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       Let's move on to the lab.




       I'm going to post a lot of different web sited to show you where I'm getting my ideals from.




       You are going to have to build some more equipment. To be used in your lab.




       But as you look around. Think back to about all the thing you have seen. All the people that I have talked about that have did this the past. They had different types of equipment. But they where all doing the same thing. Growing fungi.




       I have all ready built the equipment Gong to take picture and show you what I have. Talk about it.




       You have to have a big pot the boil every thing in.




       You have to have a glove box. It's the best way to go about this.




       But some thing that they don't show you is an Al. foil safe to grow the fungi in. It one mistake that the people who grow mushrooms are making. They tell you to grow the fungi in a dark place. But what they don't tell you is that a dark place will not protect the fungi from U.V light. Radon particles will go through walls.




       Mosses didn't make this mistake when they built the Ark of the Covenant. To covenant means to hide. What were they hiding in the box? The ark. It turns out that they weren't hiding any thing on the inside of the box from every one on the out side of the box.




       They were hiding some thing on the in side of the box from some thing on the out side of the box.?




       The light of day.




       Sun light.




       U.V. light.




       The Ark was a wooden box. That was lined on the inside and out side with gold. Gold is a first down reflecting surface. Just like. Aluminum tinfoil.




       To find a safe place to grow fungi. You would have to have a cave. Deep under ground. Or a big pyramid.




       Mosses didn't have a cave. He didn't have an aluminum foil box either. But he did have a wooden box lined with gold.




       I don't have a cave. Don't have a wooden box lined with gold. I will end up using a cord board box lined with aluminum foil. Works for me. It's cheap too. The is why they did most of their work at night. And hid the fungi from the sun light during the day




       The ark could have been used as glove box. If they put bowls of water in the box. They could control the humidity.




       It way placed on the inside of a tent. Where the built firs to keep it worm.




       They had a big pot to boil every thing.




       They cover the ark with cloth. And a water proof and light proof skin. Die blue. They made a safe room. They cleaned every thing. They wore mask over their faces. They cover there heads and body. The didn't eat or have any yeast bread. Yeast is the number one killer of fungi.




       They kept every thing covered. Petri dishes have covers. A lid.




       They where boiling beef. Beef broth agar




       What where they using for agar? The Mayan were using paper.




       Mosses didn't have any paper. Yes he did.




       Mosses wrote a book. That book was written on paper. Using cord board agar sound like such a good ideal. I am thinking about using it. Cord board is cheaper than agar too.




       It all in the broth. The drink offerings. The witches brew. There is a lot different broth that can be used for growing fungi..


















































       grow muchrooms links



http://www.google.com/search?hl=en&source=hp&q=how+to+grow+mushrooms&rlz=1R2_____en



       Larry



http://www.shroomery.org/sitemap.php




       Larry



http://www.shroomery.org/8504/Peroxide-Agar-Tek




       Larry



http://www.google.com/search?q=how+to+grow+mushroom&hl=en&sa=G&rlz=1R2_____en&prmd=v&source=univ&tbs=vid:1&tbo=u&ei=aqJ6TNimBoW8lQfLq7TsCw&oi=video_result_group&ct=title&resnum=1&ved=0CB4QqwQwAA




       Larry



http://www.shroomery.org/23/Agar



       Larry





http://www.shroomery.org/8513/Agar-sandwich-isolation-TEK



http://www.shroomery.org/8512/Cardboard-Cloning-Tek




       Larry



http://www.shroomery.org/9468/Culture-Media-for-Fungi




       Larry



http://www.shroomery.org/9427/Grocery-Store-Agar-Tek




       Larry



http://www.shroomery.org/8507/Funkybaloons-no-agar-growth-medium




       Larry



http://www.shroomery.org/5110/Sterilization-and-Pasteurization




       Larry



http://www.a1b2c3.com/drugs/pictures/mgg03.htm




       Larry



http://www.youtube.com/watch?v=1t5s2lwJgPQ



http://www.jrgenius.com/ebooks%20jrgenius.com/july2010%20to%20sort/EBOOK_Gardening%20-%20Growing%20Mushrooms%20the%20Easy%20Way.pdf


http://video.google.com/videoplay?docid=3286686859329905674#



       Larry



http://www.bom.ca/building_glovebox.htm






      There are a lot of different agars




      Agat Tek.




      agar teks




       Also make sure that your hands are washed and sterilized. I use rubbing alcohol for this but a 10% bleach in water solution will work as well.




       Washed and sterilized hands and a very clean environment is what is most important Use the glove box or a homemade HEPA system and spray everything with a 10% bleach water solution before doing your inoculations.




       Hydrate the spores 24 hours before putting on the agar.




       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 petit 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 petit dishes.




       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 strand (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 (stranded, 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




      . You can save the rest of the mycelium for later use (refrigerate), after the mycelium first forms. You can keep the dishes in a cool place to slow down the growth rate. Sun light will only affect the monokarytie mycelium. This is the time to shoot the film. This is at the point where the pattern of the hologram is formed.




       9 days to form the rhizomorphic or monokarytie mycelium. This means that the time for shooting the film has come and gone. To late to do any thing about it now. This means that the shooting has to be done in the first 6 days. Then hydrate the growth again. Then cool it down. Put in indirect sun light and fresh air. Then wait. And see what happens. The waiting game.




       They say that Ed sit around all day and watched mold grow under water. That's not true. Mold doesn't grow under water. But putting the mold has got a lot to do with what he did. There is two points at which you put the spores and mold in water. Hydrating the spores. And dunking the monokarytie (rope like) mycelium.




       Letting the cake dry out between flushes. By not misting for a few days after the flush has finished and then dunking you are imitating the natural cycle of drying out followed by sudden rehydration. In nature this rehydration will stimulate rapid fruit formation. Also, the drying out protects your cakes from molds during the waiting period as the drier surface is not as prone to contamination.




       .Makes me wander. Does rehydration cause the spores to form?




       Then it should be dunked for 24 hours. The placed in a cooler place where it has indirect sun light. And fresh air. You would have to put every thing in a Terrarium. A box that has pereilite and air and indirect sun light. And cooler.




       This will cause the mold to form dikaryotie mycelium or rysomes. What the type of mycelium that forms the fruit, which has the spores. What we want.




       Here is a little recipe that works like a charm (no agar needed)




       5.5 grams brown rice flour (home made) 14 cc's of water 1/8 tsp powdered pectin




       I use kerr half pint wide mouth jars. I add the 5.5g brown rice I ground into a fine powder then I add the roughly 1/8 tsp. pectin .then I shake it around with just the dry ingredients in then I add 14 cc's of bottled water cover with two layers tyvek and lid ring (no metal disk in the middle) and pc for 15-20 minutes at 15 psi. That’s it. Just wait for them to cool add your favorite spore and your off to the races




       Here is a little recipe that works like a charm (no agar needed)




       5.5 grams brown rice flour (home made) 14 cc's of water 1/8 tsp powdered pectin




       I use kerr half pint wide mouth jars. I add the 5.5g brown rice I ground into a fine powder then I add the roughly 1/8 tsp. pectin .then I shake it around with just the dry ingredients in then I add 14 cc's of bottled water cover with two layers tyvek and lid ring (no metal disk in the middle) and pc for 15-20 minutes at 15 psi. That’s it just wait for them to cool add your favorite spore and your off to the races




       I should add this is not a Tek I came up with, well not totally it came from a tek I read at the shroomery but it seemed to come out soupy sometimes so I decreased the water by 2 cc's it helped some but still got a few soupy ones so I decided to add a little pectin and they come out perfect hope you find as mush success with this tek as I did




       __________________ 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 Petri’s 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.




       I am an aspiring 'shroom grower myself. I have completed the single-strain isolation phase and I am ready to move on to the grain cultivation stage. After I have completed a harvest, I hope to post a list of my trials and tribulations to 'alt. drugs' including any tips or hints I discover along the way. This article contains one important tip. Read on!




       P.D.A. and M.E.A. are great alternatives to buying pre-packaged agar from somewhere like a scientific supply. I purchased 4 oz. of pre-mixed malt extract agar and spent $20 for it...and when you use 15-20 g of that mix per liter, it's not nearly as cost effective as doing it from scratch.




       Please note that most of my info comes from McKenna's definitive book: _Psilocybin: Magic Mushroom Grower's Guide_. No rights reserved, and while Terry's publisher might have something to say, I think he would be happy to see the spread of the information that leads to successful shroom cultivation.




       I mixed up a batch of PDA (Potato Dextrose Agar) from scratch. I have had great results and it's easy to make. After the recipe process, I'll explain how/where to obtain the materials and what to look for. McKenna says:




       For 1 liter total volume of liquid PDA agar you need: 250 g potatoes 15 g agar 10 g dextrose 1.5 g nutritional yeast (or yeast extract)




       Shred the unpeeled potatoes into a colander and then rinse them for thirty seconds with cold tap water. Combine the rinsed potatoes with one liter of water and gently boil for 30 minutes. Filter the resulting potato broth through muslin or cheesecloth and discard the potatoes. To the liter of potato broth add the agar, dextrose and yeast which you have previously weighed out, mixed together, and set aside. While stirring gently add in the mixed powdered ingredients. Gently boil for ten minutes or until the solution is clear. Take care to not allow the solution to boil over. Add enough water to return the total volume of the solution to one liter. Pour the solution while still hot and liquid into Petri plates, baby food jars, or slant culture tubes. Use just enough to cover the bottom of the containers, about 1/4 inch deep or a little more. The solution may be allowed to cool or sterilized immediately.




