|By Nan (Nanook) on Monday, October 22, 2001 - 12:54 am:|
Psilocyber's Syringe Tek, v1.0
NOTE: These instructions are most effective when performed in the most sterile environment available. The preferred method involves following the steps below while working in a Clean and Sterile Glovebox. There are many simple methods of glovebox construction; most are available on the web at the popular mycological culture Websites. If you do not wish to construct a glovebox, or do not have one, the following steps may be preformed with success by using the "Oven Tek." This simply involved using your oven as a way of reducing the amount of contamination present. To do this, turn your oven on its "warm" setting. Pull down the lid. You may then use the lid as a working surface or pull out one of the oven racks halfway and work on it. The theory is the rising heat from the oven causes airborne contaminations to rise as well and therefore prevents them from settling on your working surface.
Empty sterile syringes
Two quart (or larger) saucepan
One bottle of 91% isopropyl alcohol
Several paper towels
A lighter or alcohol flame
A shot glass
Procedure One: Making a sterile syringe
1. Fill your saucepan halfway with tap or distilled water (use distilled water if your tap water contains higher levels of minerals and chemicals).
2. Boil the water in the saucepan on high for a minimum of ten minutes, this should be adequate to sterilize and cleans the water of all bacteria and viruses.
3. Take your empty syringe and fill it with the boiling water. Allow it to sit for two minute with the hot water inside.
4. Purge the hot water from the syringe into a sink, not back into the saucepan.
5. Repeat steps 3 and 4 two more times. Upon the second time leave the hot water in the syringe.
6. Place the syringe in a cool draft-free place, preferable in a clean zip-lock bag
7. Allow it to cool for several hours before proceeding to Procedure Two.
Procedure Two: Transferring print spores into syringe
1. First clean your work area. This may involve wiping down all work surfaces with a diluted bleach solution and spraying the area liberally with a disinfectant such as Lysol.
2. Place the following materials in your glovebox or on the oven lid working surface: The shot glass, your cooled syringes, the bottle of alcohol, a paper towel, your print (still in zip-lock baggie) and the lighter or alcohol flame.
3. Wash hands with antibacterial soap before proceeding further.
4. Fold the paper towel up into ¼ sections and soak a corner of it with the alcohol.
5. With the alcohol soaked towel wipe the interior of the shot glass, essentially sterilizing the surface you are about to use in the transfer. Allow the shot glass to air dry, should only take a few seconds.
6. Remove the needle guard from your sterile syringe and flame sterilize the needle. Then take your alcohol soaked paper towel and wipe the needle with it to further aid in the sterilization. At this point try to avoid letting the needle touch any other surface unless otherwise instructed to do so.
7. NOTE: it is important at this point to work as quickly as possible to help combat the chances of contaminating molds and bacteria falling into your work area and thereby ruining your syringe.
8. Remove the print from its storage baggie. Unfold it to expose the spores. Lightly begin to scrape, using the needle of the syringe, a section of the print off into the shot glass. For a medium sized print it is usually adequate to scrape off a section no larger than 1/5 of the total print.
9. You will have a small noticeable collection of spores in the shot glass. Now expunge no more than half of the water from the syringe into the shot glass, lightly stirring the spores into the solution.
10. Suck the spore water solution into the syringe. You may need to expunge some more water into the shot glass and re-suck to help in capturing all the spores into the syringe.
11. Once you have the spore solution back into the syringe you should notice that the water inside has turned a darker shade of color and you may see small clusters of spores floating in the solution. This is good, you have completed the process.
12. Sterilize the needle again with the alcohol soaked paper towel, replace the needle guard and place the syringe back into your clean zip-lock bag.
13. Allow the syringe to sit for no less than 12 hours before using it in jar inoculation. This is extremely important, as the spores must be allowed to rehydrate before they can be introduced into the substrate material. Failure to allow this may result is slow or no germination.
Also See: Clean Rooms