Agar sandwich isolation TEK

[~Hippie3]

Agar sandwich isolation TEK
This method works very well for the isolation of mycelium from bacterial contaminants.


This method is briefly outlined in Growing Gourmet and Medicinal Mushrooms by Paul Stamets, but it is not illustrated in the book. This method works very well for the isolation from bacterial contaminants. Not only is the growth of bacteria inhibited by the Antibiotic agar, the mycelium is forced to penetrate through the media instead of across the surface, thus leaving the bacteria behind.

First you need the supplies:


0.3% peroxide solution - optional (10ml 3% peroxide diluted with 90ml sterile water)
Sharp scalpel or knife
Inoculation loop
Fire (for flame sterilizing the loop)
2 Petris of Antibiotic Agar. (10mg Gentamicin per 200ml Agar and 2ml 3% peroxide is illustrated here – Peroxidated agar optional). Both plates should be poured fairly thin to allow adequate room for the sandwich.




First, flame your inoculation loop and cool it by dipping it into the center of the transfer dish. Rescue the healthiest looking mycelium from the wedge, as far away from the bacterial contamination as possible. Try to get an area of 1cm square of mycelium, but do not get any agar. Scrape the mycelium free using your inoculating loop. Here scraping of mycelium from the surface of a plastic bag is illustrated.




Place the rescued mycelium in the center of the plate you used for cooling the loop (into the melted divot) after dipping into the peroxide solution. It is very important to not get any agar in the previous step so the mycelium will lie flat, allowing it to be sealed under the sandwich. Be very careful not to smear the transfer around on the plate, or spread excess peroxide across the surface. You can omit the peroxide but I use it as an additional safety measure. Be sure that if you do use peroxide, you change it between each transfer to avoid cross-contamination.




Cut a 1 inch (3cm) square from the other antibiotic agar plate you have. It is best to pour the agar thin for this plate, so you can see through it, and it is easy for the mycelium to grow through. I recommend at least 1/16th inch minimum thickness (1.5-2mm) and a 1/8th inch maximum (3-4mm). It is also important to make this agar not only thin, but soft as well. You can accomplish this by reducing the agar concentration, but I find the typical recipes to be fine for this purpose (2% agar).




Place the square piece you removed earlier onto the rescued mycelium fragment. Making sure to cover it all, and making sure all edges of the agar sandwich are contacting the lower agar. Be very careful to not smear anything around, spreading possible contamination. Basically you want to seal the contaminated mycelium between the two pieces. Seal the petri and incubate as required for your species.




After a few days mycelium will penetrate through the upper piece. It may take as long as a week or more. Once it pokes through and appears healthy, it is a good sign you now have pure mycelium (contaminant free). Now it is important to keep a careful eye on the colonization to watch for weak growth, or stalling.




(This is Hypsizygus ulmarius)

After a few more days, the mycelium will grow along the surface and crawl down the edge to the base agar. It is important to watch for even growth down onto the bottom layer. If it gets to that point and stops (or stalls), it may indicate that bacteria has crawled out from between the sandwich. Once it completely colonizes the dish, take a transfer 2-3cm away from the edge of the petri to ensure the cleanest tissue...

There you have it; it works very well to keep bacteria from crawling along with your mycelium, and reduces the chances of airborne cross-contamination when the bacteria are sealed under the agar. When working with a contaminated petri, this method will guarantee you will only need one transfer to antibiotic agar. Mycelium from this plate can inoculate standard media recipes without antibiotics or peroxide.

By ATWAR

NOTE: One can alternatively just pour fresh agar on top
of the transfered mycellia instead of getting a slice from another plate

[~Hippie3]

Multispore Mycelium Agar Culturing
A guide to get more people into agar culturing.


Materials

12 Petri Dishes or 1/2 pt canning jars (Wide-Mouth preferred) You probably won't use them all but it's a good idea to prepare a bunch at the same time.
Spores (Spore Syringe or Spore Print)
An Inoculation Loop (if using a Spore Print, not needed if you use a syringe)
Scalpel Or Knife
Plastic Wrap or Parafilm
Alcohol Lamp (Butane Lighters are OK)
Agar
Pressure Cooker
2 Test tubes or other air-tight cylindrical containers
Bleach
Lysol
Water
Formula for 44g of MYA+ for 1 liter water (As given in Growing Medicinal and Gourmet Mushrooms by Paul Stamets)
20g Fungi Perfecti Agar Agar
20g Light tan high-quality malt sugar
2g nutritional yeast
2g Gold Medal Softasilk superfine flour Preparation and Sterility
First, prepare a sterile area. This involves find an enclosed area (Small room or closet is great). The area should be draft free and at a constant temperature. Sterilize the room by washing everything down with a 10% bleach/water solution and letting the solution sit for 15 minutes. Go through the room again to remove the bleach/water solution and then spray a liberal coating on everything. Once this is done don't enter the room again until you are ready working or you will possibly be introducing contaminants into the room. Moving air is a great way to introduce contaminants into your process, when moving in your sterile area use methodical movements to minimize air currents. Make sure all fans are off, windows and vents are closed and when working in your sterile area it's a good idea to place a towel under the door to minimize any possible air currents. When you are ready to work, take a shower, brush your teeth (wear a long sleeve shirt, long pants and a hat) when working (this is to eliminate any contaminants that may fall off of you. It is also a good idea to wear some type of mask to filter your breathing.
Agar Preparation

Mix all of the ingredients listed above *, then add a liter of water to a container big enough to hold everything and something that can be safely pressure cooked and easily poured (A Quart Canning jar works). Once your mixture is made, place into pressure cooker and cook at 15psi for an hour.
Petri Dish / Test Tube Preparation

You will need to gather your pressure cooker, spores, petri dishes and test tubes before you enter the room. Once you enter the room, spray some Lysol in the air towards the ceiling and let it settle.

