Agar Culturing, Cloning, Genetic Isolation, and Master Slants for Long Term Storage

Agar is a cultivator's most powerful tool.  Agar allows us to guarantee clean inoculant that decreases colonization times vs starting from spores.  Agar also allows us to isolate and preserve substrains of our favorite species that exhibit qualities that we like.  The use of master slants even allows us to preserve our best substrains for years at a time.  For the master mycologist, agar can even be used to cross similar varieties (substrains within the same species) of mushrooms to create new mutations.  This page will detail the use of agar, and how to make agar jars to use in agar culturing.  Agar requires a sterile environment to work in, so a still air box, glove box, or flowhood will be necessary to be successful with agar culturing.  Agar cultures and slants should be stored at ambient room temperature while colonizing, then be stored in the refrigerator until ready for use.  When ready for use, remove the culture from the fridge and allow it to sit at room temperature for 24 hours before use.

Agar Culturing:  A cultivator starts an agar culture by placing spores or a tissue sample from the interior of a mushroom's stipe (cloning) onto the surface of agar.  The spores will germinate and colonize the surface of the agar.  A tissue sample (clone) will begin colonization of the agar surface within 48 hours.  Once the agar surface has been colonized, a wedge of it can be cut away with a sterile blade for use as inoculant.  A cultivator can use an agar wedge to inoculate grain jars, liquid culture jars, and start fresh agar cultures.

Genetic Isolation:  When a cultivator creates a culture from spores (and occasionally from a clone), there are multiple sets of genetics present.  When there are multiple sets of genetics present in a substrate, a cultivator never knows what to expect for results.  Agar can be used to isolate individual sets of genetics so that they can be grown out and tested for various traits, such as yield, size, color, scent, ect.  Different sets of genetics can be identified on agar by speed of growth, and thickness of the mycelial ropes formed.  When isolating genetics, a cultivator simply cuts a sample about the size of a grain of rice from the leading edge of growth, and transfers it to a new agar dish.  That culture will then be grown out, and another transfer will be made until the cultivator ends up with a dish that is uniform in growth.  The isolate can then be grown and tested.

Master Slants:  A master slant is an agar culture in a test tube.  The small surface area of the agar restricts cell divisions to help prevent senescence (the weakening of cells over generations of cell division, think of it like a person progressing into old age), and allows storage space to be used more efficiently.  A master slant is created and use just like an agar culture, but is designed to keep a culture alive for years in refrigerated conditions.

Contaminates:  Sometimes, especially with cultures from wild mushrooms, contaminates will be present.  These contaminates range from bacteria to various molds.  When trying to clean up a contaminated culture, a cultivator needs to take a piece of mycelium as far away from the contaminate as possible.  It will sometimes take multiple transfers to escape the contaminate.

MAKING AGAR JARS AND SLANTS

Agar Jars

This recipe will produce 8 agar jars.  You will need the following supplies:

9 grams Agar-Agar flakes (sold in the Asian foods isle in some supermarkets, also in health food stores)
10 grams Light Malt Extract (brewery supply store)
500ml Water
Pot, preferably with a spout
Pressure Cooker with steam rack
Aluminum Foil
8 125ml Ball Elite Collection glass canning jars (Walmart, Ace Hardware, ect)
8 Widemouth Synthetic Filter Disks (it has been suggested that the thick EZ Felt double layered is a good filter aswell)

Preparation:  Cut the easy felt to match the size of the jar lids so that it will be held in place by the jar ring over the lid.  You also need to drill 2 5/16" holes on either side of each of the jar lids.

Step 1:  Bring 500ml of water to a slow simmer, then add 10 grams of light malt extract, and 9 grams of agar-agar flakes.  The broth will look slightly chunky at first, simmer at a low boil for 10 minutes, or until most of the agar flakes have melted.

Step 2:  CAREFULLY pour 60ml of the broth into each of the 8 jars.  Place the lid on the jar upside down (the rubber seal can cause problems during the next step if you put it to the glass), place the filter over the lid, then tighten down the ring.  Leave the ring a little lose.  Cover the top half of the jars with tin foil, just like you would if making PF Tek jars, or grain spawn jars.  Place the jars into the pressure cooker above the water line.

Step 3:  Pressure cook at 15psi for 30 minutes, then let cool overnight.  The jars may be inoculated the following day.



Agar Slants

The process for making agar slants is similar as agar jars, but with a few tweaks to accommodate new hardware and to make them more effective for long term storage.  This method also needs a still air box, glovebox, or flowhood because of the nature of the test tube lids.  You will need the following supplies:

16mm x 100mm culture tubes with caps (I like the PP5 plastic type with screw on caps)
Wood craft sticks (popsicle sticks)
Light Malt Extract powder (.015g per tube)
Agar powder (.01g per tube)(will need to be ordered online)
Water (6ml per tube)
Measuring cup
Pint Jars with rings
Tyvek or similar filter (you can use the jar's actual lid if it has a fixed filter)
Cooking pot
Aluminum Foil
Pressure Cooker
Scale (for measuring agar and malt powders)
Still Air Box OR Glove Box OR Flowhood
Cooling Rack (can be any surface that will hold the tubes at an angle, a section of wire shelving with 1 side proped up with a few popsicle sticks will do nicely)

Step 1:  Break the sticks so that they'll fit into the culture tubes, leaving a visible gap between the end of the stick and the cap of the tube (the cap will take up some space on the inside of the tube).  Soak the sticks in water over night (in the cooking pot).

Step 2:  Fill each tube with 6ml of water, .01g Agar powder, and .015g Light Malt Extract powder.  Screw down the cap and shake well until mixed.  If you are making several tubes at once, you can also mix all the ingredients in a measuring cup, then pour the mixture in, or use a syringe to ensure accurate filling.  If you opt for the second method, be sure to stir the mixture between tubes as the agar powder will try to settle on the bottom of the container.  Insert one of the wet sticks into each tube, then place the cap on loosely.

Step 3:  Place tubes into pint jars, and add water to the jar until the water in the jar is even with the agar mixture in the tubes.  Place filter or lid on the jar and tighten down the ring.  Wrap the top half of the jar in foil.

Step 4:  Place jars into the pressure cooker, and heat at 15psi for 20 minutes.  Remove from burner and allow to cool until the cooker's pressure lock releases, and they are cool enough to handle without risking injury.  Place the jars inside your still air box, glovebox, or in front of your flowhood.

Step 5:  Remove the tubes from the jar and tighten down the cap before setting on the cooling rack.  Allow to cool until the agar mixture has acquired a jell like consistency.

After inoculating a culture tube, leave the cap slightly loose and wrap parafilm around it.  This will allow for gas exchange while keeping the cap in place and preventing contaminates from entering.