       There are a few different recipes for different types of agar. McKenna recommends changing agar types every few batches to prevent your Psilocybe cubensis mycelium from experiencing the "senescence" factor, or the tendency of the mycelium to "age and lose vigor." Here are some other agar recipes, mixed and used in much the same manner as PDA




      . For 1 liter total volume of MEA you need:




       20 g malt or malt extract (powdered or syrup) 25 g agar 0.1 g potassium dibasic (K2HPO) 0.1 g calcium carbonate (CaCO3) (basically powdered oyster shell)




       Add these ingredients to one liter of boiling water. After they have dissolved it is ready to be poured.




       There is another agar recipe which McKenna says is good for long-term maintenance. It is MYP or Malt-Yeast-Peptone agar. The procedure for preparing this mixture is the same as for MEA.




       For 1 liter total volume of MYP you need:




       7 g malt extract (powdered or syrup) 1 g peptone or soytone 0.5 g yeast extract 15 g agar




       A note on the ingredients. Agar (agar-agar) itself is a powdered extract of the gelatinous elements of sea-kelp. It may be found in health food stores, food co-ops or places that specialize in natural foods. It can be expensive (I have seen it for $50 a pound) but you don't need much to make a batch.




       Dextrose is CORN sugar and may be found for about $1 a pound at a beer/wine brewing supply center. Be sure to get CORN sugar and not CANE sugar (sucrose). The same goes for malt extract, easily found in syrup or powder form in a brewing supply store, or in some health food stores.




       Soytone and Peptone are commercial brands of a protein hydrosylate and can be purchased form scientific or microbiological supply houses.




       The other ingredients can be found at scientific supply houses and are definitely not on any controlled substance lists. This is one of the beauties of 'shroom cultivation as opposed to manufacture of other substances.




       Here's some info on agar sterilization. And by sterilization I am referring to the use of a pressure cooker/canner which is a substitute for a lab- grade autoclave. I don't know much about the kits you see advertised everywhere, but these sterilization-free kits tend to suffer from a high contamination rate. Even though I am relatively new to growing mushrooms, after nearly 50 cultures cultivated in baby food jars in a few weeks, I have not had one contaminated culture. You just need to sterilize properly and keep all you surfaces clean. A fume hood is nice, but not necessary: I use a polyethylene tarp draped over my table work-area. Be ruthless with the Lysol or whatever disinfectant you use...but look out for the flammable ones (like Lysol) and keep them away from open flame.




       And my tip on agar sterilization: don't overdo it! In McKenna's book, he cites how the standard sterilization time at 250 degrees (15 psi) for agar is 15 minutes. But he claims this is not long enough and recommends 45 minutes to an hour. Presumptuous of me as it is, I say NO! Several of my agar cultures were ruined by the intense heat of the pressure cooker. After they were done, they would cool but not congeal...the molecules that do the gelling had been severed by the heat. They just sort of sloshed around in clumps and actually should set into flat, glassine material (like JELL-O!). In a few experiments, I found that 250 degree, 15 psi was good, but I only needed to sterilize the cultures at this temperature for 30 MINUTES. I have had no problems with contamination so far.




       Moonflower's Cheap ---Rice Malt /alfalfa/ Brewer's Yeast Agar




       1 ea Quart Jar (Mason/Ball type with lids) Take 2 cups of rice and place in a 3 quart bowl or pan. Pour approx. 1 1/2 quarts of clean (bottled or rev osmosis) water into bowl. Stir in 1 cup of alfalfa (like in the health food stores) meal. Add 1 standard dolomite (oyster shell-crushed) tablet and allow to dissolve in resultant slop.




       Allow to sit for 2 hours/stir every 20 minutes or so. It is ok to warm the mixture to speed up the expression of nutrients, but cold soaking seems to be the best. (Takes longer, but has a better quality) The water should be about 70-80 degrees F. for best results not _chilled water.




       Strain out rice/alfalfa and throw it away.




       Mix 1 pkg of your regular baker's yeast into liquid and stir in.




       Let the mixture sit for 30 minutes to an hour or until it is obvious that the yeast are activated. (Usually a foamy looking bubble mass will be floating on top and the mixture may appear more opaque)




       Strain out and throw away solid yeast grains. Boil the resultant liquid rapidly for 5 minutes and then turn off the heat and add 18 grams of agar agar (aka red algae) that you bought at the health food store.




       Pour mixture into mason/Ball jar and put lids on (seal down).




       Sterilize in pressure cooker for 20 minutes at 15lbs and allow to cool. (This is at sea-level...people in high altitudes should consult a conversion table for equiv. pressures and times)




       You will now have 1 quart of a very good agar media for mycoculturing that will support _luxuriant_ mycelial growth. It is more than sufficient for starting spores as well and seems to produce a lot more "banding" than other media I have tried. Banding is when the mycelia (dikaryotic) grows in a pattern that resembles circles of threadlike fungal strands similar to tree rings on the Petri dish. I have seen carpophores form directly on the agar and fruit in a covered dish on this media and permeation time seems to be cut in half in grain media spawn.




       Rabbit dung Agar: ========================== Approx 3 pellets of rabbit dung or equivalent is placed in 100ml water. Agar is added, the mixture autoclaved and plates poured. ... ------------------------ ------------------------------------- Horse manure extract: ===================================== {see Fastfreds agar cookbook for alternating recipe} 2parts Hpoo 4 parts Distilled H2o Bring mixture to boil and simmer for 2 hours. Filter through cloth. Sterilization: 15 minutes at 121 degrees Celsius ------------------------------------- Oatmeal extract: ====================================== 30g oatmeal 1 liter water Wrap oatmeal flakes in cloth and T-bag Into Water. Bring to boil and simmer 2 hours. Squeeze and filter through cloth. Sterilization: 15 minutes at 121 degrees Celsius -------------------------------------- OHMA (Oatmeal Horse manure Agar): ======================================= .33 liters horse manure extract .66 liters oatmeal extract 15 grams agar




       Detailed instructions for Moonflower's Cheap ---Rice Malt /alfalfa/ Brewer's Yeast Agar: ============================================================= 1 ea Quart Jar (Mason/Ball type with lids) Take 2 cups of rice and place in a 3 quart bowl or pan. Pour approx. 1 1/2 quarts of clean (bottled or rev osmosis) water into bowl. Stir in 1 cup of alfalfa (like in the health food stores) meal. Add 1 standard dolomite (oyster shell-crushed) tablet and allow to dissolve in resultant slop. Allow to sit for 2 hours/stir every 20 minutes or so. It is ok to warm the mixture to speed up the expression of nutrients, but cold soaking seems to be the best. (Takes longer, but has a better quality) The water should be about 70-80 degrees F. for best results not _chilled water. Strain out rice/alfalfa and throw it away. Mix 1 pkg of your regular baker's yeast into liquid and stir in. Let the mixture sit for 30 minutes to an hour or until it is obvious that the yeast are activated. (Usually a foamy looking bubble mass will be floating on top and the mixture may appear more opaque) Strain out and throw away solid yeast grains. Boil the resultant liquid rapidly for 5 minutes and then turn off the heat and add 18 grams of agar agar (aka red algae) that you bought at the health food store. Pour mixture into mason/Ball jar and put lids on (seal down). Sterilize in pressure cooker for 20 minutes at 15lbs and allow to cool. (This is at sea-level...people in high altitudes should consult a conversion table for equiv. pressures and times)




       You will now have 1 quart of a very good agar media for mycoculturing that will support _luxuriant_ mycelial growth. It is more than sufficient for starting spores as well and seems to produce a lot more "banding" than other media I have tried. Banding is when the mycelia (dikaryotic) grows in a pattern that resembles circles of threadlike fungal strands similar to tree rings on the Petri dish. I have seen carpophores form directly on the agar and fruit in a covered dish on this media and permeation time seems to be cut in half in grain media spawn.




       These recipes are designed for agar maintenance of Psilocybe , but also other mushroom species. All media have a near-neutral pH and should be sterilized for twenty to thirty minutes at 15 psi pressure. 500 ml of medium will pour around 20 Petri dishes. Using a different medium from time to time will prevent the culture getting used to one medium.




       Malt Extract Agar (MEA) 10 grams light malt extract 9 grams agar agar 500 ml potable or distilled water




       Potato Dextrose (Yeast-extract) Agar (PD(Y)A) broth from boiling 150 grams sliced potatoes in 500ml water for 30 minutes(add water to 500ml) (You can use 5 g of instant potato flakes instead) 9 g agar agar 7 grams dextrose( = glucose, or 10 ml honey or corn syrup ) 1 gram brewers yeast or yeast-extract (optional)




       Amaranth Soy Agar 20 grams amaranth flour 20 grams soy flour 9 grams agar agar 500 ml potable or distilled water




       EntheoGenesis No.442 10 grams amaranth flour 10 grams brown rice flour 10 grams potato flour 10 grams soy flour 2 grams malted barley 9 grams agar agar 500 ml potable or distilled water




       Oatmeal Neopeptone Agar 40 grams oatmeal or oat flour 2 grams neopeptone (optional) 9 grams agar agar 500 ml potable or distilled water




       Modified Sabouraud's Medium 25 grams barley flour 5 grams dextrose 2 grams neopeptone (optional) 1 gram yeast extract 9 grams agar agar 500 ml potable or distilled water




       Cornmeal Dextrose Agar 25 grams yellow cornmeal 3 grams dextrose 9 grams agar agar 500 ml potable or distilled water




       Barley Malt Extract Agar 40 grams barley flour 2 grams malt extract 1 gram yeast extract (optional) 9 grams agar agar 500 ml potable or distilled water




       Dr. Pollock's Modified Agar 10 grams dried dog food (ground to flour) 10 grams amaranth flour 2 grams dextrose or malt extract 9 grams agar agar 500 ml potable or distilled water




      And more



      . There are a few things that you need to know about growing mold. The basic for it to grow are


       Sugar


       Protein


       Mineral of life


      callous water


      Heat.


