Antibiotic means "anti-life". But interestingly, fungi spores can withstand powerful doses of antibiotics while bacteria succumb easily. That is why "athletes foot" can't be cured with antibiotic creams. It could be that antibiotics come from fungi in particular. When a spore solution is made it can easily be contaminated with bacteria, the most common life form. We are surrounded by them. But with antibiotics, they are very susceptible, especially when in a solution of water and spores. The technique is quite simple. The Antibiotic is put into the spore solution where the bacteria are stopped in a variety of ways, either by cell wall destruction or protein synthesis inhibition, depending on what kind of antibiotic is used. I am no expert in antibiotics, but I know that antibiotics in concentrated form kills them dead, while leaving the fungi spores just fine. And over time, the fungi spores remain viable. And previously, it was bacteria contaminated (no spore germination - just a rotten smell and slime appeared).

But it is important to use the spore solution immediately. Over time, the cultures will still germinate and give a clean culture but you can tell something is not good. The culture is weak and it won't fruit, even though it is clean of bacteria. So use the decontaminated spore solution immediately and your results will work.

Step one: Acquire antibiotics. This is the easiest part. The best place to get a wide variety from Penicillin to Tetracycline is at your neighborhood pet store which has a good fish tank supply and of course, live fish for sale. In a particular section of the store, you can find rack fulls of a variety of antibiotics that are used for fish tank populations. They come in capsule form and are to be used directly into the fish tank water. But for our purposes, this won't work because these antibiotics are not sterile by any means. They are actually heavy with mold. Even Ampicillin you get from your doctor is heavily loaded with mold. The mold spores don't hurt you or the fish but they will destroy your attempt to culture fungi spores.

The antibiotics must be first sterilized before use in spore solutions. Heating the antibiotics is not the way, because it destroys the antibiotics. The antibiotics must be cold sterilized. This is accomplished by a very common micro biological technique ubiquitous throughout the Bio science lab world. The antibiotic is taken out of the capsule, placed into a small jar, about 10cc of water is added, the powder is mixed into solution, the solution is sucked up into a syringe and then finally, the antibiotic water is pushed through what is called a "SYRINGE FILTER". This is a small 25 mm round plastic device that sOcrews on the end of a standard leur lock syringe like the ones the Professor sells. These small round filters come individually packaged in what is called "blister packs" where they are sterile and ready to use. The antibiotic solution comes out of the syringe filter and it is sterile. The filter takes out anything bigger than .2 microns and that includes all spores, bacteria and viruses. The antibiotic molecules get through and are clean of all mold spores plus the bacteria that was in the water is dead already.

The antibiotics that I have used to great success are all available at the pet store fish tank section. Tetracycline is a powerful toxic antibiotic that colors the spore solution orange yellow. Kanamycin is a powerful antibiotic that is clear and does not show up in the spore solution. Neomycin is another clear antibiotic that has worked well. I think Tetracycline might be the best, but Kanamycin and Neomycin work just great. Penicillin is not near as good and usually fails. I have tried Streptomycin which fails half the time and gentimycin which is very weak, fails all the time. Gentimycin is an antibiotic that can withstand autoclaving and is used in agar agar (by such myco supply houses as Fungi Perfecti), but in comparison to Tetracycline, it is weak. In fact, a Doctor friend of mine says they don't use Gentimycin anymore. It's obsolete. I have used chlorpromazine and a few others, and I have found Tetracycline to be about the best, and it is always available at the fish tank store. Plus, it is the only one that colors the water yellow orange (not pretty) and it is the most expensive (less than $10 per pack of a dozen or so pills at the fish tank store).

There are two sides of the equation. The first side is basically non sterile and the second side is sterile. In the first side, the antibiotic is put into non sterile water (distilled), which is sucked up into the syringe ready for injection through the syringe filter into the targeted spore solution. The second side is the clean side, which is the syringe filter itself, the needle if used and the spore solution (which is as clean as possible prior to the antibiotic treatment).

Supply list - non sterile side of the equation

1. Tetracycline - Kanamycin - Neomycin capsules
2. 10 cc syringe
3. Distilled water
4. small mixing jar
5. mixing rod

Supply list - sterile side of the equation

1. .2 micron syringe filter (25 mm around) - available from science supply catalogs.
2. syringe needle (optional)
3. spore solution in jar (clean as possible)

On the non sterile side, just start clean. All the contaminants will be taken out by the syringe filter. You needn't autoclave anything except the needle if it is used on the syringe filter in the injection of the sterile antibiotic fluid. First, open the antibiotic capsule, and empty the entire contents into a waiting small clean jar. With the non sterile syringe, inject 10cc of distilled water into the jar and with a rod mix the antibiotic until dissolved. It might not dissolve completely, but that is OK. The syringe filter will do the rest.

