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Nan (Nanook)
Posted on Monday, October 22, 2001 - 12:51 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

"Pure Culture Technique"

The most difficult part of the psilocybin mushroom cultivation is the observance of the rules of pure culture technique. These are the sanitary codes of mushroom cultivation. There are many varieties of bacteria and fungal spores in our environment; floating in the air, clinging to our hands and clothing, exiting our mouths every time we exhale. Extreme measures must therefore be taken to keep these out of our cultures, which they would rapidly overrun. The following points should be diligently observed. Work in a clean, uncluttered, dust free room. Immediately before work wash the work table and spray the room with disinfectants (Lysol). Scrub arms, hands and nails with a disinfectant soap. Wear simple clothing, a freshly cleaned short-sleeved T-shirt is ideal. Gargle with antiseptic mouthwash and cover the mouth and nose with a clean cloth or disposable surgery mask. Cover the hair (if you have any) with a surgical cap or shower cap. Allow no drafts in the room. Close all doors and stuff all door jams. Let no flies, animals or unnecessary people in the room. Let only sterilized equipment touch the medium or inoculum. Don't lean over your work. Avoid all swift movements that may cause a draft. Be neat and do not permit anyone to enter the room while work is in progress. See: Clean Room


"Sterilization"

All utensils used in the cultivation of mycellia must be sterilized by heat before use. Glassware must be boiled in water for 30 minutes. Metalware used repeatedly must be held in a flame until glowing and then allowed to cool before making contact with any cultures. All medium containers must be sterilized after the medium has been poured into them. This process is known as autoclaving.

Containers, no more than 1/2 full with medium are placed in a large pot of water (not too much, you don't want the jars bouncing around). The lids of these jars must be loose enough to allow escape of internal pressures, otherwise the jars might crack. Using high heat, bring the water to a boil and cover the pot with a lid. Reduce heat to medium and let boil for 30 minutes, this should be long enough to destroy any foreign spores or lifeforms. Any higher temperature or longer period of time would cause the dextrose sugars in the medium to caramelize and this would inhibit growth and the psilocybin production of the mycelium. When the 30 minutes are finished remove the pot from the heat and leave covered until the pot is warm to the touch. Remove the jars and tighten the lids. Discard any jars that may have cracked during this sterilization process. Keep all jars at room temperature for 3 days to see if any foreign molds develop. If they do develop, discard the medium in the contaminated jars and clean and sterilize such jars before using again.


"Preparation Of Media"

PDA (Potato Dextrose Yeast Agar)

Wash 9 ounces of unpeeled potatoes and slice them 1/8" thick. Wash these several times in cool tap water until the water is clear. Drain these slices in a colander and rinse once in DISTILLED water. Cook the potato slices in 2 cups of DISTILLED water until tender. Strain the liquids through a piece of cloth in a strainer and collect the liquids. Rinse the potatoes 2 more times using 1 cup of DISTILLED water each time, add these rinse waters to the collected strained liquids and discard the potatoes.

Add enough DISTILLED water to the collected liquids to make 1 quart (4 cups). Bring the collected liquids to a boil in a pot and add 15 grams of agar, 15 grams of dextrose and 1.5 grams of nutritional yeast (you can purchase all three of these items in about any Health Food store). The agar should be added slowly to avoid boiling over. While the liquid is still hot, pour it into 1/2 pint canning jars until about 1/4 full. Sterilize these jars of agar medium in boiling water as described earlier under "Sterilization".

PDY (Potato Dextrose Yeast Broth): PDY Broth is made in exactly the same manner as PDA except the agar is omitted. One quart canning jars are filled 1/2 full of this broth and sterilized as described earlier. [Tip: Karo Tek]


"Starting The Culture"

A single spore print contains millions of spores and any one of these will germinate in an agar medium to form a mycelium (the root system of the mushroom plant).

Using the Pure Culture Technique, remove the lid carefully from one of the 1/2 pint jars of agar you prepared (and watched for 3 days). Hold the spore print at a sharp angle over the open jar and using the tip of a sterilized scalpel or X-acto knife, scrape a small portion of the spores into the agar jar and replace the lid. As a rule of thumb, if you can see any of the spores fall onto the agar, that's enough. There is no reason to waste a whole spore print on just one agar jar.

