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@ -11,26 +11,24 @@ Contamination occurs when other microorganisms take advantage of the gratuity yo
There is always a risk that bacteria and mold will invade, competing with your precious mushroom makers.
Many guides do everything they can to reduce this risk.
My approach gives you methods that are still effective but easy and cheap to recover from contaminated spawn.
My approach gives you methods that are still effective but easy and cheap to recover from contamination.
# Do It Thrice
As a result of the above hubris we triple our efforts.
- Make at least two agar slants or liquid cultures for each sample we wish to test.
- Make at least two liquid cultures for each sample we wish to test.
- Make at least two colonization jars for each sample we wish to colonize.
- Make one extra of the above as a control for testing your sanitation.
- Make one extra liquid culture and colonization jar as a control for testing your sanitation.
It is OK.
Bird seed, coconut husk, and sugar water are inexpensive.
All other equipment can be reused.
Saving a control will help you test for contamination introduced through your technique.
If all fares well you can always end up using it.
Spawn medium refers to any nutrient rich growing surface, such as seed grain or culture media.
`Spawn medium` refers to any nutrient rich growing surface, such as seed grain or simple sugar.
# Finding Samples
@ -53,9 +51,9 @@ Eventually you will want to make your own cultures from your harvested mushrooms
There are three general stages to cultivating mycelium.
1. [Culturing](#culturing), [wiki](https://en.wikipedia.org/wiki/Microbiological_culture)
1. [Culturing](#mycelium-cultures), [wiki](https://en.wikipedia.org/wiki/Microbiological_culture)
- Using simple sugar and agar as a surface to test sample mycelium for viability and contamination.
- Using simple sugar solution to test a sample of mycelium for viability and contamination.
2. [Colonization](#colonization), [wiki](https://en.wikipedia.org/wiki/Colony_(biology)#Microbial_colonies)
@ -108,25 +106,23 @@ Slow or hold your breath when inoculating spawn medium to avoid shaking.
Remember that mistakes happen and they look pretty cool when they do.
# Culturing
# Mycelium Cultures
Any samples of mycelium are potentially contaminated.
Even using a microscope and visually checking the entire sample is tedious, error prone, and impractical.
A small culture will allow you to grow a test batch of your sample without for a jar of [grain spawn](#grain-spawn) to show signs of contamination.
A small culture will allow you to grow a test batch of your sample without for a jar of [grain spawn](#grain-spawn-and-substrate) to show signs of contamination.
Agar gives mycelium and contamination a surface to grow in large enough colonies to view with the naked eye.
A week of growth is usually long enough to produce colonies which can be identified and transferred to [liquid cultures](#liquid-cultures).
While petri dishes are part of the usual trappings of cultivating microorganisms, they are fairly high maintenance and better suited for a laboratory environment.
[Agar slants](#agar-slants) provide the same functionality without the need to seal and re-seal with parafilm.
The preferred [culture medium](#culture-media) is [liquid cultures](#liquid-cultures).
[Liquid cultures](#liquid-cultures) provide a similar micro climate as [agar slants](#agar-slants) with the added benefit of being a much easier for inoculating [grain spawn](#grain-spawn).
The preferred culture medium is [liquid cultures](#liquid-cultures).
[Liquid cultures](#liquid-cultures) provide a similar micro climate as agar slants with the added benefit of being a much easier for inoculating [grain spawn](#grain-spawn-and-substrate).
The disadvantage of liquid culture is they are a little difficult to identify contaminations.
You may use [agar slants](#agar-slants) to isolate mycelium from contamination before transferring to [liquid cultures](#liquid-cultures).
Skipping testing on [agar slants](#agar-slants) works well enough as long as you give [liquid cultures](#liquid-cultures) extra time for bacteria to become visible while suspended in water.
You may use agar slants to isolate mycelium from contamination before transferring to [liquid cultures](#liquid-cultures).
This guide does not cover their use.
Skipping testing on agar slants works well enough as long as you give [liquid cultures](#liquid-cultures) extra time for bacteria to become visible while suspended in water.
# Culture Media
# Liquid Cultures
A simple sugar like light corn syrup is dissolved in distilled water to provide mycelium calories for reproduction.