Suck the antibiotic water into the syringe. Leave about one cc of air in the syringe for good control of the outflow.

Now we go into the sterile zone. If you don't have your own spore print taken PF style according to the PF TEK, you will have to use a print gotten from somewhere. When this is the task, you must use an isolation hand box and using sterile equipment like a forceps (to handle the spore print) and a sterile exacto knife blade (to scrape the spores off the print) and a waiting sterile PF style jar with a little water in it (based on the syringe making tek in the PF TEK book). Many books already written describe how to do it, but they are all really the same. It is self evident how to do it. One just has to get the spores off the print and into a waiting sterile jar with water in it. The jar lid has already been fixed with the proper sized needle or access holes that are covered with good tape or tin foil.

Once that is done, have the jar with the hydrated spores ready. Follow the PF TEK on that (PF spore syringe making tek). Next, with the syringe loaded with the antibiotic solution (non sterile), peel off the back of the syringe filter "blister pack" (kind of like preparing a bandage). With the back peeled off, hold the syringe filter in the blister pack (by the sides) and screw on the syringe with the antibiotic solution and remove the filter from the blister pack holder. If a needle is to be fixed onto the syringe filter, remove the sterile needle from its tin foil and fix it to the syringe filter. Don't touch the needle tip, but you can handle the needle by the hub if you want to. Don't touch anything that the solution will come in contact with. Next, remove the tape covering the needle or access hole in the lid of the spore print jar, insert the tip of the syringe filter (or optional needle) into the hole in the lid of the spore solution jar and slowly but firmly press on the plunger. There will be a short pause while the solution begins to move through the filter, but then it will start dripping out the end of the syringe filter and into the waiting spore solution in the jar. The fluid will be a clear orange color (Tet) or clear (Kana or Neo). There will be no particles in the antibiotic fluid as it comes out of the syringe filter. Once you do this, this is actually fun to do and very easy.

When the antibiotic fluid is emptied out of the syringe, withdraw the syringe filter tip or needle, put some fresh tape on the lid hole, swirl the spore solution around a bit and put it safely on a shelf for a couple of days. Actually, once the antibiotic goes into the spore solution, the spore solution is ready to use, but to be sure, give it a couple of days of rest.

If the procedure is done correctly, and no mold contaminants are introduced during the procedure, the spore solution will be cured of bacteria.

To test the spore solution, inject a jar with an agar nutrient layer on the bottom or a slurry of brown rice powder. If the fungi appears a few days later and looks pure and clean, success is yours. But, there might be another problem, and that would be mold - green, blue etc.

If there is mold in your culture, it will be growing along with the pure white fungi mycelium, usually next to it. The next step is very easy. Go on to the PF micro peroxide brown rice cloning tek to clean up the mold through sub culturing with peroxide and on to the shrooms, PF style.

The original idea comes from Rush Wayne's cultivation manual "GROWING MUSHROOMS WITH PEROXIDE". The following is a simplification of the idea down to the smallest degree, using brown rice powder, peroxidated water dilution's and small jars.

MATERIALS

1. Small jars with lids
2. Brown rice powder
3. Regular store bought Hydrogen Peroxide (3%) antiseptic solution (usually in a brown bottle)
4. Dissection knife and long needle (exacto etc)
5. Living mushroom or fungus, or culture of fungus

If the previous antibiotic tek is used and there is mold growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi. The mold growing near the good fungi will shower it with spores, but this peroxide tek will usurp the contaminant spores and clean the good mycelium for further cultivation - PF style. This tek can be a follow up to the antibiotic tek, or can be used to clone a mushroom outright. Both purposes work just fine.

As a preliminary, start reasonable clean, but the following is to be done in the open air, without sterile implements or containers.

Mix the peroxide antiseptic and water at a 20% ratio. Example - 2cc peroxide and 8cc water. Place an amount of brown rice powder into the jar and add some of the peroxidated water to get a slurry, or very wet condition. The diluted peroxidated water sterilizes the medium. For extra experiments, you should try more potent peroxide contents in the brown rice peroxide slurry medium (30% - 40% - 50%). It is so easy, it can't hurt to experiment and you can benefit from it.