       Sugar is the food of mold. = maple syrup


       Protein = DNA is made of protein


       Minerals of life = the maple syrup has minerals in it.


       Callous = what cell walls are made of = jell-o = agar


       Water = distilled water


       Heat = the right heat at the right time.



      El-Cheap - O. I know that you can go out and buy all the thing that you need. But to do it the same way they did it a long time a go. But if you use a little high tech stuff. It would help. I have been reading up on how to grow mold. A lot of info out there.



       First you will have to go out into the woods and find you some shower mold. Mildew.



       This stuff is the all time winner and steal champion. When it comes to molds. It will grow on all most any thing. Glass even. Bath room tile.



       There are a lot of different kinds of mildew. The one we are looking for is the one that is black, doesn’t have long mycelium. Paper thin. In order to use mold as film to form a hologram. It can’t have long high rising mycelia. It has to lay flat. Some times you can find it growing on cement walls. Where there is ha been water flowing off or over it. It has to be sensitive to sun light. Look on the dark side of building or rock cliffs. It will not grow in sun light. Sun light can kill this kind of mildew. It will look like a black stain on the wall.



       It is what they call a water mold. It grows under water. It can be grown in a broth made of water. This mold can grow on wood. Wood is fiber. It’s the type of mold that grows in the digestive systems of animals and insects that eat wood or grass. Or fiber. !0 parts of carbine to very one part of gluten. Very little glucose. Or so called sugar.



       Ed grew his mold under water. When I first heard this. I thought. Mold doesn’t grow under water. I better check this out. Sure enough. There is such a thing that is called water mold. Why? It can grow under water. It’s a kind of mildew. How much water does it take to put the mold under water? Not much. A very thin layer. Just wet.



       I have been up on the net studying other people's technique. In order to find out the mistakes that I have been making. Found a lot of mistakes that I have made. This is the number one reason why I can’t get it to form a worm hole. Mistakes many mistakes.



       The people that grow mushrooms. Tell me that the number one reason why your growth has flailed is contamination. You have to learn to do this in (mushroom are a fungi.) a sterile environment.



       If you look at Ed’s, Mosses'. The Egyptians’’ and the Mynas’ equipment. It looks like they are growing mushrooms. They were growing fungi all right. But it wasn’t mushrooms. It was mildew.




       Manna was a fungus.




       There is a picture on the walls in Mexico of a woman pulling a vine through her tong. There is a bowl before her where she is catching the blood. But it is also catching the saliva from her mouth. There can be seen in the picture, a string ray spike. And some paper.




       The blood has protein and gluten in it. And her saliva has an enzyme that can change gluten into sugar. The food of mold.




       But the bowl has some paper in it. I couldn’t under stand why the paper. Then when I read on the net, about how you can use gray cardboard to clone mycelium. It all make since.




       Paper wasp and dung beetles have mold in their digestive systems. The helps them digest the fiber of wood and grass. Digest fiber. There is a picture on the walls of Egyptian where they are getting the juice out of a dung beetle.




       All this means is that I’m on the right track.




       Back to growing mold. When you find the mold that you want. Take a Q-tip. (Cotton sob.) And touch the mold in the center of the growth. Put it in a plastic sandwich bag and seal it. Take nit back to your lab. The reason why you touch it in the center of the growth is because there is less chance of picking up some other germs on the sob.




       Isolation.




       That is where you are trying to grow the mold that you want and not any other bacteria yeast or some other kind of mole. Just mildew. Only mildew. The one the only mildew.




       It’s hard to do. But there are ways to do it that are easy. One way is streaking. This is where you take a red hot wire. Cool it down. Touch it in the center of you mold sample. The one you got from the wild. You open up your Petri dish with agar. And draw a line across one side. Just like a pant brush. The longer you drag it across the wall. The less paint is left in the brush. This is what we want the wire with the sample to do. We want the mold sample to thin out. Run out of gas. Where it runs dry. Out of sample. WE heat the wire again and let it cool down. And where the first line ran dry. We draw a line with the wire a cross the first line. And make the second line as long as we can by drawing a wave pattern across the Petri dish. A back and forth wavy line. Put the lid back on and put it in a warm place for about weak, 87 degrees f.




       You can use baby food jars. Cheap. You eat the baby food. And save the jars. Apple sauce is good eating. Clean them out when through. You can use one of the jars to make an alcohol heat burner. To heat the wire up with. You make the wire by sticking the wire in a wooden handel. Some people like a straight wire some like a wire that has a hook or loop at the end. Just nail a big hole in the top and make a cotton wick to stick through the hole.




       Now you need to build a glove box. That’s nothing more than a cardboard box. (Dumpster diving. [El Cheap-O] is where I get mine.) Need some duct tape. Plastic bags. A glass plate. Lysol disinfectant.




       Cut a hole in the top for the glass plate to lie over. Duct tape one end of the glass plate to the box for a hinge. Cut holes in the sided for your hands to fit through. Tape some plastic bags to the holes. Put ever thing in the box. Disinfect it all. Closed the gall lit. And you’re ready to go. You have to nail a hole in the center of the top of the lid and stick cotton in it. So that it can’t build up pressure.




       The agar we are going to use to isolate the mole is called water agar. All it has in it is agar and water. 20 g. of agar and 1000 ml of distilled water. About the only thing that will grow on this weak form of agar. Is mildew. And even mildew doesn’t grow very well. Real slow growth.




       Your suppose to cook it in a presser cooker to sterile the agar. But I don’t have a pressure cooker. I’m going to try to use the microwave over. You would have to put the agar and water in a tall jar to keep it from boiling over.




       If you cook some thing in water at 212 degrees for 18 minutes. They say it should be sterile. Maybe.




       I would cook it for 45 minutes. Still a maybe.




       Let cool down. Pour in to baby jars. Put the lid on. Lean them on their sides. When they are cold and solid. Place them in a cabinet for two weeks and see of any thing is growing on them If nothing is growing on them It would be OK to use them to isolate your mold in the glove box.




       After you streak one jar and grow it. You get another jar. Heat the wire and take a new sample from the first jar, to the new jar and streak it. Then let it grow. Do this one more time. By the time that you have done this three times. You should have just one kind of mold growing in your jar.




       I found out some thing new on the net called sandwich with mycelium. This is where you take some agar. Heat the wire and make a little pot whole in the agar. Take a good clean sample from you last jar. Dip it into a weak solution of hydrogen peroxide. Then place the sample in the pot hole in the agar. Take a second thin sheet of agar and place over the top of it. The only way out of the hole is fore the mycelium to grow through the thin layer of agar. What comes out of the top is germ free. USDA clean.




       But if what they are telling me about the use of hydrogen peroxide is true. That It will kill bacteria and spores. But not live mycelium. You can make an agar with H.P. And take a live sample of mycelium and clone it from an old plate to the new plate of agar with H.P in it. And it will stay free from any other germs. Because the h. P. in the agar will kill any bacteria and any new mold spore like yeast or some other mold. And your live mycelium still will grow. Just what we are looking for.




       This means that if we turn the top mirror up side down. So that it is like a bowl. We could pour a thin layer of the H.P. agar in the center of the mirror and use it as our film.




       So how do we get the mold over to the mirror? Sandwich any one?




       If we take a flat board and place a thin rubber mat on top of it. Then place a sheet of plastic saran rap on top of that. Then while the agar is still worm and in a liquid state. Pour a thin layer of agar in the center of the saran rap. But a clean jar on top of it and let it cool. The rubber mat acts like a seal to keep the jar closed. Wait two weeks is see if it is germ free. If it is then put a sample of you mold in the center of it and wait three days, just long enough for the mycelium to spread out. Find a place where it is growing solidly. You can take and cut a disk shape thing layer out of it and place on the agar in the mirror. When the mycelium catches holt. Take the disk sandwich off. And you should have a solid growth of mold that blankets the whole film area.




       Put the top mirror back on the rack.




       But to keep the top mirror germ free for six days. Maybe more. I would have to place the lens rack in a glove box. With a glass plate window in the top of it all.




       What I’m going to do is take my five gallon orange bucket. Turn it up side down. And put a glass plate in the bottom of the bucked. Which would be pointing up now. The lid has a seal in it. With out changing any thing to much. I should be able to keep my mold in a closed environment. From start to finish.




       I’ll take pictures of it all when I get it finished. Which has to be soon. If any thing is going to happen this summer.




      I’m going to use three different kind of agar.




       The first is used to isolate the sample.




       20 g. of agar and 1000 ml of distilled water




       The next kind is the same as the first with some stuff added.




       20 g. agar 970 ml of distilled water Two table spoons of spit Two drops of maple syrup Two drops of soy sauce 1/4 package bakers yeast




       Because you are putting bakers yeast in the agar. It has yeast in it. You will have to let sit a whilein warm water, so that the yeast will germinate. Then cook it for a long time. It would be best to cook it in a pressure cooker. Sterilize for 20-30 minutes at 15 psi.




       The agar that I’m going to use for film in the mirror. Will have hydrogen peroxide added to it.




      20 g. of agar 970 ml. of distilled water Two table spoons of spit One teaspoon of maple syrup Two drops of soy sauce 1/4 package bakers yeast Add 3% hydrogen peroxide the amount of what distilled water is. 30 ml.




       Cook the agar but don’t add the hydrogen peroxide till after cooking.




       Let cool down to 140 degrees F. Mix in the 30 ml. of hydrogen peroxide




       Pour in mirror before it cools down to 120 degrees F. Put in glove box. Let cool.








      agar teks




       Also make sure that your hands are washed and sterilized.




      I use rubbing alcohol for this but a 10% bleach in water solution will work as well.




