|Posted on Monday, October 22, 2001 - 12:24 am:||
[Note from Nan: Obviously I did not write this. Nor have I tested it. Don't stick your nose into contammed jars and sniff. This can be cleaned up with H2O2]
Uncle Lazlo's EZ Mycelium Syringe Tek
Here's a way to get into cloning with a technique that does not require a Blender , Agar, a HEPA Filter, or even a Glovebox. It allows you to make the most of the knowledge you have gained using the PF Tek or any of the other sealed jar teks, and allows you to select strong mycelial growth for cloning. I and several others have reported good results using it.
This technique makes use of a piece of colonized substrate from an unopened, uncontaminated PF-style jar. You will remove a small piece of substrate, place it in a jar of sterile water, agitate the water to distribute mycelial cells through it, and draw a syringe from that jar. You will need one or more sterile syringes for this tek. If you have previously used ("dirty") syringes, you can sterilize them by autoclaving for 20 minutes at 15 psi; or, if you have no autoclave, you can boil them.
I have no glove box and no HEPA filter. I do, however, own a "poor man's laminar flow hood." In other words, I warm up the old Kenmore oven, pull out a rack, and work in the warm updraft of air, which basically keeps airborne contaminants from falling onto the work surface. If you keep your hands and tools clean, you should get acceptable results working this way. If you have a HEPA filter or glove box, by all means, use it.
OK OK... Here is the real poop: Oven Tek Sux. The flow dynamics theory is wrong. The best place for sterile work is in a Glovebox. You can shoot jars in a clean still room. I left Oven Tek here because the tek is too firmly entrenched to remove (see links below) but none of the staff here uses it nor recommends it. Period. - Nan
I prepare my water jar by putting a few jagged pieces of broken glass into a half pint jelly jar and filling the jar with distilled water.You can punch a single hole in the jar lid, near the lip of the jar, through which you can insert a hypodermic needle. If you decide to do this, you can put foil over the mouth of the jar and screw the lid over it, so that, later, you can just push the needle through the foil with little risk of contamination.
I place the lid loosely on the jar (rubber seal in place) and heat it at 400F for an hour. You must let it cool before you can use it. I suggest that it be at room temperature before you proceed. When your sterile water is cool, lay out your materials on the oven rack. You should have your sterile syringe, scalpel, colonized jar, and water jar ready to go. Working quickly, use the scalpel to remove a chunk of colonized substrate from the rice jar. It is not necessary that the jar be completely colonized. Do not, however, take your sample from a contaminated jar - - this is NOT a way to save a bad batch of jars. Believe me, I've tried it!
Try to pick a section of mycelium that looks stringy (rhizomorphic) rather than cottony. I like to push the scalpel straight down into the jar twice so as to cut a wedge shaped piece from the perimeter of the cake. You can use a metal fork to remove the chunk - - sterile, of course. The chunk of colonized rice/verm, maybe the size of a sugar cube, should go immediately into the jar of sterile water.
Next, tighten the jar lid and shake the solution vigorously for a minute or so. You will now see the purpose of the glass shards, as they help break up the chunk of mycelium. When the water begins to look milky, let the solids - - vermiculite, rice grains, etc. - - settle to the bottom.
At this point, you should be able to draw a syringe of mycelium solution. Don't worry if the syringe clogs: when it does, just press the plunger down to clear the needle. As you draw up the milky water, try to pull up as much white mycelial tissue as possible. You'll be able to see it floating around. The more of it you get, the better. When you have drawn as many syringes as you need, I suggest that you use them as soon as possible. Some people report that mycelium in distilled water can last for weeks. I prefer to use the solution within a day.
So there you go. A very fast way to make mycelial inoculate without all that blender rigmarole and all the hassles associated with agar plates. Inject this solution into sterile grain to make spawn, or shoot it directly into PF-style jars. I've seen impressive growth in as little as 48 hours, though, at times, it may take several days for growth to appear. I will warn you that, if you do not see growth within one week, the likelihood of bacterial comtamination is high. One whiff of an open jar will tell the story.