Overview: With this method we are going
to create a clean and contaminant-free spore filled syringe. The syringe will be
filled with heated and sterilized water and allowed to cool. The spores will
then be transferred from spore print into the syringe solution using a cleaned
and sanitized shot glass. At the completion of this Tek you will have created a
spore solution syringe ready for use in any microscopy application.
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 or in front of a laminar flow hood. If you
do not wish to construct a Glovebox, or do not have one, the following steps
have been performed with success by working on a thoroughly cleaned and
sanitized countertop. Liberal use of spray disinfectant and diluted bleach
solution is recommended for cleaning and sanitizing the work area.
Empty sterile syringes
(or larger) cooking pot
One bottle of 91% isopropyl alcohol
Several paper towels
A lighter or alcohol flame
A shot glass
Sterile spore print
Making a sterile syringe
1. Fill your cooking pot halfway with tap or distilled water (use
distilled water if your tap water contains higher levels of minerals and
2. Boil the water
in the pot on high for a minimum of ten minutes, this should be adequate to
sterilize and cleans the water of all bacteria, viruses, and mold spores.
3. Take your empty
syringe and fill it with the boiling water. Allow it to sit for two minutes with
the hot water inside. (If your syringe was sterility packaged, skip steps 4 and
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
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 cleaned and sanatized 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 to further aid in the
sterilization. Try to avoid letting the needle touch any other surface unless
otherwise instructed to do so.
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 become slightly tinted 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 in microscopy application
or inoculation for edible varieties. The older the print used, the more
"dehydrated" the spores will become. For proper microscopy observation or
germination, the spores will need to be allowed to rehydrate.