- [ ] food scale, grams
@ -138,7 +134,7 @@ Skipping testing on [agar slants](#agar-slants) works well enough as long as you
- [ ] light corn syrup
- [ ] diammonium phosphate with urea (optional)
- [ ] marbles or magnetic stirring rods
- [ ] aluminum foil
@ -148,11 +144,6 @@ Imperial | Metric
-------- | ------
16 oz | 450 ml
## Liquid Cultures
Mixing liquid culture is similar to [agar slants](#agar-slants) without needing to cool the test tubes in any particular position.
A simple sugar like light corn syrup is dissolved in distilled water to provide mycelium calories for reproduction.
Ingredient | Mason Jar | Ratio
---------- | --------- | -----
water | 300 ml | 100 ml
@ -166,64 +157,22 @@ simple sugar | 15 g | 5 g
2. Microwave with a safe cover until boiling, about 1 minute.
3. Add 15 grams of light corn syrup to warm water and stir for 1 minute with a sanitary utensil.
3. Add 15 grams of light corn syrup to warm water.
- Optionally add no more than 1 gram of diammonium phosphate (dap) with urea for yeast nutrients.
4. Cover jar opening with aluminum foil and [pressure cook](#autoclave) for 25 minutes at 70 kPa (10 psi).
4. Insert a marble or magnetic stirring rod for stirring the solution once inncoulated.
5. Cover jar opening with aluminum foil and [pressure cook](#autoclave) for 25 minutes at 70 kPa (10 psi).
Allow to cool to room temperature.
5. Replace aluminum foil with mason jar lid, screwing closed tightly to prevent spilling.
6. Replace aluminum foil with mason jar lid, screwing closed tightly to prevent spilling.
6. Store in a dark cool place until inoculation.
7. Store in a dark cool place until inoculation.
7. Daily shake the mason jars daily to release carbon dioxide and oxygenate the water.
8. Daily shake the mason jars daily to release carbon dioxide and oxygenate the water.
Unscrew the cap and slightly lift the lid to allow the carbon dioxide to escape.
## Agar Slants
- [ ] agar
- [ ] 30 to 50 ml test tubes with screw cap, [autoclavable](#autoclave)
Agar agar is a plant gelatin that solidifies at room temperature, providing a physical surface that mycelium to colonize.
When mixed with a simple sugar like light corn syrup this provides mycelium calories for reproduction.
Malt Extract Agar (MEA) is available premixed fairly cheap online.
Mixing 2:3 grams ratio of light corn syrup to agar powder is good for saving money by bulk.
Scale the following recipe as needed to fill 1/3 of each test tube or 10 ml:
Ingredient | Mason Jar | Ratio
---------- | --------- | -----
water | 300 ml | 100 ml
agar | 9 g | 3 g
light corn syrup | 6 g | 2 g
0. [Sanitize Your Workspace](#sanitize-your-workspace).
1. Fill a 16 oz mason jar with 300 ml water on a food scale.
- Grams is the same as milliliters of water at [STP](https://en.wikipedia.org/wiki/Standard_conditions_for_temperature_and_pressure).
2. Microwave with a safe cover until boiling, about 1 minute.
3. Add 9 g of agar and 6 g of light corn syrup to the warm water and stir well for 1 minute with a sanitary utensil.
- Optionally add no more than 1 gram of diammonium phosphate (dap) with urea for yeast nutrients.
4. While the MEA solution is still warm, fill a sanitary syringe with the solution.
5. Fill a sanitary test tube one third (1/3) capacity and replace the screw cap.
6. Place test tubes in an empty mason jar and [pressure cook](#autoclave) for 25 minutes at 70 kPa (10 psi).
7. Carefully remove each test tube and place on a flat surface with the screw cap slightly elevated on a dish towel.
- Once cooled the agar should solidify into a slanted surface for inoculation.
8. Store in a dark cool place until inoculation.
Do not freeze.
## Autoclave
[Autoclave](https://en.wikipedia.org/wiki/Autoclave) is a generic term for pressurized steam chambers for sanitization and sterilization.
@ -240,16 +189,15 @@ Autoclavable tools are safe to bring to 120 celsius for extended durations witho
2. Insert items to be autoclave on to the steam rack.
3. Press `Steam` option and set the appropriate time.
3. Press `Steam` or `Canning` option and set the appropriate time.
4. Turn vent valve to close.
5. Once time has completed allow to passively depressurize.
Venting the valve early may be necessary for working with agar.
