Recipe for Malt Extract Agar (1 liter)
20 grams Malt Extract
2 grams Yeast
15 grams Agar
The dry ingredients are put in a flask and the water is added. This is sterilized for 30 minutes at 121 degrees Celsius.
The flask should be twice as large as the volume of the medium that is sterilized to prevent it from boiling over and should preferably have a narrow opening. The opening is stuffed with polyfill, hydrophobic cotton or capped with Tyvek . This is covered with tinfoil.
Pouring petri dishes
Condensation on the lids can be prevented by:
pouring petri dishes when agar has cooled to 40-50 degrees Celsius;
stacking the dishes with warm agar;
putting something hot on the top dish of the stack (a mug with hot water for example).
Dishes should preferably be poured in a glove box or HEPA filtered environment.
Incubation of agar cultures
Petri dishes should be taped around the edges with
polyethylene cling wrap. This prevents both dehydration and the
introduction of contaminants. A small roll, cut from a big roll with a
sharp knife is very useful.
Dishes are Incubated upside down so that droplets of condensation will not fall on the agar surface but will remain on the lid until they evaporate.
Keep track of how often a strain is subcultured. Do not subculture for more than 5 petri dishes or you will risk losing your strain to degeneration. A sure sign of degeneration is the formation of zones of fluffy mycelium. These cultures should preferably not be used. If there's no choice but to use them, use only the rhizomorphic zones. See:Incubation
For storage of cultures agar slants are used. The agar is prepared by boiling in a pan and then pouring it into test tubes (a big syringe is useful for this). The tubes are loosely capped and sterilized for 25 minutes in the pressure cooker. The tubes should be taken out before the agar solidifies and put under an angle so that upon solidifying the agar has larger surface for mycelial growth.
The tubes can be inoculated by dropping a small piece of colonised agar into the tube. The tube is loosely capped and sealed with cling wrap. The piece of agar can now be forced downwards onto the agar by tapping the tube it with your hand.
After two weeks of growth the tubes can be put in the fridge where they will remain viable for up to 5 years. Every year tubes are taken out and subcultured in petri dishes to check if they are still viable. After prolonged storage cultures may show some sectoring (the formation of flufffy zones in the mycelium) so only healthy rhizomorphic mycelium is subcultured. See:Storing Strains on Agar
Sporeprints are best taken on a smooth surface that allows easy picking up of the spores with an inoculation loop. Most clean smooth materials will work fine as long as they are clean. Most store bought dry products (paper, tinfoil) that are packaged can be considered clean.
Some strains and species do not easily drop their
spores. Often sporulation can be stimulated by increasing the humidity.
This is especially important when sporeprints are made from small caps.
The caps themselves would dry pretty quickly and once they are dry, they
will not deposit any spores.
For species such as Psilocybe mexicana and Psilocybe tampanensis a small plastic container that seals well is used some of the stems of the mushrooms are put in this as well to help raise humidity.
Not all strains drop their spores immediately after the veil tears, so it's best to wait until spores can be seen on the stem near the cap.
The mushroom is picked and put on clean tinfoil. With a sharp knife or scalpel the cap is cut off and the cap is put on the printing medium (paper, tinfoil, petri dish). The container is closed and after 24 hours the cap is removed. A perfect sporeprint should be the result. Store the sporeprints individually sealed in a ziplock type baggie. These can be stored for years in a cool dry location.
Substrates for Psilocybe cubensis
Almost any type of grain can be used to grow this species on but in our opinion rye grain is the best choice. Other grain types and mixtures do not evenly absorb moisture during sterilization and have to be preboiled.
Rye (in dry form) can be combined with water and then sterilized without the need of preboiling. This saves a lot of time and hassle. When the jars or bags are still hot they need to be shaken to disperse the wet bottom kernels with he dryer top kernels. Upon cooling moisture will spread evenly throughout the substrate and the end result can be shaken easily.
Other grains have to be pre boiled in plenty of water. Boiling time depends on the type of grain that is used. Mixtures with small kernels often need less time than those consisting of bigger ones. Kernels that have burst open are not good, and too many of these may give problems with bacterial contamination later on. After boiling the grain is drained and filled into jars or bags. These can then be sterilized.
Substrates for Panaeolus cyanescens and Panaeolus tropicalis
Although literature suggests that pasteurized straw can be used yields are poor compared to using a substrate that contains dung. People have reported good success with pasteurized cow and horse dung. We prefer to sterilized the substrate in spawn bags. A mixture of straw or grass seed and dung seems to work best. Vermiculite can be added to improve structure and water holding capacity.
A high yielding substrate can be prepared with the
7 parts dried cow dung
7 parts soaked grass seed Instead of grass seed soaked straw can be used as well
2 parts fine vermiculite
2 parts coarse vermiculite
Ingredients are mixed in dry form (except the soaked grass seed or straw) and water is added until it is saturated. Squeeze out the moisture and sterilised the mixture in spawnbags for 2 hours. These bags can be inoculated from rye spawn.