      Washed and sterilized hands and a very clean environment is what is most important Use the glove box or a homemade HEPA system




      and spray everything with a 10% bleach water solution before doing your inoculations.




      Hydrate the spores 24 hours before putting on the agar. 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 petir 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 petir dishes.




      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.




      You can save the rest of the mycelium for later use (refrigerate), After the mycelium first forms. You can keep the dishes in a cool place to slow down the growth rate. Sun light will only effect the monokarytie mycelium. this is the time to shoot the film. this is at the point where the patteren of the hologram is formed.




      9 days to form this rhizomorphic or monokarytie mycelium. This means that the time for shooting the film has come and gone. To late to do any thing about it now. This means that the shooting has to be done in the first 6 days. Then hydrate the groth again. Then cool it dowm. pluse indriect sun light and fresh air. Then wait. And see what happens. The waiting game. They say thatEd sit around all day and watched mold grow under water. That's not true. Mold doesn't grow under water. But putting the mold has bgot alot to do with what he did. There is two points at which you put the spores and mold in water. Hydrating the spores. And dunking the monokarytie mycelium.




      Letting the cake dry out between flushes. By not misting for a few days after the flush has finished and then dunking you are imitating the natural cycle of drying out followed by sudden rehydration. In nature this rehydration will stimulate rapid fruit formation. Also, the drying out protects your cakes from molds during the waiting period as the drier surface is not as prone to contamination.




      .Makes me wander. Does rehydration cause the spores to form?




      Then it should be dunked for 24 hours. The placed in a cooler place where it has indreect sun light. and freash air.You would have to put every thing in a Terrarium. Abox that has pereilite and air and indrect sun light. And cooler.




      This will cause the mold to form dikaryotie mycelium or rysomes. What the type of mycelium that forms the fruit, which has the spores. What we want.




       heres a little recipe that works like a charm (no agar needed)




       5.5 grams brown rice flour (home made) 14 cc's of water 1/8 tsp powdered pectin




       i use kerr half pint wide mouth jars. i add the 5.5g brown rice i ground into a fine powder then i add the roughly 1/8 tsp. pectin .then i shake it around with just the dry ingredients in then i add 14 cc's of bottled water cover with two layers tyvek and lid ring (no metal disk in the middle) and pc for 15-20 minutes at 15 psi. thats it just wait for them to cool add your favorite spore and your off to the races




      i should add this is not a tek i came up with, well not totally it came from a tek i read at the shroomery but it seemed to come out soupy sometimes so i decreased the water by 2 cc's it helped some but still got a few soupy ones so i decided to add a little pectan and they come out perfect hope you find as mush success with this tek as i did




       __________________ 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.




       I am an aspiring 'shroom grower myself. I have completed the single-strain isolation phase and I am ready to move on to the grain cultivation stage. After I have completed a harvest, I hope to post a list of my trials and tribulations to 'alt.drugs' including any tips or hints I discover along the way. This article contains one important tip. Read on! P.D.A. and M.E.A. are great alternatives to buying pre-packaged agar from somewhere like a




      scientific supply. I purchased 4 oz. of pre-mixed malt extract agar and spent $20 for it... and when you use 15-20 g of that mix per liter, it's not nearly as cost effective as doing it from scratch. Please note that most of my info comes from McKenna's definitive book: _Psilocybin: Magic Mushroom Grower's Guide_. No rights reserved, and while Terry's publisher might have something to say, I think he would be happy to see the spread of the information that leads to successful shroom cultivation.




      I mixed up a batch of PDA (Potato Dextrose Agar) from scratch. I have had great results and it's easy to make. After the recipe process, I'll explain how/where to obtain the materials and what to look for. McKenna says: For 1 liter total volume of liquid PDA agar you need: 250 g potatoes 15 g agar 10 g dextrose 1.5 g nutritional yeast (or yeast extract) Shred the unpeeled potatoes into a colander and then rinse them for thirty seconds with cold tap water. Combine the rinsed potatoes with one liter of water and gently boilf for 30 minutes. Filter the resulting potato broth through muslin or cheescloth and discard the potatoes. To the liter of potato broth add the agar, dextrose and yeast which you have previously weighed out, mixed together, and set aside. While stirring gently add in the mixed powdered indgredients. Gently boil for ten minutes or until the solution is clear. Take care to not allow the solution to boil over. Add enough water to return the total volume of the solution to one liter. Pour the solution while still hot and liquid into petri plates, baby food jars, or slant culture tubes. Use just enough to cover the bottom of the containers, about 1/4 inch deep or a little more. The solution may be allowed to cool or sterilized immediately.




      There are a few different recipes for different types of agar. McKenna recommends changing agar types every few batches to prevent your Psilocybe cubensis mycelium from experiencing the "senescence" factor, or the tendency of the mycelium to "age and lose vigor." Here are some other agar recipes, mixed and used in much the same manner as PDA. For 1 liter total volume of MEA you need: 20 g malt or malt extract (powdered or syrup) 25 g agar 0.1 g potassium dibasic (K2HPO) 0.1 g calcium carbonate (CaCO3) (basically powdered oyster shell) Add these ingredients to one liter of boiling water. After they have dissolved it is ready to be poured.




      There is another agar recipe which McKenna says is good for long-term maintenance. It is MYP or Malt-Yeast-Peptone agar. The procedure for preparing this mixture is the same as for MEA. For 1 liter total volume of MYP you need: 7 g malt extract (powdered or syrup) 1 g peptone or soytone 0.5 g yeast extract 15 g agar




      A note on the ingredients. Agar (agar-agar) itself is a powdered extract of the gelatinous elements of sea-kelp. It may be found in health food stores, food co-ops or places that specialize in natural foods. It can be expensive (I have seen it for $50 a pound) but you don't need much to make a batch.




       Dextrose is CORN sugar and may be found for about $1 a pound at a beer/wine brewing supply center. Be sure to get CORN sugar and not CANE sugar (sucrose). The same goes for malt extract, easily found in syrup or powder form in a brewing supply store, or in some health food stores. Soytone and Peptone are commercial brands of a protein hydrosylate and can be purchased form scientific or microbiological supply houses.




       The other ingredients can be found at scientific supply houses and are definitely not on any controlled subtance lists. This is one of the beauties of 'shroom cultivation as opposed to manufacture of other substances.




       Here's some info on agar sterilization. And by sterilization I am referring to the use of a pressure cooker/canner which is a substitute for a lab- grade autoclave. I don't know much about the kits you see advertised everywhere, but these sterilization-free kits tend to suffer from a high contamination rate. Even though I am relatively new to growing mushrooms, after nearly 50 cultures cultivated in baby food jars in a few weeks, I have not had one contaminated culture. You just need to sterilize properly and keep all you surfaces clean. A fume hood is nice, but not necessary: I use a polyethylene tarp draped over my table work-area. Be ruthless with the Lysol or whatever disinfectant you use...but look out for the flammable ones (like Lysol) and keep them away from open flame.




       And my tip on agar sterilization: don't overdo it! In McKenna's book, he cites how the standard sterilization time at 250 degrees (15 psi) for agar is 15 minutes. But he claims this is not long enough and recommends 45 minutes to an hour. Presumptious of me as it is, I say NO! Several of my agar cultures were ruined by the intense heat of the pressure cooker. After they were done, they would cool but not congeal...the molecules that do the gelling had been severed by the heat. They just sort of sloshed around in clumps and actually should set into flat, glassine material (like JELL-O!). In a few experiments, I found that 250 degree, 15 psi was good, but I only needed to sterilize the cultures at this temperature for 30 MINUTES. I have had no problems with contamination so far.




      Moonflower's Cheap ---Rice Malt /alfalfa/ Brewer's Yeast Agar 1 ea Quart Jar (Mason/Ball type with lids) Take 2 cups of rice and place in a 3 quart bowl or pan. Pour approx. 1 1/2 quarts of clean (bottled or rev osmosis) water into bowl. Stir in 1 cup of alfalfa (like in the health food stores) meal. Add 1 standard dolomite (oyster shell-crushed) tablet and allow to dissolve in resultant slop. Allow to sit for 2 hours/stir every 20 minutes or so. It is ok to warm the mixture to speed up the expression of nutrients, but cold soaking seems to be the best. (takes longer, but has a better quality) The water should be about 70-80 degrees F. for best results not _chilled water. Strain out rice/alfalfa and throw it away.




       Mix 1 pkg of your regular baker's yeast into liquid and stir in. Let the mixture sit for 30 minutes to an hour or until it is obvious that the yeast are activated. (usually a foamy looking bubble mas will be floating on top and the mixture may appear more opaque) Strain out and throw away solid yeast grains. Boil the resultant liquid rapidly for 5 minutes and then turn off the heat and add 18 grams of agar agar (aka red algae) that you bought at the health food store. Pour mixture into mason/Ball jar and put lids on (seal down).




       Sterilize in pressure cooker for 20 minutes at 15lbs and allow to cool. (this is at sea-level... people in high altitudes should consult a conversion table for equiv. pressures and times) You will now have 1 quart of a very good agar media for mycoculturing that will support _luxuriant_ mycelial growth. It is more than sufficient for starting spores as well and seems to produce a lot more "banding" than other media I have tried. Banding is when the mycelia (dikaryotic) grows in a pattern that resembles circles of threadlike fungal strands similar to tree rings on the petri dish. I have seen carpophores form directly on the agar and fruit in a covered dish on this media and permeation time seems to be cut in half in grain media spawn.




       Rabbit dung Agar: ========================== Approx 3 pellets of rabbit dung or equivalent is placed in 100ml water. agar is added, the mixture autoclaved and plates poured. ...