# Culture Inoculation
Once you autoclave your [culture media](#culture-media) and they have cooled to room temperature it is ready for mycelium.
Once you autoclave your [liquid cultures](#liquid-cultures) and they have cooled to room temperature it is ready for mycelium.
Depending on the source of the sample there are two ways to inoculate, [cloning](#cloning) from a mushroom or using [spore samples](spore-samples).
Newbies should start with [cloning](#cloning) grocery store mushrooms.
@ -264,7 +212,7 @@ Piercing the mushroom tissue with a hollow needled syringe is enough to collect
0. [Sanitize Your Workspace](#sanitize-your-workspace).
- Sanitize the outside of each [culture medium](#culture-media).
- Sanitize the outside of each [liquid culture](#liquid-cultures).
1. Cut the stem off the mushroom with a sanitary knife.
@ -272,11 +220,11 @@ Piercing the mushroom tissue with a hollow needled syringe is enough to collect
3. Plunge a hollow needle tip of the syringe into the center along the length of the stem to collect a small sample.
4. Gently push the plunger on the syringe to use air pressure to put the sample into an open [culture medium](#culture-media).
4. Gently push the plunger on the syringe to use air pressure to put the sample into an open [liquid culture](#liquid-cultures).
5. Replace the screw cap on the [culture medium](#culture-media) and store in ambient room light at room temperature.
5. Replace the screw cap on the [liquid culture](#liquid-cultures) and store in ambient room light at room temperature.
6. Daily release the pressure within the [culture medium](#culture-media) container by unscrewing the cap.
6. Daily release the pressure within the [liquid culture](#liquid-cultures) container by unscrewing the cap.
## Spore Samples
@ -290,22 +238,22 @@ Spores can then be added to a syringe of sterile water for ease in shipping.
0. [Sanitize Your Workspace](#sanitize-your-workspace).
- Sanitize the outside of each [culture medium](#culture-media).
- Sanitize the outside of each [liquid culture](#liquid-cultures).
1. Using a lighter, heat the spore syringe needle until it glows red. Allow to cool.
2. Place a single drop of water from a spore syringe on the [culture medium](#culture-media).
2. Place a single drop of water from a spore syringe on the [liquid culture](#liquid-cultures).
3. Replace the screw cap on the [culture medium](#culture-media) and store in ambient room light at room temperature.
3. Replace the screw cap on the [liquid culture](#liquid-cultures) and store in ambient room light at room temperature.
### Spore Print
An inoculation loop maybe useful to apply the spore print samples to a [culture medium](#culture-media).
An inoculation loop maybe useful to apply the spore print samples to a [liquid culture](#liquid-cultures).
Alternatively use a sanitary sharp knife or scalpel to apply the spore print samples.
0. [Sanitize Your Workspace](#sanitize-your-workspace).
- Sanitize the outside of each [culture medium](#culture-media).
- Sanitize the outside of each [liquid culture](#liquid-cultures).
1. Lightly scrape spores off a print delivery medium with a sanitary sharp.
@ -314,42 +262,21 @@ Alternatively use a sanitary sharp knife or scalpel to apply the spore print sam
3. Collect spores on the loop by gently rubbing against the loose spores.
4. Insert loop into an open [culture medium](#culture-media) and gently apply spores.
4. Insert loop into an open [liquid culture](#liquid-cultures) and gently apply spores.
5. Replace the screw cap on the [culture medium](#culture-media) and store in ambient room light at room temperature.
5. Replace the screw cap on the [liquid culture](#liquid-cultures) and store in ambient room light at room temperature.
6. Daily release the pressure within the [culture medium](#culture-media) container by unscrewing the cap.
6. Daily release the pressure within the [liquid culture](#liquid-cultures) container by unscrewing the cap.
## Wood Plugs
# Reading Liquid Cultures
Mycelium samples are often sold as colonized grain or wood plugs.
Usually these can be used to inoculate [grain spawn](#grain-spawn) directly.
Cultures are used to store these samples long-term.
[Liquid cultures](#liquid-cultures) should show growth within the first week after inoculation and should be monitored for contamination weekly.
Use sanitary tweezers to place a sample in spawn medium.
Mycelium are snowy white with grey, blue, or brown tints depending on the species.
