multiple flushes.

The first technique I like best, because it is simple and fool proof. That is to cut them and close the monotub afterwards. This way the new pins will form on substrate that previously had no pins and you do not have to take the whole thing into a bathtub. Best if using a casing layer because mushrooms receive extra moisture from the casing like the treees that get it from the ground. Also a casing layer prevents evaporation, keeping the moisture where you want it. Downside to cutting the mushrooms is that the whole thing is more suseptable to contamination, as you can see in the image above.
The cut mushrooms will be covered with fresh mycelium protecting itself from bacteria and mold but also regulating the moisture levels inside the monotub. When you cut them, the cut location is very sensitive to contamination and often ends up with green mold on them. So be sterile when cutting them.

The second method is soaking the cake (cake is the name of the substrate when fully colonized). You can fill up your bath tub or similar with water. At this stage, risk of contamination is low, however be wary of Fungus gnats and mold in the bathroom.

Soak 12 hours fully submerged (use a weight to keep the substrate submerged)

Soaking

When finished,drain the water and make sure you let fresh water run over the cake before taking it out. This to reduce the risk of bacterial contamination.

The deterioration in quality during the last flush is often surprising. In many cases this is caused by mushrooms maturing instead of actually growing. This indicated not enough food.
Moisture management is an important aspect here. Do not add new substrate or honey or any other food to the existing substrate. People have tried and none have succeeded.

The only way to influence the amount of flushes is the depth of your substrate. The thicker the more flushes.

Printing

Two caps on sterile aluminium foil

Fresh spore print on aluminium (water droplets still visible)

Only 24 hours are needed to complete a print.

Easiest is to buy the hair perm (salon) aluminum foil. Those are 10 cm thick so less cutting. Normally these are used to color hair.

You place the aluminium foil in a small or large flat clear storage box. You do not want too much air in there so a flat box is better. Place the mushroom caps on the edge for 24 hours and place a drop of water on each cap. After 24 hours, remove the caps and you have the print. Now you have to dry the print. This is done in a still air box in a warm dry room for 6 hours.

Then you vacuum seal the spore print and store them in a fridge at 5 C (or 40F). Make sure you do not freeze the spores, they will die. If you have been sterile and they are dry, the spores will last 10 years.

Spore swaps:

Swap the mushrooms with a sterile Cotton Tip Applicator and vacuum seal them. Option is to dry them above an open oven - make sure the air is not too hot (<40C) The oven air is sterile.

Making spore vials:

A simple printing method which involves filling some small containers with a small amount sterile distilled water, then flipping the lids over and placing a mushroom cap on it to drop spores, then flip the lid back over onto the container and shake it. Instant dark spore solution with very little contamination possibility. Use about 1cc/ml or less per 10-12cc/ml syringe (1cc spore solution 9-11cc water)

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Useful links:

https://vikingspore.co.uk/

https://www.beaconhillmushrooms.co.uk/

https://www.mushrooms.com