       ------------------------ ------------------------------------- Horse manure extract: ===================================== {see Fastfreds agar cookbook for alternating recipe} 2parts Hpoo 4 parts Distilled H2o Bring mixture to boil and simmer for 2 hours. filter through cloth. Sterilization: 15 minutes at 121 degrees celsius




       ------------------------------------- Oatmeal extract: ====================================== 30g oatmeal 1 liter water Wrap oatmeal flakes in cloth and T-bag Into Water. Bring to boil and simmer 2 hours. squeeze and filter through cloth. Sterilization: 15 minutes at 121 degrees celsius




       -------------------------------------- OHMA (Oatmeal Horse manure Agar): ======================================= .33 liters horse manure extract .66 liters oatmeal extract 15 grams agar




       Detailed instructions for Moonflower's Cheap ---Rice Malt /alfalfa/ Brewer's Yeast Agar: =============================================================




      1 ea Quart Jar (Mason/Ball type with lids) Take 2 cups of rice and place in a 3 quart bowl or pan.




      Pour approx. 1 1/2 quarts of clean (bottled or rev osmosis) water into bowl.




       Stir in 1 cup of alfalfa (like in the health food stores) meal.




      Add 1 standard dolomite (oyster shell-crushed) tablet and allow to dissolve in resultant slop.




      Allow to sit for 2 hours/stir every 20 minutes or so. It is ok to warm the mixture to speed up the expression of nutrients, but cold soaking seems to be the best. (takes longer, but has a better quality) The water should be about 70-80 degrees F. for best results not _chilled water.




      Strain out rice/alfalfa and throw it away.




      Mix 1 pkg of your regular baker's yeast into liquid and stir in.




      Let the mixture sit for 30 minutes to an hour or until it is obvious that the yeast are activated. (usually a foamy looking bubble mas will be floating on top and the mixture may appear more opaque)




      Strain out and throw away solid yeast grains. Boil the resultant liquid rapidly for 5 minutes and then turn off the heat and add 18 grams of agar agar (aka red algae) that you bought at the health food store.




      Pour mixture into mason/Ball jar and put lids on (seal down).




      terilize in pressure cooker for 20 minutes at 15lbs and allow to cool. (this is at sea-level...people in high altitudes should consult a conversion table for equiv. pressures and times)




       You will now have 1 quart of a very good agar media for mycoculturing that will support _luxuriant_ mycelial growth. It is more than sufficient for starting spores as well and seems to produce a lot more "banding" than other media I have tried. Banding is when the mycelia (dikaryotic) grows in a pattern that resembles circles of threadlike fungal strands similar to tree rings on the petri dish. I have seen carpophores form directly on the agar and fruit in a covered dish on this media and permeation time seems to be cut in half in grain media spawn.




       These recipes are designed for agar maintenance of Psilocybe , but also other mushroom species. All media have a near-neutral pH and should be sterilized for twenty to thirty minutes at 15 psi pressure. 500 ml of medium will pour around 20 petri dishes. Using a different medium from time to time will prevent the culture getting used to one medium.




       Malt Extract Agar (MEA) 10 grams light malt extract 9 grams agar agar 500 ml potable or distilled water Potato Dextrose (Yeast-extract) Agar (PD(Y)A) broth from boiling 150 grams sliced potatoes in 500ml water for 30 minutes(add water to 500ml) (You can use 5 g of instant potato flakes instead) 9 g agar agar 7 grams dextrose( = glucose, or 10 ml honey or corn syrup ) 1 gram brewers yeast or yeast-extract (optional)




       Amaranth Soy Agar 20 grams amaranth flour 20 grams soy flour 9 grams agar agar 500 ml potable or distilled water




       EntheoGenesis No.442 10 grams amaranth flour 10 grams brown rice flour 10 grams potato flour 10 grams soy flour 2 grams malted barley 9 grams agar agar 500 ml potable or distilled water




       Oatmeal Neopeptone Agar 40 grams oatmeal or oat flour 2 grams neopeptone (optional) 9 grams agar agar 500 ml potable or distilled water




       Modified Sabouraud's Medium 25 grams barley flour 5 grams dextrose 2 grams neopeptone (optional) 1 gram yeast extract 9 grams agar agar 500 ml potable or distilled water




       Cornmeal Dextrose Agar 25 grams yellow cornmeal 3 grams dextrose 9 grams agar agar 500 ml potable or distilled water




       Barley Malt Extract Agar 40 grams barley flour 2 grams malt extract 1 gram yeast extract (optional) 9 grams agar agar 500 ml potable or distilled water




       Dr. Pollock's Modified Agar 10 grams dried dog food (ground to flour) 10 grams amaranth flour 2 grams dextrose or malt extract 9 grams agar agar 500 ml potable or distilled water




       Grey Cardboard (instead of agar) The goodb stuff Here are the detailed steps for making cardboard plates. Note that you can also use small jars in place of Petri dishes. 1) Measure about 100 mls. of tap water into a small jar. 2) For nutrients, , measure another 100 mls. of tap water into a second jar and add one drop of ordinary soy sauce to the water, and a quarter teaspoon (1.25 mls.) of molasses or light malt powder. 3) Find some gray cardboard, the thicker the better, preferably gray on both sides. Trace a Petri plate onto the cardboard with a pencil and cut out several disks to fit into your plates. 4) Weigh one of your disks and record the weight. Multiply this weight by a factor of 1.3 as a rough guide (you may need to experiment with the amounts here), and add the resulting weight of tap water or nutrient solution to each disk in its Petri plate. (Remember, 1 ounce of water equals 28.35 grams; one gram equals one milliliter.) Example: Suppose my disks weighed 0.17 ounces each. Multiplying 0.17 by 1.3, I get 0.22 ounces. There are 28.35 grams in an ounce, so 0.22 ounces x 28.35 equals 6.3 grams. That means I'll add 6.3 milliliters of solution to each disk. 5) Close up the disks in the plates, and let the water or nutrient solution soak in. 6) Pressure-sterilize the jar of plain water, and the Petri plates with moistened newsprint disks inside, for 10 minutes at 15 psi (allowing the cooker to equilibrate steam for 10 minutes before putting on the pressure regulator). 7) Cool the cooker, and remove the plates and jar of plain water.




       8) When the water has cooled, add 3.3 mls. 3% peroxide to the jar, using a pasteurized pipette, to give you a final concentration of about 0.1% peroxide in sterile water. 9) Add about one third of the initial weight of the cardboard as 0.1% peroxide to each disk. Let the solution soak completely into the disks. They are now ready to use. [23]




       A. Classic Favorite Recipes Potato Dextrose Agar (PDA)(FDA M127) Potato Dextrose Yeast (PDY) Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93) Malt Extract Agar with Yeast (2%)(MEAY) Sabouraud's Dextrose Broth and Agar (SabDex or SDA)(FDA M133)




       B. Special Blends "Karo Water" (Corn Syrup Broth) "Dextrose Tek" Liquid Media (Corn Sugar Broth) Honey Water (Mycotopia Honey Tek) Malt-Yeast-Peptone Agar (McKenna's MYP) Malt Yeast Peptone Agar (Stamets MYP) Grey Cardboard (instead of agar) Oatmeal Flake Agar Moonflower's Rice Malt-Alfalfa-Brewer's Yeast Agar MycoPsycho's Liquid Mycelial Culture Broth [Malt base] Ragadinks Liquid Medium Amaranth Soy Agar EntheoGenesis No.442




       C. Food Based Recipies and Variations Corn Meal Agar (CMA) Cornmeal Dextrose Agar Potato Flake Agar Potato Starch Agar Potato Flake Agar with Yeast Potato-Carrot Agar Barley Flour Malt Extract Agar Barley Flour Modified Sabouraud's Oatmeal Agar (OA) Oatmeal Agar A Oatmeal Agar [B] V-8 Oatmeal Agar V8 Medium Bean Agar Faba Bean Dextrose Agar (FDA) Pea Agar Cabbage Agar Dr. Pollock's Modified [Dog Food] Agar




       D. Other Media and Alternate Formulations Potato-Glucose Agar 1 Potato-Peptone Medium Potato-Peptone-Yeast Agar (PPYA)




       ------ Malt Agar (MA)(FDA M185)[aka 2% MEA] Difco Malt Extract Broth (FDA M94) Malt Extract Agar for Yeasts and Molds (MEAYM)(FDA M182) Malt Extract Peptone Agar Raper & Thom MEA (RTMEA) ISP 2 Medium (Malt, Yeast, Glucose)




       ------ Yeast Extract Agar (YEA)(FDA M181) Yeast Glucose Agar Glucose and Yeast Extract Agar Glycerin Yeast Agar




       ------ Manure Tincture Manure Agar




       ------ Water Agar (aka Starved Agar) Gelatin Agar (GA)(FDA M54) Plate Count Agar (SMA)(aka Standard Methods Agar)(FDA M124) Nutrient Agar (FDA M112) Starch Agar (FDA M143)(Nutrient agar with starch) Starch-Yeast Agar 1/5 Starch-Yeast Agar Long-term Preservation Medium (FDA M85) Peptone Meat Agar (Meat Water)




       E. Common Solutions Gentamicin Sulfate Solution (FDA M57)




       F. Sources




       -------------------------------------------------------------------------------- Section A: Basic recipes




       -------------------------------------------------------------------------------- Potato Dextrose Agar (PDA)(FDA M127) 200 g Potato infusion [dilute to 1L total, ~4g solids] 20 g Dextrose 20 g Agar 1 L Distilled water (dH2O)




       To prepare potato infusion, boil 200 g scrubbed, sliced(unpeeled) potatoes in 1 liter distilled water for 30 min. Filter through cheesecloth, saving effluent, which is potato infusion (or use commercial dehydrated form). Mix in other ingredients and boil to dissolve. [Dilute to obtain 1 L final volume] Autoclave 15 min at 121�C. Dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. Final pH, 5.6 � 0.2.