# Reading an Agar Slant
Colors like yellow, red, green, and black usually indicate contamination.
[Agar slants](#agar-slants) should show growth within the first week after inoculation and should be monitored for contamination weekly.
Mycelium are snowy white with grey and blue tints as normal.
Any other colors and you are dealing with contamination.
Contamination is not the end of the world.
If enough mycelium growth has occurred that a sample can be collected without touching the contamination then it can be transferred to fresh [agar slants](#agar-slants) and tested again.
Otherwise, a successful agar slant colony is used to create [liquid culture](#liquid-cultures).
0. [Sanitize Your Workspace](#sanitize-your-workspace).
- Sanitize the outside of each [culture medium](#culture-media).
1. Using a lighter, heat a sharp knife or scalpel until it glows red. Allow to cool.
2. Carefully cut a small sample of the mycelium colonized agar off.
3. Insert agar sample into an open [liquid culture](#liquid-cultures) to apply spores.
4. Replace the screw cap on the [liquid culture](#liquid-cultures) and store in ambient room light at room temperature.
Dispose of the contaminated solution being mindful of any stirring rods or marbles you might have inserted.
# Colonization
@ -360,7 +287,7 @@ This guide does not address these special requirements.
I leave it up to you to research your mycelium species.
Mushroom growers have had some success with the much quicker method of inoculating store bought sterilized whole brown rice.
These are usually sold as
These are usually sold as:
> Instant Microwavable Whole Grain Brown Rice
@ -368,147 +295,113 @@ For your first time I recommend store bought sterilized whole brown rice to lear
Simply cut a small hole in a corner of the sterile container and follow the instructions for [spawn inoculation](#spawn-inoculation).
Tape the small hole closed.
You should, however, provide mycelium a variety of [grain spawn](#grain-spawn) and [substrates](#bulk-substrate) between generations of colonies so the strain does not lose it's ability to digest different sources of nutrition.
These general instructions are the same for most [grain spawn](#grain-spawn) but the most accessible source of grain spawn is `wild bird seed`.
You should, however, provide mycelium a variety of [grain spawn and substrate](#grain-spawn-and-substrate) between generations of colonies so the strain does not lose it's ability to digest different sources of nutrition.
These general instructions are the same for most [grain spawn](#grain-spawn-and-substrate) but the most accessible source of grain spawn is `wild bird seed`.
# Grain Spawn
## Grain Spawn and Substrate
Nutrient rich grain treated and sanitized to feed mycelium.
Vitamin and mineral rich substrated treated and sanitized to provide mycelium a growing surface.
- [ ] 16 oz wide mouth mason jars with lids
- [ ] wild bird seed with millet
- [ ] colander
- [ ] coconut coir
- [ ] 8 liter, approximate, bucket and lid
- [ ] aluminum foil
- [ ] electric pressure cooker, stove top, or [autoclave](#autoclave)
Wild bird seed is dusty and full of bacteria and molds.
Because the seeds are dry the bacteria has likely [endosporulated](https://en.wikipedia.org/wiki/Endospore), making it resistant to sanitization.
Soaking the bird seed will clean it up a bit and encourage the endospores to reproduce, leaving them weaker to [autoclave](#autoclave).
Imperial | Metric
-------- | ------
16 oz | 450 ml
1 cup | 250 ml
Wild bird seed is dusty and full of bacteria and molds.
Because the seeds are dry the bacteria has likely [endosporulated](https://en.wikipedia.org/wiki/Endospore), making it resistant to sanitization.
Soaking the bird seed will clean it up a bit and encourage the endospores to reproduce, leaving them weaker to [autoclave](#autoclave).
Earlier it was mentioned that mycelium want a variety of [grain spawn](#grain-spawn-and-substrate) and [substrates](https://en.wikipedia.org/wiki/Substrate_(biology)) between generations of colonies so the strain does not lose it's ability to digest different sources of nutrition.
This guide uses coconut husk (coir) mixed with [grain spawn](#grain-spawn-and-substrate) for colonization as a substrate.
You can also add coffee grounds, sawdust, straw, and rice husks for variety.
Take caution to only change one thing at a time between generations or you risk overwhelming the mycelium.
Be mindful of commercial gardening coir as they are treated with [endophyte](https://en.wikipedia.org/wiki/Endophyte) to protect plant roots.