Incubating bags produce a lot of heat so watch out for overheating of the substrate!
Substrates can be effectively sterilized by
pressurized steam. A pressure cooker or autoclave are perfectly suitable
for this purpose.
The required time for sterilization depends on a variety of factors the most important being:
the amount of substrate in one container (bag/jar/flask);
total amount of substrate in the cooker/autoclave;
solid or liquid substrate.
The more substrate is filled into the containers the longer they should be sterilized. Tightly packed pressure cookers require longer sterilization than empty ones. Liquids (agar, water) require briefer sterilization then solids such as rye or birdseed.
Sterilization time is measured from the moment the pressure cooker or autoclave has reached 121 degrees Celsius (1,0 kg/cm2, 15 psi).
200 ml jar rye 40 minutes
200 ml jar grass seed 40 minutes
800 ml jar rye 60 minutes
800 ml jar grass seed 60 minutes
Spawnbag rye (4 liter) 120 minutes
Spawnbag grass seed (5 liter) 150 minutes
Spawnbag dung mixture (3 liter) 120 minutes
Agar slants 25 minutes
Agar (in bottle) 30 minutes
Water (in bottle) 30 minutes
Scalpel, syringes etc. 20 minutes
Large bag casing soil 60 minutes partial sterilization!
Many species benefit from a layer of "casing" soil on top of the colonized substrate. The process of putting such a layer on is called casing.
10 parts peat
5 parts vermiculite
2 parts limestone
The ingredients are mixed in dry form and water is added until the casing soil is almost saturated. The object is to get as much water as possible in without turning it to mud. If too much water has been added some more dry ingredients can be added to compensate.
The casing soil needs to be heat treated before use.
Some people have reported success with untreated soils but it seems a big
risk to take. There are two practical options: partial sterilization in a
pressure cooker and partial sterilization in a microwave.
The wetted soil is put in bag or jars and is 'sterilized' for 1 hour in the pressure cooker or for 25-30 minutes in a microwave at full power. Extra water should be added when a microwave is used.
When it had cooled down the soil is ready to use. If it looks a little dry you can add some clean water to it before applying it to colonized substrate.
The colonized substrate is broken up and poured into trays. The casing mixture is applied and the tray is covered. A normal depth for a casing layer is 1.5-3 cm. Panaeolus cyanescens and Panaeolus tropicalis also need a casing layer but it must be very thin as these species seem to have trouble growing through it.
Some species can form so called "sclerotia" in the
casing soil and inside of the substrate. Psilocybe mexicana and Psilocybe
tampanensis are the two most important species.
Sclerotia are hardened mycelial strucutures that are formed by the mycelium as a survival mechanism to protect it from floods, forest fires etc. Sclerotia contain 30% dry matter where mushroom only contain 9-10%. Interestingly the sclerotia of the two mentioned species also contain psilocybin and/or psilocin.
Most strains and substrains of these species form sclerotia in the casing soil of cased trays. As a substrate millet and grass seed can be used.
In vitro sclerotia (where the substrate remains in the bags or jars it's growing in) are formed by the "A" strain of mexicana and by some substrains of the Psilocbe tampanensis. Sterilized grass seed seems most suitable substrate. It can be prepared by combining the dry grass seed with water in jars or bags followed by steam sterilization.
Recipe (800 ml jars)
110 grams grass seed
180 ml water
These jars should be sterilized for 1 hour.
Recipe (5 liter spawn bags)
4 liters grass seed
2 liters water
These bags should be sterilized for 2 1/2 hours.
The substrate is inoculated and shaken after a few days. After some time sclerotia will start to form. Tampanensis forms then only against the glass or inner bag surface, mexicana forms them throughout the substrate. After 3 months (mexicana) or 6 months (tampanensis) growth has halted and the sclerotia can be harvested.
One the substrate (and casing layer) are colonized
it's time to expose the substrate to fruiting conditions. The three most
important changes should be:
a drop of temperature by a few degrees Celsius;
lowering of CO2 concentration;
exposure to light for a few hours a day.
Not all strains are as sensitive as others. In general the more fleshy and stout strains of Psilocybe cubensis are somewhat more difficult to get to pin than the more slender/thinner strains. Difficult to fruit strains can be 'cold-shocked' by putting the substrate in the fridge for 24 hours before putting them in the fruiting chamber.
Panaeolus cyanesecens and tropicalis are very sensitive to high CO2 concentrations (much more than Psilocybe cubensis). If they do not receive enough fresh air, the mushrooms will grow tall and spindly and will abort at an early stage.
if the conditions are not right for the species being cultivated often the mycelium will continue to colonize the casing surface without forming any pinheads. The casing becomes a hard closed surface that will not absorb water. If this happens the casing surface should be scratched open with a clean fork and conditions should be corrected to allow fruiting. See:Pinning