       Medium should not be re-melted more than once. Medium powder is available commercially but may require supplementing with extra agar to a final concentration of 20 g/liter. To BBL or Difco dehydrated medium, add 5 g of agar. The broth is clear to slightly opalescent and yellowish in color. [1][2]




       Potato Dextrose Yeast (PDY) 200 g Potato, infusion from [dilute to 1L total] 20 g Dextrose 2 g Yeast extract 20 g Agar 1 L Distilled water (dH2O) Gently boil for ten minutes or until the solution is clear. Autoclave 15 min at 121�C. [13]




       Malt Extract Agar (3%)(MEA)(General Microbiology)(FDA M93) 30 g Malt extract 20 g Agar 1 L Distilled water Boil to dissolve ingredients. Avoid overheating, which causes softening of agar and darkening of medium color. Autoclave 15 min at 121�C. Dispense 20-25 ml into sterile 15 x 100 mm petri dishes. Final pH, 5.5 � 0.2. This medium is recommended as a general maintenance medium. [1]




       Malt Extract Agar with Yeast (2%)(MEAY) 20 g extra light malt extract 2 g yeast 15-20 g agar 1 L water [10]




       Sabouraud's Dextrose Broth and Agar (SabDex or SDA)(FDA M133) 40 g Dextrose 10 g Polypeptone or neopeptone 1 L Distilled water




       Dissolve completely and dispense 40 ml portions into screw-cap bottles. Final pH, 5.8. Autoclave 15 min at 118-121�C. Do not exceed 121�C.




       For Sabouraud's dextrose agar, prepare broth as above and add 15-20 g agar, depending on gel strength desired. Final pH, 5.6 � 0.2. Dispense into tubes for slants and bottles or flasks for pouring plates. Autoclave 15 min at 118-121 �C. [1]




       Sabouraud Dextrose (SabDex) Agar is used for the isolation, cultivation, and maintenance of saprophytic and pathogenic yeasts and fungi.




       SabDex Agar is an excellent substitute for Malt or Potato Dextrose Agar, when used by mushroom cultivators to propagate mushroom mycelium.




       Sabouraud Dextrose Agar was described by Sabouraud in 1892 and was used for the identification of fungi based on their morphological characteristics. Sabouraud Dextrose Agar is a standard medium used to support the growth of yeasts and molds. It supplies peptone as the protein source and dextrose as the carbohydrate source for nourishment. Bacterial suppression occurs due to the low pH. This media is especially suited for the primary isolation of fungi from normally sterile sites such as cerebrospinal fluid (CSF).




       Later, Emmons modified the medium by decreasing the dextrose content and adjusting the pH closer to the neutral range. This modification enhances sporulation and is particularly useful for the subculture of fungi that so not develop fruiting structures on other media, and so is useful in their identification. It also serves as a good holding medium for stock cultures. [3]




       -------------------------------------------------------------------------------- B. Special Blends




       -------------------------------------------------------------------------------- "Karo Water" (Corn Syrup Broth) 1 Teaspoon Light Corn Syrup (Karo Syrup or store brand Light Corn Syrup) 100 ml Purified Water Mix well until dissolved, sterilize for 20-30 minutes at 15 psi. [17]




       "Dextrose Tek" Liquid Media (Corn Sugar Broth) 1 Teaspoon Powdered Dextrose (corn sugar) 75 ml water [17]




       Honey Water (Mycotopia Honey Tek) 40 g Honey (or roughly 1 tablespoon per pint of water) 1 L Water The correct mixture for optimum results is 4% sugars (honey) by weight and 96% water. Water weighs 1 gram per cc/ml so if you use 100 ml as total weight, then 96 grams/ml/cc of water is mixed with 4 grams of honey, etc. Sterilize for 30 min at 121�C (15 psi). [14]




       Malt-Yeast-Peptone Agar (McKenna's MYP) 7 g malt extract (powdered or syrup) 1 g peptone or soytone 0.5 g yeast extract 15 g agar [11]




       Malt Yeast Peptone Agar (Stamets MYP) 20 g extra light malt extract 1 g yeast extract 1 g peptone 15-20 g agar 1 L water [10]




       Grey Cardboard (instead of agar) Here are the detailed steps for making cardboard plates. Note that you can also use small jars in place of Petri dishes.




      1) Measure about 100 mls. of tap water into a small jar.




      2) For nutrients, , measure another 100 mls. of tap water into a second jar and




      add one drop of ordinary soy sauce to the water, and a quarter teaspoon (1.25 mls.) of molasses or light malt powder.




      3) Find some gray cardboard, the thicker the better, preferably gray on both sides. Trace a Petri plate onto the cardboard with a pencil and cut out several disks to fit into your plates.




      4) Weigh one of your disks and record the weight. Multiply this weight by a factor of 1.3 as a rough guide (you may need to experiment with the amounts here), and add the resulting weight of tap water or nutrient solution to each disk in its Petri plate. (Remember, 1 ounce of water equals 28.35 grams; one gram equals one milliliter.) Example: Suppose my disks weighed 0.17 ounces each. Multiplying 0.17 by 1.3, I get 0.22 ounces. There are 28.35 grams in an ounce, so 0.22 ounces x 28.35 equals 6.3 grams. That means I'll add 6.3 milliliters of solution to each disk.




      5) Close up the disks in the plates, and let the water or nutrient solution soak in.




      6) Pressure-sterilize the jar of plain water, and the Petri plates with moistened newsprint disks inside, for 10 minutes at 15 psi (allowing the cooker to equilibrate steam for 10 minutes before putting on the pressure regulator).




      7) Cool the cooker, and remove the plates and jar of plain water.




      8) When the water has cooled, add 3.3 mls. 3% peroxide to the jar, using a pasteurized pipette, to give you a final concentration of about 0.1% peroxide in sterile water.




      9) Add about one third of the initial weight of the cardboard as 0.1% peroxide to each disk. Let the solution soak completely into the disks. They are now ready to use. [23]




       Oatmeal Flake Agar 75 g Oatmeal flakes 20-25 g Agar 1 L water Stir for 5-10 minutes then filter out the larger particles by pouring it through some mesh, save the broth. [Then add the agar] This is the best medium for Panaeolus cyanescens I've ever encountered. Beware, this medium will be less firm than the other recipes so extra agar has to be added to compensate. [22]




       Moonflower's Rice Malt-Alfalfa-Brewer's Yeast Agar 1 cup alfalfa, infusion from 2 cups rice, infusion from 1 standard dolomite (oyster shell-crushed) tablet 1 pkg of regular baker's yeast Prepare infusion using approx. 1 1/2 quarts of clean water. Mix in the alfalfa and rice, then allow to soak for 2 hours at room temp with occasional stirring. Filter or strain before adding the infusion. Prepare yeast by activating in water for around 30 minutes, then strain out solid yeast grains. Sterilize in pressure cooker for 20 minutes at 15lbs. This media is reported to produce more "banding" than other media. Will support luxuriant mycelial growth, it is also more than sufficient for starting spores. [19]




       MycoPsycho's Liquid Mycelial Culture Broth 1/2 tsp malt (2.5 ml) 3/4 cup water 1. procure a half-pint jar w/ lid & ring and also a spore syringe. 2. drill/punch a hole in the center of the lid large enough for the syringe. 3. mix 3/4 cup of water with 1/2 tsp.(2.5 ml.) malt. 4. put lid & ring on and put aluminum foil over the top of that and then Pressure Cook (PC) for 20 min. @ 15 psi. , when finished let cool to below 90 degrees F. 5. once cool, setup your sterile environment (flow hood, glove box or even your oven will work) with an alcohol lamp, extra alcohol in a dish, paper towels/napkins, small round bandaid and lastly the spore syringe. 6. remove the foil from the jar, shake the needle and then sterilize the point with the alcohol lamp (get it red hot), cool the needle with a wipe & some alcohol and then inject 1-2 cc/mil spore solution into the jar. 7. once injected wipe the needle w/alcohol and put the guard back on it. 8. cover injection site w/ round bandaid (thanks for that tip magash!). 9. put the jars in your incubator set at 82-84 degrees F. for 4-14 days, you should notice growth by then. 10. sterilize empty syringes in your PC for 15-20 @ 15 psi. 11. remove the bandaid from the jar, sterilize your needle again and then suck myc solution into empty syringe(s) for use on PF style jars or substrate bags. [15]




       Ragadinks Liquid Medium 16.5 g dextrose 1.5 g yeast 500 ml tap water [16]




       Amaranth Soy Agar 20 g amaranth flour 20 g soy flour 9 g agar 500 ml potable or distilled water Sterilize for 20-30 minutes at 15 psi. [20]




       EntheoGenesis No.442 10 g amaranth flour 10 g brown rice flour 10 g potato flour 10 g soy flour 2 g malted barley 9 g agar 500 ml potable or distilled water Sterilize for 20-30 minutes at 15 psi. [20]




       -------------------------------------------------------------------------------- Section C: Food Based Recipes and Variations




       -------------------------------------------------------------------------------- Corn Meal Agar (CMA) -------------------- 2 g Corn Meal, Infusion from [filter] 15 g Agar 1 L dH2O [4]




       Cornmeal Dextrose Agar ---------------------- 25 g yellow cornmeal 3 g dextrose 9 g agar 500 ml potable or distilled water [20]




       Potato Flake Agar ----------------- 20.0 g Potato Flakes 10.0 g Dextrose 15.0 g Agar 1.0 L Demineralized Water Potato Dextrose Agar and Potato Flake Agar are formulations developed to promote sporulation of fungi. The potatoes contained in these media provide nutritious bases for luxuriant growth of fungi. Both media contain dextrose as a growth stimulant. pH 5.6 +/- 0.2 @ 25�C [7]