Within 3 to 5 days you should see what resembles mold growing across the surface of the agar. What you are looking for is a snow white growth, this is the mycelium of the Psilocybe Cubensis mushroom plant. If any other color appears it is a foreign fungus and isn't wanted. If the only color that appears is snow white, then consider yourself lucky, but if different colors appear you have to use the "Pure Culture Technique" to snag a small piece of the pure white mycelium from the contaminated agar jar (I use flame sterilized tweezers points) and transfer it to a new agar jar where it can grow uncontaminated.

When the mycelium has almost covered the surface of the agar in the jar (3 to 5 days) it is time for inoculation into the 1/2 filled broth jars you have previously prepared and watched for 3 days.


"Inoculation Into Broth"


Now is the time to transfer the mycelium growth from the agar jar into the 1/2 filled one quart jars of PDA broth. Using the "Pure Culture Technique", transfer a small amount of the pure white mycelium (about the size of a match head) from the agar medium into one of the jars of prepared broth that you have prepared and observed for 3 days for foreign growth. Tighten the lid and shake the jar, this assures that the liquid broth has plenty of oxygen distributed throughout and also helps break up the mycelium for faster growth. Loosen the lid on the jar slightly and set on a shelf at room temperature. Continue with each jar of broth until all prepared jars have been inoculated. Every 2 days tighten the lid on the jar(s), shake to distribute oxygen and break up the growing mycelium, reloosen lid(s) and put back on shelf. In the beginning (the first few days) you will see what looks like white clouds in the liquid broth, this is the young mycelium plant. Within 10 to 12 days this will turn into a thriving mass of mycelium root system that is ready for harvest. All you do at this time is open the jar(s) and pour into a colander to drain. What you should have is 2 to 4 ounces of white mycelium per jar (wet weight). Since this mycelium is the same chemical composition as the mushroom fruit itself, you can prepare it in the same way you would prepare the mushroom.

"Using Spore Prints"

One of the things I don't understand is how someone can obtain a pure and uncontaminated sporeprint. Since the mushroom fruits grow above ground they are constantly surrounded by air that is full of foreign molds and spores by the millions and it seems to me that when this "polluted" air passes over the mushroom, which is very moist, some of these foreign contaminates will stick to the underside of the mushroom and therefore will contaminate the prints taken from that mushroom. I have received a lot of mail as to how someone can have success starting their cultures with one of these contaminated sporeprints and this is my reply;


'CONTAMINATED SPOREPRINTS'
I use what I call a double-agar technique. I know a lot of people who shudder at the thought of using an agar method of cultivation but once they have tried it they find how easy it is they use it all the time. The instructions for making your own agar can be found in my "Pure culture Technique" section under the heading "Media Preparation".

It is very easy to make and will help you cut your contamination loss almost to zero. When you cook up your agar solution you need to make enough for at least two jars (the instructions are for 4 so that is better). Once you have the agar solution in the jars and have autoclaved them (sterilized) you can scrape a small amount of spores from your spore print onto the surface of the agar in "one" of your prepared jars, place the lid back on the jar tight, set the jar on a shelf in your closet and wait 3 to 5 days for the mycelium to start growing across the surface of the agar.

What you are looking for is a "Snow White" growth of fungus, any other color is a contaminate and needs to be destroyed. Now what you need to do is, using the "Pure Culture Technique", open the agar jar and snag a piece of the white fungal growth and transfer it to the second agar jar where it will grow uncontaminated and discard the initial agar jar you began with. This second jar will now grow the snow white mycelium only and can be used to Inoculate many (about 24) substrate jars. [or PF Tek]

I have been using this double agar technique for many years now and I only lose 1 or 2 jars out of 100 to contamination. In making the agar solution, if you can't find any Dextrose where you live you can try using any unrefined or organic sugar in its place, just don't use plain old white sugar because it will fail you.
by Shroom Wizard, I believe

Also See: Long Term Storage : Agar Culture Info : Saving Strains on Agar : Sanitizing FAQ : Shroom Glossary