These endophytes do not protect our mushroom roots and will compete for resources like any other contamination.
0. [Sanitize Your Workspace](#sanitize-your-workspace).
1. Fill 16 oz wide mouth mason jars with 1 cup of bird seed for each final jar of grain spawn.
0. Hydrate the coconut coir brick following the packaging instructions.
2. Cover bird seed with tap water, lid jar, and soak for 15 minutes.
0. Mix the coconut coir and wild bird seed in the bucket at a 50/50 ratio.
3. Strain the bird seed with a colander and rinse with water.
0. Fill with water until moisture is visible on the surface of the mixture and soak overnight.
- Pick out any unwanted bits like twigs or mush.
0. Split evenly to each 16 oz wide mouth mason jar, about 1 cup.
- 1 hour to dry, stirring occasionally to expose moisture.
0. Cover jar opening with aluminum foil and [pressure cook](#autoclave) for 2 hours.
4. Split evenly to each 16 oz wide mouth mason jar, about 1 cup.
0. Lightly screw lid over the foil to secure it in place without sealing the jar and allow to cool to room temperature.
5. Cover jar opening with aluminum foil and [pressure cook](#autoclave) for 2 hours.
0. Store in a dark cool place until inoculation.
6. Lightly screw lid over the foil to secure it in place without sealing the jar and allow to cool to room temperature.
7. Store in a dark cool place until inoculation.
# Spawn Inoculation
## Spawn Inoculation
- [ ] 50 to 100 ml syringe with hollow needle, [autoclavable](#autoclave)
0. [Sanitize Your Workspace](#sanitize-your-workspace).
- Sanitize the outside of each jar of [grain spawn](#grain-spawn) and [liquid culture](#liquid-cultures).
- Sanitize the outside of each jar of [grain spawn](#grain-spawn-and-substrate) and [liquid culture](#liquid-cultures).
1. With a sanitary syringe extract 1 ml of [liquid culture](#liquid-cultures) and replace the screw top.
1. Gently stir the [liquid culture](#liquid-cultures) and with a sanitary syringe extract 1 ml of [liquid culture](#liquid-cultures), replace the screw top.
2. Inject syringe into the jar of [grain spawn](#grain-spawn) by carefully piercing the aluminum foil straight down along the inside of the jar.
2. Inject syringe into the jar of [grain spawn](#grain-spawn-and-substrate) by carefully piercing the aluminum foil straight down along the inside of the jar.
3. Replace the mason jar lid by flipping it so the seal is facing up and lightly screwing on the rim.
4. Store in ambient room light at room temperature.
# Reading a Spawn Jar
## Reading a Spawn Jar
Similar to [reading an agar slant](#reading-an-agar-slant), [grain spawn](#grain-spawn) should show growth within the first week after inoculation and should be monitored for contamination weekly until it is fully colonized, 2-3 weeks.
Similar to [reading liquid cultures](#reading-liquid-cultures), [grain spawn](#grain-spawn-and-substrate) should show growth within the first week after inoculation and should be monitored for contamination weekly until it is fully colonized, 2-3 weeks.
Mycelium are snowy white with grey and blue tints as normal.
Mycelium are snowy white with grey, blue, or brown tints depending on the species.
Any other colors and you are dealing with contamination.
Colors like yellow, red, green, and black usually indicate contamination.
Minor contamination is not the end of the world, many colonies can recover.
If the jar is fully colonized following a contamination and doesn't smell like rotten apples it can still produce mushrooms.
Once the grain in the jar is covered with feathery white mycelium, give it one more week before moving the cakes to [bulk substrate](#bulk-substrate).
Once the grain and substrate in the jar is covered with feathery white mycelium, give it one more week before [fruiting](#fruiting).
# Fruiting
Getting mycelium to fruit is a labor intensive process.
Fruiting isn’t necessary for the production of organic compounds.
You could use food grade grain like steel cut oats or brown rice in [colonization](#colonization) then skip the fruiting stage by waiting until it is fully colonized and dehydrate the mycelium.
It is much more appetizing to prepare and consume the fruiting bodies.
If you've ever been walking in the woods after a light rain you've probably seen mushrooms peaking up.
This is because mycelium sense that water is evaporating and wish to take advantage of the updraft to spread their spores.
