Underground

UNDERGROUND

Truffle growing kit

Instructions:

DO NOT REMOVE THE MICROFILTERED CAP FROM THE SUBSTRATE BAG.

1) Kit includes:

Microfiltered bag with growth substrate and injection port.

5 ml liquid culture vial.

Sterile alcohol swab.

Sterile syringe.

2) You will need:

Gas flame (a blowtorch type lighter goes very well).

Clean hands and a clean area to work in.

The spores you want to inoculate in the kit.

3) Inoculation:

You have two options for inoculating the truffle kit:

A; spores:

You can inject the spores directly into the growing substrate. This option is a little less work, but it takes longer for the kit to fructify and not always enough spores germinate. If you choose this option go directly to step 1 (see below).

B; liquid culture:

For faster colonization and higher yields, you should first inoculate and colonize a vial of liquid culture and then inject the liquid culture into the growing substrate bag. Spores that produce truffles may require more time to germinate on grain than spores that produce mushrooms, so the liquid culture can serve as a shortcut. If this is the option you have chosen follow steps B1 to B10, otherwise go directly to point 1).

B1) If the liquid culture medium is refrigerated, remove it from the refrigerator and store it at room temperature 24 hours before use.

B2) Wash hands thoroughly (use of antibacterial soap is optional). Dry with a clean towel (e.g. paper towels).

B3) Remove the vial from the bag and place on a clean table in a clean, debris-free area.

B4) Apply alcohol over the injection port of the Liquid Culture Vial and any other site intended for injection (e.g. spore vial or Liquid Culture Vial). Use a clean swab for each product to be wetted in order to reduce mutual contamination. Do not exhale directly onto the injection sites.

B5) Remove the syringe and needle from the bag. Remove the plastic cover protecting the needle (turn clockwise and pull).

Do not allow the needle to come into contact with any object, otherwise it will have to be re-sterilized. If a syringe from any other supplier is used, it must also be sterilized.

The needles can be sterilized with a flame. It is desirable that the needle be red hot, taking care not to overheat and melt the area where the needle is attached to the plastic. Always allow the needle to cool before injecting.

B6) If using a spore or colonized Liquid Culture vial, shake vigorously to mix the suspension. Hold the vial at an angle, inject with the syringe, and draw up the desired amount of suspension.

B7) Inject several drops of culture suspension (i.e., colonized liquid from the culture vial) or between several drops and 1 ml of spore suspension (depending on the density of the spore solution) into the Liquid Culture Vial. Withdraw the needle very slowly, otherwise the pressure may cause some of the liquid to be sprayed out-which is not a problem.

B8) Wipe the injection port of the Liquid Culture Vial with a clean swab moistened with alcohol; allow to dry, and store vertically in an airtight plastic bag.

B9) Vigorously shake the vial once a day for ten seconds and store away from sunlight. Incubate the mushroom species at the appropriate temperature (generally 23º to 27º C). A lower temperature delays the germination and colonization time of the spores. However, too high a temperature (above 27°C) increases the risk of contamination.

Note: Temperature requirements depend on the species.

B10) Growth is similar with both spores and Liquid Culture. However, growth with spores takes longer than with the latter.

Spore suspension: Spores generally take 24-72 hours to germinate, and within 3-7 days colonization of the liquid medium should be observable growing in the lower two thirds of the vial.

Note: MycoMate® Liquid Culture Vials require very few spores for growth.

Liquid Culture: Growth is evident within 24 hours of transferring the mycelium from a colonized Liquid Culture vial to fresh Liquid Culture vials. Fresh vials are generally colonized after several days (depending on species, variety, and temperature).

Observation is most effective when the vial is held carefully against the light, avoiding shaking. The sediment in the culture medium can become agitated if you are not careful enough, making it more difficult to identify growth (this is not a problem, as the sediment will settle back to the bottom after a while). White threads of mycelium (fungus) will float to the surface as you lift the vial. There should be a small cloudiness around the mycelium, but the entire vial should not be opaque. The area around the cloudiness of the mycelium should be reasonably clear. If the vial is slowly lifted and yet appears completely opaque it is possible that it is contaminated. Allow the vial to stand undisturbed for several hours and check again to rule out that it was sediment from the bottom that obscured the vial. This can be used as soon as there is healthy growth or after one or two weeks when the vial shows increased colonization.

Note: When one to two thirds of the vial has been colonized it should be used or stored under refrigeration for later use (see below).

B11) When colonization is complete the vial can be used to inoculate other MycoMate® Liquid Culture Vials (repeating steps B1 through B10), MycoMate® Edible Mushroom Kits or Sclerotia (repeating steps B1 through B6).

1) Hold the injection port of the substrate bag with one hand, lift the two plastic tabs with the other hand and carefully tear the plastic cover protecting the injection port. Apply alcohol with a clean swab and allow to dry.

2) Remove the syringe and needle from the bag, remove the plastic cover protecting the needle (turn clockwise). Do not allow the needle to come into contact with any object, otherwise you will have to re-sterilize it.

The waters can be sterilized with a flame, it is desirable that the needle is red hot, being careful not to overheat and melt the area where the needle is attached to the plastic, let the needle cool before injecting. Avoid exhaling on the needle or injection area.

3) Push the syringe needle through the injection port of the substrate bag.

Spores: Use the injection port to guide the needle and inject 1 to 2 ml at each corner and parallel to the sides of the substrate bag. Include several drops on the substrate itself. The more uniform the injection on the substrate the faster the colonization will be.

Liquid culture: Use the injection port to guide the needle and inject 1 to 2 drops in each corner and parallel to the sides of the substrate bag. Allow the solution to drip over the sides of the substrate bag. Include several drops on the substrate itself. Each drop of culture represents a growth point, so the more uniform the injection into the substrate the faster the colonization will be.

4) Colonization:

The appropriate temperature for incubating the substrate bag is between22°Cand24°C.

Spores: Within several days to two weeks the spores will germinate (small cottony-looking clumps) and the fungus will colonize the bag over the course of several weeks. If germination is not evenly distributed throughout the bag over the next 14 to 22 days, you can accelerate colonization by carefully mixing the contents of the bag. Do this only once, so that the fungi can complete colonization.

Liquid culture: Within 24 to 72 hours the fungus will come to life and within one to two weeks will colonize the substrate, depending on the species and variety.

5) Harvesting:

Although you can start harvesting truffles earlier, it is advisable to wait at least 12 to 24 weeks before harvesting. The longer you wait, the higher the yield and the larger the truffles will be. The speed of colonization, the yield and its characteristics vary from one variety and species to another.

The Underground will produce more than 100 grams of fresh truffles in a few months.