       Potato Starch Agar ------------------ 30 g potato starch, soluble 20 g dextrose/glucose 15 g agar, pure (omit for liquid media) 1000 mL (d)H2O The pH is adjusted following autoclaving to prevent agar hydrolysis by acid. [20]




       Potato Flake Agar with Yeast ---------------------------- 20 g potato flakes 10 g glucose 1-2 g dried yeast 20 g agar 1 L water [18]




       Potato-Carrot Agar ------------------ grated potato 20.0 g grated carrot 20.0 g Agar 20.0g Tap water 1000.0 ml Boil potato and carrot in 1000.0 ml of water for 1 h, filter, add water to the initial volume, adjust pH to 7.0 - 7.1 and add agar. Sterilize at 121�C for 30 min. [6]




       Barley Flour Malt Extract Agar ------------------------------ 40 g barley flour 2 g malt extract 1 g yeast extract (optional) 9 g agar 500 ml potable or distilled water Sterilize for 20-30 minutes at 15 psi. [21]




       Barley Flour Modified Sabouraud's --------------------------------- 25 g barley flour 5 g dextrose 2 g Polypeptone or neopeptone (optional) 1 g yeast extract 9 g agar 500 ml potable or distilled water Sterilize for 20-30 minutes at 15 psi. [21]




       Oatmeal Agar (OA) ----------------- 60.0 g Oatmeal [filter] 12.5 g Agar [may require more] 1.0 L dH2O Cook oatmeal 5-10 minutes then filter the liquid into another container using cheesecloth or a metal strainer with a tight mesh. Dilute liquid to 1L, add agar, and heat with swirling until solids dissolve. This is reported to be a good media for cultivating Panaeolus cyanescens. [5]




       Oatmeal Agar A -------------- Oatmeal 20.0g Agar 20.0g Tap water 1000.0 ml pH 7.2. [6]




       Oatmeal Agar B ---------------- Oats 30.0g Agar 15.0g Tap water 1000.0 ml Keep oats on a water bath at 58�C for 1 h, filter through 2 layers of gauze, dilute to 1000.0 ml and add 15.0 g agar. [6]




       V-8 Oatmeal Agar ---------------- 50 ml V-8 juice 25 g Cream of oats 20 g Agar 1 L Water Be careful to use a container much larger then the volume of medium, i.e., prepare a 500 ml medium in 2 liter flasks or it will tend to boil over no matter how slowly it is cooled down. [20]




      V8 Medium --------- 50 ml V8 Juice .2 g CaCO3 20 g of agar 1 L Water The commercial V8 juice is occasionally used for tissue cultures of edible mushrooms. It should be noted that most mushrooms prefer neutral to slightly acid range of medium, that is, a pH of about 5.5 to 6.5. However the straw mushroom, Volvariella volvacea, prefers a high pH medium, 6.8 to 7.8. Therefore, it is important to make sure the acidity or pH of the medium is correct for a particular mushroom. Here be careful to use a container much larger then the volume of medium, i.e., prepare a 500 ml medium in 2 liter flasks or it will tend to boil over no matter how slowly it is cooled down. [20]




      Bean Agar --------- Beans (peas or pulse) 100.0 g [infusion from (filter)] K2HPO2 0.5g Sucrose 10.0g Agar 20.0g Water 1000.0ml Prepare infusion from beans. Sterilize at 121�C for 30 min. [6]




      Faba Bean Dextrose Agar (FDA) ----------------------------- 200 g faba bean seeds or 400 g of faba bean leaves [infusion from] [autoclave and filter to obtain faba infusion] 20 g of dextrose 18 g agar 1 L water Method: a. Weigh out 200 g of faba bean seeds or 400 g of faba bean leaves in a 1.5 1 flask. Add 1L of water, and autoclave at 15 psi. for 30 minutes. b. Pass the autoclaved beans through a sieve, add 18 g of agar, heat, and stir till dissolved. c. Add 20 g of dextrose, stir till dissolved, and make up the volume to 11 with tap water. d. Autoclave at 15 psi. for 20 minutes, cool to about 40�C, and pour into petri dishes (normally 40 petri dishes/1). This medium is used for propagation of B. fabae, A. fabae, and A. tenuis. [12]




       Pea Agar -------- 100 g Yellow peas 0.5 g K2HPO2 10.0 g Sucrose 20.0 g Agar 1.0 L Tap water Boil peas in 1000.0 ml of water, filter through gauze, add water to the initial volume; add phosphate, sucrose, and agar. Sterilize at 121�C for 30 min. [6]




       Cabbage Agar ------------ Cabbage 50.0g glucose 20.0g Peptone 10.0g Agar 20.0g Tap water 1000.0 ml Boil 50.0 g of cabbage in 1000.0 ml of water, filter cabbage, adjust the volume of broth to the initial value. [6]




       Dr. Pollock's Modified [Dog Food] Agar -------------------------------------- 10 g dried dog food (ground to flour) 10 g amaranth flour 2 g dextrose or malt extract 9 g agar 500 ml potable or distilled water Sterilize for 20-30 minutes at 15 psi. [20]




       -------------------------------------------------------------------------------- Section D: Other Media and Alternate Formulations --------------------------------------------------------------------------------




      Potato-Glucose Agar 1 --------------------- grated potato 200.0 g glucose 20.0g Agar 20.0g Tap water 1000.0 ml Boil potatoes for 1 h in 1 L of water, filter through gauze. add water to the initial volume, add glucose and agar. Sterilize at 105�C for 30 min. [6]




      Potato-Peptone Medium --------------------- Potato decoction 200 ml [infusion from 200g potato] Yeast extract 1.0 g Peptone 5.0g Agar 30.0g Distilled water 800.0 ml [6]




      Potato-Peptone-Yeast Agar (PPYA) -------------------------------- Potato decoction 200 ml [infusion from 200g potato] 200.0 ml Peptone 5.0g Yeast extract 1.0 g Agar 25.0g Distilled water to 1000.0 ml pH 8.0. [6]




      Malt Agar (MA)(FDA M185)[aka 2% MEA] ------------------------------------ 20 g Malt extract, powdered 20 g Agar 1 L Distilled water Mix ingredients, steam to dissolve agar and sterilize for 15 min at 121�C. Temper medium to 45�C and pour plates under aseptic conditions. This medium is recommended as a general maintenance medium. [1]




       Difco Malt Extract Broth (FDA M94) ---------------------------------- 6.0 g Malt extract base 1.8 g Maltose, technical 6.0 g Dextrose 1.2 g Yeast extract 1.0 L Water Final pH, 4.7 � 0.2. [1]




       Malt Extract Agar for Yeasts and Molds (MEAYM)(FDA M182) -------------------------------------------------------- 20.0 g Malt extract, powdered 20.0 g Glucose 1.0 g Peptone 20.0 g Agar 1.0 L Distilled water Mix ingredients, heat to dissolve agar and sterilize at 121�C for 15 min. Temper media to 45�C and pour plates under aseptic conditions. Dehydrated MA is commercially available, but since several MA formulas exist, check for the correct composition. Final pH 5.4. [1]




       Malt Extract Peptone Agar ------------------------- 30 g Malt extract 3 g Soya peptone 15 g Agar 1 L Distilled water Adjust pH to 5.6. Sterilize at 121�C for 10 min. [8]




       Raper & Thom MEA (RTMEA) ------------------------ To 2% MEA add: Glucose 10g Soy Peptone 5g [9]




       ISP 2 Medium (Malt, Yeast, Glucose) ----------------------------------- Malt extract 10.0 g Yeast extract 4.0 g Glucose 4.0g Agar 15.0g Distilled water 1000.0 ml pH 7.2. [6]




       Yeast Extract Agar (YEA)(FDA M181) ---------------------------------- 10.0 g Proteose peptone 3.0 g Yeast extract 5.0 g NaCl 15.0 g Agar 1.0 L Distilled water Adjust pH to 7.2-7.4. Autoclave at 121�C for 15 min. [1]




       Yeast Glucose Agar ------------------ Yeast extract 5.0 g Glucose 10.0g Peptone 5.0g Agar 20.0g Distilled water 1000.0 ml pH 7.2. Sterilize at 121�C for 15 min. [6]




       Glucose and Yeast Extract Agar ------------------------------ Glucose 20.0g Yeast extract 10.0 g CaCO2 20.0g Agar 17.0g Distilled water 1000.0 ml [6]




       Glycerin Yeast Agar -------------------- Yeast extract 5.0 g Glycerin 50.0g (also called glycerine or glycerol) CaCO2 1.0g Agar 20.0g Distilled water 1000.0 ml [6]




       Manure Tincture --------------- Cow manure (fresh) 1.0 kg Distilled water 3000.0 ml Boil, squeeze through gauze into a bottle and dilute 3 to l. [6]




       Manure Agar ----------- Horse manure 100-125 g Agar 25.0g Distilled water 1000.0 ml




      Boil manure in 1000.0 ml of water for 10 min, then keep for 16-20 h, filter through 1-2 layers of filter paper, adjust to the initial volume, add agar. Sterilize at 121�C for 15 min. [6]




       Water Agar (aka Starved Agar) ----------------------------- Agar 20.0g Distilled water 1000.0 ml Sterilize at 121�C for 15 min. [6]




       Gelatin Agar (GA)(FDA M54) -------------------------- 4 g Peptone 1 g Yeast extract 15 g Gelatin 15 g Agar 1 L Distilled water Suspend ingredients with constant stirring to prevent scorching gelatin, and boil to dissolve gelatin and agar. Adjust to pH 7.2 � 0.2. Autoclave 15 min at 121�C. Cool to 45-50�C. Pour plates. [1]




       Plate Count Agar (SMA)(aka Standard Methods Agar)(FDA M124) --------------------------------------------- 5.0 g Tryptone 2.5 g Yeast extract 1.0 g Dextrose 15.0 g Agar 1.0 L Distilled water Heat to dissolve ingredients. Dispense into suitable tubes or flasks. Autoclave 15 min at 121�C. Final pH, 7.0 � 0.2.