Tear the shroom apart and with an exacto knife, excise a small fragment about the size of a match head. With a long needle, knock or scrape the shroom fragment off the exacto blade and place the fragment on the surface of the peroxidated brown rice slurry. Or if you are using a culture of mycelium that may be contaminated with mold, cut out a small piece of the white mycelium with the exacto knife blade and do the same - scrape the fungi fragment of the blade onto the surface of the peroxidated brown rice slurry. If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.

Screw the lid tightly onto the peroxide slurry jar and put the jar in a warm place for growth. The culture can be opened and exposed for observation or experimentation without danger to the growing medium and culture.

Under magnification (10x), the fragment of mycelium appears to "melt". It also turns blue, as if it were killed. Within a few days, tiny white hair like tendrils (hyphae) will appear on the "melted blue" fungus. It will grow and fill the culture jar.

To use the culture for further inoculations, add 20% peroxidated water to the culture, mix the brown rice and fungus, and with a syringe, draw up water and inoculate PF jars, with the standard PF spore syringe technique. This is done nonsterily also because the peroxidated water will kill contaminant spores. But flame sterilize the needle before injecting into the sterile PF substrate. Instead of flaming the needle, an even better way to sterilize the needle (outside of the needle) is to wipe the needle with a tissue soaked with rubbing alcohol. Let the needle briefly dry of the alcohol before injecting. Use the culture well before it grows in. That way, it is much easier to get a usable slurry for the syringe without clogging.

As an addition, try more potent peroxidated water ratios. For instance, a 50% ratio works also. That would be half peroxide antiseptic and half water. Increase peroxide content until the mycelium doesn't survive. Then back off the amount of peroxide and use a near death peroxide load for guaranteed clean results. But a 20% peroxide to water ratio seems to be perfect.

If you are working with previously made plate or slurry cultures that have mold or bacteria slime growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi excised out of the culture. Make sure you don't have any growing mold or bacteria in the good mycelium because the peroxide won't kill it (as it doesn't kill the good fungi). If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.

The only problem is syringe needle clogging. As a remedy, do not allow the culture to fully grow in or get thick. Keeping the culture "thin", allows a good breakdown of the mycelial fragments for use in a syringe. At first, doing the tek can be messy, but learning is quick and easy. Finding and using needles as big as possible is important.

But another route is very possible and actually preferable. Using long glass bulb pipettes are very good to use, but here, one must customize and "tweak" the teks a bit (one must be careful when injecting PF style jars with the bigger pipettes and not to breach the top vermiculite contaminant barrier). Also, flaming the glass pipettes can sometimes break the glass. In this case, always sterilize the outside of the pipette with the rubbing alcohol. Also, when releasing the bulb, it will inflow air. Do this inside the jar where the air is clean and protected by the vermiculite contaminant barrier. Or, after injecting, keep the bulb squeezed and withdraw it, and then refill it with fresh peroxidated mycelium for another injection, resterilize the outside of the pipette with rubbing alcohol, and inject, and so forth. When doing this, use small calculated amounts of mycelium slurry to keep from over doing it. Bulb pipettes are actually better to use than needles and syringes because they don't have much problem with clogging as compared to needles and syringes with mycelial slurries. But if the tek is followed closely and the cultures are not allowed to grow in and get thick, the 18 gauge needles with 10cc syringes work OK.

Hydrogen Peroxide is a powerful antiseptic. The solution of Hydrogen Peroxide bought in a drug store is 3% Hydrogen Peroxide and 97% water. Even at this low concentration, and with further dilution's, the germ killing is potent. But that germ killing power only works for micro fungi spores and bacteria endospores. A micro-organism that has germinated into its secondary form (mycelium), is safe from the antiseptic power of diluted Hydrogen Peroxide. But the ungerminated spores, bacteria endospores, and microbes are all susceptible. If there is bacteria that is growing (germinated), it will not succumb to the peroxidated water (just as the fungi mycelium is not succumbing). Also, any mold that is growing will survive. A clean fragment of shroom flesh or mycelium from a mold contaminated culture has germs all over it, but only in the spore or endospore form. They won't germinate in the peroxidated medium and water or on the recovering mycelia.

The tek is like a tightrope act. The mycelium that is cultured in the peroxide enriched medium can survive and grow, but it is not clean. Any spores or bacteria that are "piggy-backing" on the mycelium and not in contact with the peroxidated medium can come to life if given the opportunity.

After the culture of mycelium grows, it can be rehydrated with more peroxidated water. The spores and bacteria endospores that are "piggy-backing" on the mycelium will die. The mycelium in its fully secondary form, will survive the new peroxidated solution, "cleaned".

Professor Fanaticus