Lay out all 12 petri dishes on your work area.

!!!WARNING!!!

Make sure your pressure cooker is SAFE to open (all pressure is released) BEFORE opening it!

Open your pressure cooker and remove the container that is holding the agar. Using quick yet methodical motions remove the top of each petri dish and fill each about 1/2 way with the agar mixture and replace the covers. A better method is to only slightly open the tops of the petri dishes and pour through the opening that is created on the side. The key is that you want to disturb the air as little as possible, so as to minimize the possibility of contamination. You will also want to fill each test tube about 1/4 - 1/3 full. Next wrap the sides of the petri dishes (tops of test tubes) with parafilm or plastic wrap (either is fine).

Once this is done let the agar filled petri dishes / test rubes cool to room temperature. Remember when entering or exiting the sterile area try to disturb the air as little as possible. Lay the test tubes diagonally on something (slanted) so that when the agar dries it is dried slanted as well.
Inoculation and Incubation

Inoculation

Once your petri dishes have cooled to room temperature it is time to inoculate them. If you are using a spore print you will want to re-hydrate the spores for 24 hours before continuing, you can do this by placing the spores in a dish of sterile water for 24 hours with plastic wrap covering it (to minimize contamination) leave the dish in your sterile area. If you are using spore syringes make sure to shake them well before continuing Once your spores are ready flame sterilize your needle and squirt some spore solution through to cool it or flame sterilize your inoculation loop. Open the tops of the petri dishes slightly and inject/scrape some spores into the center of the agar plate. Repeat this (sterilizing your needle/loop between dishes) until you have 3 completed petri dishes.
NOTE:

It is not necessary to use a lot of spores, if you can visibly see the spores in the agar you probably already have too much. Spores are tiny, the smallest speck that you can see is hundreds of spores.
Incubation

It is now necessary to let the spores incubate. Place the agar filled petri dishes that have been inoculated in a dark, warm (86 F) place for a week. After a week check on them, they should show signs of growth. Over the next week or two you will start to see white mycelium forming and growing out from the center of the dish. During this phase you will probably notice that there are different types of mycelium growing. Some may seem strandy (rhizomorphic) and some may seem cottony (tomentose). The characteristic of different types of mycelium growing on the same dish is called Sectoring, this is normal and expected. Keep an eye on the dishes and look for mycelium that looks especially fast growing and that is very rhizomorphic (strandy, rope like), this is the mycelium that we want. You do not want to let the mycelium reach the sides of the dishes, as the sides of the dishes are prime spots for contaminants to be lurking. Once the dish is 3/4 colonized it is time to go onto the next step.
Contamination Check, Strain Isolation and Slants

Contamination

This is the part that a lot of people are interested in. To get the best possible fruiting and yields from our precious mycological wonders we want our mycelium to be the strongest and most virile. You will want to check each dish for anything that is non-white (or a slight off white), be on the lookout for green, blue, pink or brown these are most likely contamination. Avoid sections of the dish that are contaminated (better yet toss/sterilize the dish and start over with it).
Strain Isolation

Now, go through each dish and since you were keeping an eye on the fastest growing mycelium (right?) you want to look for the spots where mycelium colonized quickly, is thick and prolific and is rhizomorphic. This is where a flow hood or glove box comes in handy, it is useful but not a requirement (reduces contamination). If you do have a glove box of flow hood make sure you do all transfers in this environment. Get a previously prepared petri dish and a colonized petri dish. Sterilize your scalpel or knife until red-hot and allow it to cool. Carefully open the colonized petri dish and cut a slice from the most promising section of the mycelium roughly the size of a dime (rhizomorphic, prolific, fast colonizing, contaminant free), now carefully remove the slice of mycelium and close the petri dish cover. Open the pre-prepared dish and place the slice of mycelium in the center and close. Repeat these steps for all other good cultures. You can save the rest of the mycelium for later use (refrigerate), toss it or create more dishes. With the newly inoculated dishes you will want to follow the basic steps in Inoculation and Incubation however since the spores are already incubated, colonization times will be reduced. You want to continue this process (colonization, selecting promising mycelium, and transferring) until there is no sectoring apparent. Once this is accomplished you now have a pure strain of mycelium that is virile, rhizomorphic and healthy.
Slants

You will want to now cut a slice of mycelium from the pure strain you have created and transfer it to the test tubes, then refrigerate. This is called a slant which you can use at a later date to get back to a pure strain if necessary (without repeating all of the steps above). You can then use the remaining mycelium in the petri dish(es) to inoculate your favorite substrate. This will save time as the spores have already been incubated. Growth should be apparent in a couple of days, with full colonization in under 2 weeks.

[cap]

thank you!

[Bobcat]

Very nice- I hope more people get involved with agar. Don't be scared!