In our fruiting containers we want to mimic that as much as possible with a spray bottle and a light breeze.
In our fruiting containers we want to mimic that as much as possible without exposing the substrate to contamination.
This can be accomplished by opening a fully colonized [grain spawn](#grain-spawn) jar and misting the mycelium cake with water daily.
The mushrooms are light sensative, to prevent them grow inside the jar we cover the glass with aluminum foil.
However, exposing more surface area give the mushrooms more room to sprout.
# Bulk Substrate
- [ ] coconut coir
- [ ] take-out containers, black opaque, polypropylene
Earlier it was mentioned that mycelium want a variety of [grain spawn](#grain-spawn) and [substrates](https://en.wikipedia.org/wiki/Substrate_(biology)) between generations of colonies so the strain does not lose it's ability to digest different sources of nutrition.
Once you have practice making [grain spawn](#grain-spawn) you'll want to mix 25% of the substrate chosen for this step to give the mycelium time to produce the right enzymes during colonization.
This guide uses coconut husk (coir) mixed with [grain spawn](#grain-spawn) for colonization as a substrate.
You can also add coffee grounds, sawdust, straw, and rice husks for variety.
Take caution to only change one thing at a time between generations or you risk overwhelming the mycelium.
Many commercial gardening coir are treated with [endophyte](https://en.wikipedia.org/wiki/Endophyte) to protect plant roots.
These endophytes do not protect our mushroom roots and will compete for resources like any other contamination.
**Coconut coir must be [autoclaved](#autoclave) prior to use.**
Take-out containers are available at most grocery or box retailers.
They must have black opaque bottoms with clear lids, usually made from polypropylene.
You can tell if they are polypropylene when they are advertised as dishwasher and microwave safe.
Polypropylene products also carry a recycling #5 designation.
0. Soak the coconut coir brick for 15 minutes or following the packaging instructions.
1. Fill mason jars with the hydrated coconut coir and [pressure cook](#autoclave) for 1 hour and cool to room temperature.
2. Layer the bottom of the take-out containers with 2 cm of coconut coir.
3. Open and invert a fully colonized [grain spawn](#grain-spawn) jar and firmly but gently tap the mycelium cake out into the open take-out container.
Gently break up the cake with a sanitary utensil until the mycelium covers the layer of coir.
4. Layer the top of the mycelium layer with 2 cm of coconut coir.
5. Return the lid to the take-out container and store in ambient room light at room temperature.
# Fruiting Chambers
## Fruiting Chambers
- [ ] clear plastic tubs with lids, shoebox or larger
- [ ] hygrometers
Check the mycelium in the take-out containers once a week for contamination.
Once the top surface of the coconut coir has been covered by feathery white mycelium it is ready to fruit.
0. Remove lid from take-out container and place within a clear plastic tub.
0. Cover the glass mason jars with a sheet of aluminum foil, remove lid exposing the colony to the air.
1. Place a sanitary hygrometer onto the tub where it is visible.
0. Place jar within a clear plastic tub with the jar on its side.
2. Replace tub lid and store in ambient room light at room temperature.
0. Place a sanitary hygrometer onto the tub where it is visible.
0. Once tub is full of sideways open jars, replace tub lid and store in ambient room light at room temperature.
## Daily
@ -519,8 +412,6 @@ It is necessary to open at least daily to provide fresh air and to clear out the
0. If needed, gently mist the mycelium cake and surrounding coconut coir a spray bottle of water until humid.
1. Fan the tub with the lid for a few moments then replace the tub lid.
Each mushroom is different generally harvest mushrooms when their gills are exposed but before they dump their spores and make a mess.
# Checklist
@ -543,24 +434,18 @@ Each mushroom is different generally harvest mushrooms when their gills are expo
- [ ] light corn syrup
- [ ] diammonium phosphate with urea
- [ ] agar
- [ ] 30 to 50 ml test tubes with screw cap, [autoclavable](#autoclave)
- [ ] marbles or magnetic stirring rods
- [ ] flame or lighter
- [ ] wild bird seed with millet
- [ ] colander
- [ ] 8 liter, approximate, bucket and lid
- [ ] aluminum foil
- [ ] coconut coir
- [ ] take-out containers, black opaque, polypropylene
- [ ] clear plastic tubs with lids, shoebox or larger
- [ ] hygrometers