      For viable yeasts and molds, dispense 20-25 ml portions into sterile 15 x 100 mm petri dishes. [1]




      Nutrient Agar (FDA M112) ------------------------ 3 g Beef extract 5 g Peptone 15 g Agar 1 L Distilled water Heat to boiling to dissolve ingredients. Dispense into tubes or flasks. Autoclave 15 min at 121�C. Final pH, 6.8 � 0.2. [1]




       Starch Agar (FDA M143)(Nutrient agar with starch) ------------------------------------------------- 23 g Nutrient agar (FDA M112) 10 g Potato starch 1 L Distilled water Heat to dissolve agar in 500 ml water. Dissolve starch in 250 ml water. Combine and dilute to 1 liter. Autoclave 15 min at 121�C. Note: add 3 g agar to Difco's dehydrated starch agar. [1]




       Starch-Yeast Agar ----------------- Yeast extract 2.0 g Starch (soluble) 10.0 g Agar 20.0g Tap water 1000.0 ml pH 7.3. [6]




       1/5 Starch-Yeast Agar --------------------- Yeast extract 0.4 g Soluble starch 2.0 g Agar 20.0 g Distilled water 1000.0 ml pH 7.3. [6]




       Long-term Preservation Medium (FDA M85) --------------------------------------- 3 g Yeast extract, 0.3% 10 g Peptone 30 g NaCl 3 g Agar 1 L Distilled water Heat to dissolve ingredients. Dispense 4 ml portions to 13 x 100 mm screw-cap tubes. Autoclave 15 min at 121�C. Cool and tighten caps for storage. No pH adjustment is necessary. [1]




      Peptone Meat Agar (Meat Water) ------------------------------ Peptone 10.0g NaCl 5.0g [optional] Agar 20.0g Meat water 1000.0 ml Preparation of meat water: comminute 500 g of meat free of bones, fat and tendons, add 1000.0 ml of tap water and leave for 12 h at room temperature or in a thermostat at 30�C, or for 2 h at 37�C. Then squeeze the meat through gauze or cloth and boil the filtrate for 5 min. The proteins are denatured. Filter the cooled down mass through a cotton-wool filter and add water to the initial volume. pH 7.2 - 7.4. Sterilize at 121�C for 30 min. [6]




       -------------------------------------------------------------------------------- E. Common Solutions --------------------------------------------------------------------------------




      Gentamicin Sulfate Solution (FDA M57) ------------------------------------- 5.00 g Gentamicin sulfate 1.00 L Distilled water Sterilize by filtration through 0.20 �m membrane. Store at -20�C. [1] Chloramphenicol 0.2g/liter of media survives autoclaving.




       -------------------------------------------------------------------------------- F. Sources -------------------------------------------------------------------------------- [1] Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. [2] EMD Chemicals [aka Merck] [3] PML Microbiologicals [4] Sigma Fine Chemicals [5] Sigma Fine Chemicals, instructions by FastFred [6] VKM Media Catalog 2003 [7] remel [8] DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Germany [9] Xenova Limited, UK [10] Paul Stamets (GGMM and/or TMC) [11] Terence McKenna [12] Screening Techniques for Disease Resistance in Faba Beans [13] FastFred [14] Mycotopia Honey Tek [15] MycoPsycho (Shroomery) [16] Ragadinks (Shroomery) [17] Nan (Nanook) [18] Pinback (Shroomery) [19] Moonflower (???) [20] Unknown (Shroomery) [21] Unknown (Shroomery) edited by FastFred [22] [i]Unknown (Shroomery), comments by Una[/i] [23] Rush Wayne's Peroxi Manual Volume II (Via Hippie3)(Mycotopia) [24] USDA Complete Guide to Home Canning




       Grey Cardboard (instead of agar)




       Here are the detailed steps for making cardboard plates. Note that you can also use small jars in place of Petri dishes.




       1) Measure about 100 mls. of tap water into a small jar. 2) For nutrients, , measure another 100 mls. of tap water into a second jar and add one drop of ordinary soy sauce to the water, and a quarter teaspoon (1.25 mls.) of molasses or light malt powder. 3) Find some gray cardboard, the thicker the better, preferably gray on both sides. Trace a Petri plate onto the cardboard with a pencil and cut out several disks to fit into your plates. 4) Weigh one of your disks and record the weight. Multiply this weight by a factor of 1.3 as a rough guide (you may need to experiment with the amounts here), and add the resulting weight of tap water or nutrient solution to each disk in its Petri plate. (Remember, 1 ounce of water equals 28.35 grams; one gram equals one milliliter.) Example: Suppose my disks weighed 0.17 ounces each. Multiplying 0.17 by 1.3, I get 0.22 ounces. There are 28.35 grams in an ounce, so 0.22 ounces x 28.35 equals 6.3 grams. That means I'll add 6.3 milliliters of solution to each disk. 5) Close up the disks in the plates, and let the water or nutrient solution soak in. 6) Pressure-sterilize the jar of plain water, and the Petri plates with moistened newsprint disks inside, for 10 minutes at 15 psi (allowing the cooker to equilibrate steam for 10 minutes before putting on the pressure regulator). 7) Cool the cooker, and remove the plates and jar of plain water. 8) When the water has cooled, add 3.3 mls. 3% peroxide to the jar, using a pasteurized pipette, to give you a final concentration of about 0.1% peroxide in sterile water. 9) Add about one third of the initial weight of the cardboard as 0.1% peroxide to each disk. Let the solution soak completely into the disks. They are now ready to use. [23]




       and add one drop of ordinary soy sauce to the water, and a quarter teaspoon (1.25 mls.) of molasses or light malt powder.




       soy sauce is protin ==== peptone molasses is corn syrup




       malt has a emzine that turns starch into sugar. spit. like out of your mouth has a emzine that turns statch into sugar.




       gluton is starch,




       8) When the water has cooled, add 3.3 mls. 3% peroxide to the jar, using a pasteurized pipette, to give you a final concentration of about 0.1% peroxide in sterile water. 9) Add about one third of the initial weight of the cardboard as 0.1% peroxide to each disk. Let the solution soak completely into the disks. They are now ready to use. [23]




       Use water agar for isolation of spores




       Water Agar (aka Starved Agar) ----------------------------- Agar 20.0g Distilled water 1000.0 ml Sterilize at 121C for 15 min. [6] spraqy bottle for steraisatiom 10 % bleach and water. Prepare a mixture of 1 oz of bleach with 10 oz of water. Put this mixture in the small spray bottle.




       Glove box===== cord board box with glove holes and plastic glass plate in top Alcohol spray botttle and burner baby food jar. paper towles coffee filters. duct tape baby food jars microwave oven. clorix bleach. hydrigen proxid make wire loop get mold samples from wild mildue sireng nideals alomanm foil plastic rap perlite rocks ph strips one spray can of lysol




      so peroxide will not guard against mold spores that have already germinated. But perhaps the best attribute of peroxide is that it will kill all bacteria, so you will never have a problem with bacterial contamination on peroxide enriched agar. As soon as is possible without burning yourself, move the jar of molten agar to the tabletop, open the lid, and insert a clean thermometer into the agar. Sine hydrogen peroxide decomposes at 140 degrees fahrenheit, you need to let the temperature of the agar drop below that before you add the peroxide. Otherwise it will decompose into water and oxygen and be effectively useless. This is why the peroxide is added after sterilization. I have found that my agar begins to solidify at around 120 F so fill a syringe with peroxide and when the temperature falls to 130F, add one ml to the molten agar and stir it in with the thermometer. Then remove the thermometer and pour your plates, stacking them as you go. I can usually pour nine or ten plates with this amount of agar. If you want to make a larger or smaller amount, simply adjust the amounts of water, agar, malt, and peroxide, keeping all the ratios the same close up your container of agar and sterilize for 45 minutes.




      I sterilize mine by letting it sit in boiling water in a covered pot, but you could use a pressure cooker if you wanted to. While it is sterilizing, thoroughly clean your plates if you haven't already done so, and stack them on a tabletop. As soon as is possible without burning yourself, move the jar of molten agar to the tabletop, open the lid, and insert a clean thermometer into the agar. Sine hydrogen peroxide decomposes at 140 degrees fahrenheit, you need to let the temperature of the agar drop below that before you add the peroxide. Otherwise it will decompose into water and oxygen and be effectively useless. This is why the peroxide is added after sterilization. I have found that my agar begins to solidify at around 120 F so fill a syringe with peroxide and when the temperature falls to 130F, add one ml to the molten agar and stir it in with the thermometer. Then remove the thermometer and pour your plates, stacking them as you go. I can usually pour nine or ten plates with this amount of agar. If you want to make a larger or smaller amount, simply adjust the amounts of water, agar, malt, and peroxide, keeping all the ratios the same.




       seems to tolerate amounts ranging from 1 ml to 7 ml per liter for this mix.(Wayne recommends from 6-10 ml of the 3eroxide per liter of agar medium).
The more you use, the longer "adjustment" period for mycelium to adapt to the peroxidated agar. As a general rule, the least amount necessary is best. But since your mileage may vary, you may modify as to what works for you.
There tends to be a "recovery period" that the mycelium goes through when transferred to agar containing peroxide, especially after the first transfer. The mycelium is slow during this recovery period, as it is building up the enzymes needed to break down the peroxide so it can digest the malt (in MEA). After that, the mycelium quickly picks up speed and grows just as quickly as it would if peroxide were not present. I've found this recovery period to usually last only a day or two.




      


       More to come.




       Larry





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