Inoculation of Substrate Jars

Last updated 09/06/09

Spore/Brew Injection 

 

Perform this next procedure within the Glove Box!

Material needed:

Masking Tape (Not included in kit)

One dozen pre-made and sterilized substrate jars. 
(DO NOT OPEN THE LIDS OR ATTEMPT TO STERILIZE IN A PRESSURE COOKER).

Keepers Brew OR Spore Syringe 
(IF YOU JUST GOT YOUR KIT YOU ONLY NEED TO CONCERN YOURSELF WITH THE SPORE SYRINGES or Edible Mushroom Cultures)

91% Alcohol

Lysol

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Before moving on you should 'disturb' the spore solution within each syringe by turning the syringe upside down and right side up for about 3-4 minutes.  This should cause most of the spores to dislodge themselves from the side and bottom of the syringe.

Your kit will or should include one dozen pre-made substrate jars.  (Newer kits, November 20th and after, will have these doz. pre-mixed and sterilized substrate jars)

YOU DO NOT HAVE TO PLACE ALL 24 JARS INTO THE GLOVE BOX AT ONCE.  Do 9 or 12 at a time.  Just make sure you place clean tap over the holes before pulling them out and continuing on.

 

Wipe each jar lid with alcohol and place them, and the syringe, into the Glove Box.  Also place an alcohol soaked paper towel and a roll of Masking Tape inside the Glove Box.  Place the Plexiglas lid on the Glove Box and plug in the fan.  Take Lysol and squirt the white filter which is located on the opposite side of the fan (outside of the Glove Box) for two full seconds.

WAIT FOR 5 MINUTES BEFORE GOING ON TO THE NEXT STEP!!!

I want you to understand the importance of the position of each jar as they are held during the spore inoculation of the Substrate jars.  Hold the jar at a 45 degree angle.  When you plunge the NEEDLE into the jar point the needle against the inside of the glass.  Holding the jar at a 45 degree angle, causing the tip of the needle to 'touch' the side of the glass, allows for the .25cc of spore solution to run from top of the jar to the bottom.  This will cause the side of the jar (substrate) to colonize from the top to the bottom and allow for easy viewing as the mycelium grows.  If there is any contamination within the mycelium you should be able to see it quickly. 

Note:  Be careful not to inject too much into one single hole.  It can be very easy to squeeze too hard and dump a full cc into a single hole.  This would would suck because you are NOT to use more then one FULL CC into any single jar.  So if you dumb a full cc into one hole you have to live with it and move onto the next substrate jar.

Here we go....

Put your gloves on and spray a few squirts of Lysol onto them.  These gloves can be a little difficult to work with at first.  It is not 100% necessary to wear them during the Inoculation Stage, but will be VERY necessary in future techniques.  If you decide not to use the gloves you must wash your hands with Antibiotic.  Now carefully place your hand threw the holes (the flaps should lift upward) and into the Glove Box.

Within the Glove Box you are too carefully remove all of the masking tape from each jar and place it to one side of the Glove Box...to be discarded later.  

Gently shake the syringe, carefully holding the cap of the needle with your forefinger so it does not fly off, for about 10 seconds and remove the green cap and place it to the side in order to cover the needle once again when finished.  

Note: You will notice that your syringe has 12 cc's marked on the side of the syringe.  For every 1 cc you are to inoculate 4 holes (.25 cc) = ONE JAR.  Thus, one 12 cc spore syringe should inoculate twelve 1/2-pint jars. 

Remove black, or white, protective tape.  Twist the green cap until it is in place.  If not, you will open the syringe and risk contaminating your spores!  Take of the protective green covering off of the needle and quickly (CAREFULLY) wipe the needle with the alcohol cotton ball and place the syringe off to one side.

Carefully place the needle into one of the four holes as described above.  

Slowly squeeze out about .25cc (a quarter of a cc) of spore solution into each of the four holes.  Please notice that the spore solution is running down the side of the jar from the top to the bottom.  After you inoculated all four holes of one substrate jar you must wipe the needle with the alcohol which insures that IF there is a contaminate lurking on the needle it will not be able to spread to the next jar.

After ALL of the jars have been inoculated, place the green cover of the needle back over the needle and move the syringe to one side.

NEW!
Turn jar after inoculation!

Here is a little trick that I have learned recently.  Place the jars in your hands just after inoculation and quickly flick your wrist so the substrate in the jar moves a little. Try to get the substrate to 'rotate' to the other side of the jar.  That will GREATLY speed up the process due to the fact it spreads the spores throughout the jar.

 

Get your Masking Tape and begin placing a piece of it over EACH AND EVERY HOLE.

Take everything out of the Glove Box and move on to setting up your incubator (Step #2) or repeat this process if you have more jars to inoculate.  

Note:  Any unused spore solution can be kept in a cool place for up to 6 months.  After six months the spores can become less viable and the mycelium might not spread as quickly as one would like.

Note:  Inject ALL of the pre-sterilized substrate jars with spore syringe ASAP.  Any pre- sterilized substrate jars that have not been injected with a spore syringe is susceptible to contamination in the near future.  
Store the jars at your own risk!

Note:  Sometimes the needle will get jammed with substrate upon entry into the jar.  If this happens take the needle out and wipe it with the alcohol soaked paper towel until you see nothing on the tip of the needle and try again. 

Continue immediately to Step #2