Preperation of Agar

Preperation of Agar – Medium in Petri Dishes
(Level 3 – mushroom expert)


The following formula makes 1 litre of “MEA” – malt extract agar medium, convenient for most species of cultivatable mushrooms. Especially for working with spores, antibacterial agar media is recommended. Ready to use formulas are offered, that only need to be mixed with water.


Recommended equipment:

20 g agar-agar
20 g malt extract
2 g dry yeast
1 g peptone
1 litre drinking water

Erlenmeyer flask
Sterile disposable Petri dishes or glass Petri dishes
Measuring container
Scale
Parafilm
Latex gloves
Face mask and hairnet
Disinfectant solutions for surfaces and hands
Pressure cooker
Sterile working area: Glove Bag/Glove Box or
sterile air flow (HEPA-filter, Laminar Flow Hood)


Mixing the substrate

Pour the ingredients into the Erlenmeyer flask along with hand-hot water, close the lid and shake well until mixed thoroughly. The maximum level of filling the flasks is reached at two-thirds of their volume. Remove the lid and wrap some aluminium around the flask's neck forming a tube which you bend down at its free end. This attached duct will ban condensate water to enter the unlocked flask during sterilisation.

Sterilisation of the agar medium

Agar medium should be sterilised for 45 minutes using a pressure cooker. Overlying a 2-3 centimetre water level at the bottom of the cooker a separating insert keeps the flasks above the water line. Assure yourself that there is no contact between water and the upright standing flasks, decant some water if necessary. (Mind the handling instructions given by manufacturers of your pressure cooker!)

Get the lid locked and put the cooker on a hotplate. Sterilise for 45 minutes, counting from the moment as the pressure gauge reaches its highest stage (for household pressure cookers). If you've got a professional pressure steriliser at your disposal sterilise at 121°C/ 259°F/ 15 psi/ 1.05 bar.

Attention: Do not exceed temperatures specified above for a longer span of time. By overheating the nutrients, the culture medium can degrade and the solutions colour darkens from yellowish to brownish. If so, the medium is useless. Substrates which are composed of other ingredients may have different colours.

After 45 minutes of sterilisation, let the cooker cool off in front of a HEPA-filter or in a clean place. During pressure equalisation air is sucked in; a clean and wet cloth over the cooker's cover filters the aspired air, if you do not use a HEPA-filter.





Pouring the Petri dishes

Wear protective clothing, disinfect working area and hands. Do not open the cooker before the pressure gauge indicates 0. Continue processing from now on because the agar medium is only in its liquid stage above a certain temperature. (If you work with glass Petri dishes, keep in mind, they have to be sterilised too.)

Pour about 25-30 ml warm agar-medium into a Petri dish with 90 mm in diameter, which gives 35-40 Petri dishes per 1 litre agar-medium. For cooling down leave them in a place of low microbiological contamination in front of a HEPA-filter or inside a Glove Box.

When the agar is hardened and cooled to room temperature, the Petri dishes are ready for inoculation. If you do not use all casted Petri dishes, you can seal up the remaining Petri dishes with Parafilm, wrap them in plastic foil or bag and keep them in a clean refrigerator for up to 4 weeks. (35.6-39.2°F / 2-4°C)


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References:

„Mycelium running/ How mushrooms can help save the world“, Paul Stamets; Ten Speed Press, Berkeley/Toronto;
„The Mushroom Cultivator: A Practical Guide to Growing Mushrooms at Home", Paul Stamets, Agarikon Press; First Edition edition (December 1983);
„Growing Gourmet and Medicinal Mushrooms”, Paul Stamets, Ten Speed Press, Berkeley/Toronto;

Recommended books: