Phase One: (Step 8) Inoculation of Grain Jars

 

Disclaimer: What are Your Intentions For Growing or Using?

Transport the Grain Jars To Your Clean Room 

Before you start this part, visualize what is on the table in your clean room, and what you are going to do. 

 The isopropyl alcohol is incredibly pungent so you need to be very efficient, very clean, and very quick about this. 

1. Take off clothing if you put some back on from the last time you entered the clean room.
   
2. Put on the hair net.
   
3. Put on a mask. 
4. Wash your hands.
5. Put on your gloves. 
6. Get the second alcohol sprayer. One is already in your clean room, but you left one out by the grain jars.
7. Spray down a paper towel generously with alcohol. The paper towel should be very wet with the 70% isopropyl alcohol. 
8. Rub down the isopropyl alcohol container and the sprayer head with the wet paper towel.
   
9. Spray alcohol on the gloves. Rub the gloves together, getting the alcohol all around, like you are disinfecting your hands... well because you are! You are disinfecting those gloves.
10. Be very generous with the amount of alcohol you are using. You need it on every surface for 3 minutes to truly kill all the germs and spores.   
    
11. Spray down the box you are going to put the grain jars in. Remember to spray the bottom of it as well, that is where you will be holding the box, to transport the jars of grain. Use a paper towel to spread the alcohol around well. I know this isn't perfect. The alcohol will be sucked into the cardboard, and so you can't be perfect about disinfection. We are just doing our best.
    
12. Alcohol your gloves again. 
13. Spray alcohol on the outside of your PC, where you are going to touch to open it. Let the alcohol sit on the PC for 3 minutes before opening it, to assure disinfection.
    
14. I only open the PC, once I am ready to take them into the clean room. Again, this is to decrease the chances of contamination. 
    
15. Open the PC, and pull out all the grain jars. They should look close to something like this picture below. You can see in this picture that they put the top three grain jars on a second canning rack. You don't need to do this. A canning rack (the large metal circle thing) only needs to be on the bottom, so the jars do get overheated by directly touching the bottom of the pot, which is directly touching the heating element. 
16.  Put the grain jars in the box. Leave the tin foil on them. This will keep them protected from any spores and germs. 
17. Pick up the box full of the grain jars, and take them to the clean room. 
    
18. Open the door to your clean room gently. Close the door gently. 
19. Place the grain jars on the table. 
20. Put the box aside, or on the ground. 
21. You know the drill... use paper towels to clean the bottle of alcohol and the sprayer handle. Use alcohol on the gloves. 
    
22. Get the alcohol sprayer, and spray down everything again on the table, including the outside and top of the grain jars. Remember the tin foil should still be on those grain jars. You will take the tin foil off right before inoculation. 
23. So spray the tinfoil super well. along with the whole grain jar.
    
24. Spray, spray, spray! Spores probably have landed on everything. You need to kill those spores! Spray everything on the table.
25. Spray and wipe down the table where you are going to be working.
    

Let's Talk About Needles

Before inoculation time, we need to chat about needles. I see people do really strange things with needles in mycology. 

I used to be an infusion registered nurse. So I know my needles!

When a needle comes in a package, it is sterile. Inside that package, every part of that needle is sterile. Zero germs. 

The cover of the needle is sterile, the needle is sterile, the hub of the needle is sterile (that yellow thing in the picture above). 

The hub of the needle will twist on perfectly onto the end of the syringe, called the luer lock. You will twist the hub onto the luer lock, and it locks it into place... thus the name. 

The luer lock and the hub are sterile. Once they are turned and locked into place, then all the sterile parts are locked away inside the syringe. That's a good thing.

 

 

DO NOT clean the needle with alcohol when you pull it out of the package. 

The needle is already sterile! In fact, in trying to disinfect the needle, you will actually contaminate it. Your gloves are not sterile, they are clean. They have been disinfected with alcohol, but that is not as awesome as sterile. 

You want to keep that needle sterile!

Do not re-use your needles or use fire to sterilize them. They are super cheap. Just buy a new needle the next time you want to grow. 

Some people think that you need a new needle every time you access a new port on a spawn bag or grain jar, or that you need to sterilize the needle with fire in between the jars. That's not needed. 

Again, think about this logically. You have just disinfected the jars and grain (went through the PC). If one jar is contaminated then all of them are. It's the same grain in the same jars that all went through the exact same process. If one is contaminated then all of them are.

Some people say this will decrease contamination to use a new needle with each jar, or to burn the needle in between jars. I think otherwise. I think messing with the needle will increase contamination with the increased time that it's exposed to air. 

You DO NOT need to use fire to sterilize a needle if it has never been used. Heating the needle to that degree takes extra time, and you will also risk killing your spores if the needle has not cooled enough when you use it. 

Additionally please remember... fire and 70% isopropyl alcohol do not mix. 

The alcohol is incredibly FLAMMABLE!!  

You don't want to start a fire. Look how easy the alcohol lights on fire in this video!!!

Use one syringe with one needle and then throw the needle away. This will decrease chances of contamination.

I do use a new needle for each syringe, but NOT for each jar of grains.

I think changing needles that often would increase chances of contamination, touching the luer lock (the top of the syringe) on accident or contaminated air getting spores on it.

I use 4 cc of spore syringe fluid for one grain jar, or one 10cc (or ml) syringe for 3 different jars. 

Jar one gets 4 cc, jar two gets 4 cc, and jar three gets two cc of the first syringe. 

The second syringe I put 2 cc in jar three, 4 cc in jar 4, and 4 cc in jar 5. 

Jar three will have the greatest opportunity to get contaminated. That jar has spores from two different syringes being put in there. So if one of the two syringes are contaminated then that jar will also be.

That's just what I do. I've seen on videos that some people will only put 1 cc in one jar. 

Personally, I wanted to make sure that there were enough spores to get the mycelium growing. The 4 cc worked for me, so that's what I'm going to stick to. The spore syringes are really not that expensive, so do what you think is right and the least risky for you. 

I was able to do it this way with 0% contamination. 

 

Inoculate The Grain Jars With Mushroom Spores

All that hard work... you are finally to the last steps! Good job! 

This next part comes super easy for me. I used to be a nurse, so I'm very good at understanding what is sterile (NO GERMS), what is disinfected (SOME GERMS), and what is dirty (Has normal amount of germs, but it can still look like its clean). I will explain this as I go, hopefully in a way that is easy for you to understand. 

As you look at your table you should have on there 

• Paper towels
• Grain jars
• Spore syringes
• Needles
• Gloves (4 pair) 
• Alcohol prep pads - 40 of them
• 70% isopropyl alcohol sprayer

Again, go over all of this process in your mind, many times before you enter your clean room to do it. The alcohol in the air is really hard to breath. You want to spend as little time in your clean room as possible. 

Everything has been sprayed down and that alcohol is now disinfecting everything that you have sprayed it on. It really needs about 3 minutes to complete that process. 

Preparing the Syringes - Putting the Needles on the Syringes

Pick up the spore syringe. You will see there is a cap at the end of the syringe. In the picture below you will see that they are red. The caps can come in all sorts of different colors like blue, black, clear, green, etc.

This cap twists off. It can be incredibly tight. So you want to loosen it to make sure it can come off. 

Don't take off the cap yet! You just want to turn it SLIGHTLY to make sure that it can come off, then tightening it lightly again. 

Turn it to the left to loosen it. Rightly tightly lefty loosely. 

Under the cap of the syringe it is sterile. You want to keep it sterile, so that's why you won't want the cap off, until you can immediately attach the syringe to it. 

Additionally, once the cap is off, there is potential for anything in the air to land INSIDE the syringe. The cap is covering the entrance to the syringe.

Once you know that the cap is loose put the spore syringe back down on the table. 

Pick up the package of one needle. You want to open the package on the side of the needle that has the needle hub (see picture below). The package will have an easy open part on that side of the needle.

 

You don't want to pull the whole package back yet. Be VERY CAREFUL not to touch the hub of the needle. That is sterile and you want to keep it that way. 

The only thing that will touch the hub of the needle is the luer lock on the syringe. That is the very tip of the syringe. The leur lock, makes it so the hub of the needle will turn onto the syringe, and lock it all very tightly, so there won't be any leaks when the fluid in the syringe passes through the needle.

So in one hand you have your open needle package, and now the other hand will pick up your spore syringe. The needle package will naturally stay closed even after you open it a little bit. That's a good thing. You want the least amount of exposure as possible on the needle hub.

Take the cap off the spore syringe, being very careful not to touch the leur lock. 

Open the needle package and attach the hub onto the luer lock. It turns on there, just like the cap did. To tighten it on there, turn left. 

This video shows the needle and syringe part very well. She does a lot of other weird things you don't need to do.

If you also notice, she doesn't loosen the cap of the syringe first. You can see that she struggles doing this with also holding the needle as well. That is why I suggest you loosen it first. 

She says "keep the syringe upright to avoid any spilling"

Nothing will spill from the syringe unless you push the plunger and spray it out. 

You keep the syringe upright, so you are not contaminating it. It's very easy to touch the end of the syringe - the luer lock on stuff, so you want to hold it in your hands, upright. 

Start watching at 2:41 and go until until 3:23

 

DO NOT WIPE OFF THE NEEDLE WITH ALCOHOL!!! 

She does that right at 3:24. This can contaminate everything!

I've already explained this above in the needle section. You are actually contaminating the needle by cleaning it with alcohol. 

The needle is sterile. Sterile is the step above disinfection. 

Alcohol can disinfect, not sterilize. 

Sterilization means there is 0% of any kind of contamination. Disinfection means that most of the stuff has been killed.

The needle is sterile, don't bring it down the ladder of contamination by wiping it with anything. 

Keep the cap on the needle until you are going to use it. Once the cap is on the needle, then everything that is sterile is inside the syringe and inside the needle. You are now good to place it on the table. 

The needle has the cap on. The syringe will look like the picture below. It will have the needle now attached to it. 

The table is considered a "dirty" area. It's been disinfected, but you do not want sterile stuff hanging out open on that space.  

The outside of the syringe is also "dirty" but disinfected. 

The needle, under the cap, is sterile. 

So prepare all of your spore syringes, by putting the needles on. 

After the needles are all on, take the spore syringes and give them a good shake. Spores are often heavier than the sterile water they are in. You want to make sure that the spores are throughout the whole syringe.  

Let's talk a second about what the spore syringes will look like. Some look yellowish like the picture above. I've seen some that have black spots in them (probably from chunks of spores off a spore print). I've seen some with a clear fluid, but if you hold it up to the light you can see some stuff floating in there.

The ones that I got looked completely clear! I couldn't believe it! I thought for sure I would see something in that syringe, but I didn't. Honestly, I wondered if I had been tricked, that I was just sent some water in a syringe.

Nope! My syringes worked perfectly, and I got a great harvest. 

So just because it looks like it's only water, it's most likely not! 

If you have a microscope, you could put a little of the fluid on a slide, and look for yourself. I don't have that equipment, so I just prayed that it was going to work! Thank you God for helping me, because it did work.

Inject The Spores Into The Grain Jars

You are ready for the last and final steps! YAY!!!

You are going to inoculate three jars at a time, with one syringe. If you have several types of mushrooms you are growing, you will want to keep close track of what syringe is used on what jar. Just keep the empty syringe next to the jar you inoculate, if you need to keep track. 

You will label the jars AFTER the inoculation is done. You need to get this done ASAP, so you can breath! You need to get out of that clean room, and air the clean room out. 

1. Disinfection.... AGAIN! I know we've done this so many times, but it's important. There are millions of spores all over the place. Even with all the precautions we've taken, there are still some around... so use that alcohol liberally!
2. Disinfect the sprayer and the alcohol bottle by spraying a paper towel with alcohol and wiping down the bottle. 
   
3. Disinfect your gloves again by spraying with alcohol. 
4. Disinfect the tin foil on top of the grain jars by spraying with alcohol.  
5. Disinfect the spore syringe, wiping with a paper towel, and/or spraying with alcohol. You want to disinfect all sides of the syringe you will be touching. 
6. Do NOT open the cover on the needle. You can spray and wipe down the outside cover, but remember that the actual needle under the cover is sterile. 
   
7. Spray over the alcohol wipes, to try and disinfect the outside part of the box and the wipes themselves. 
8. Take the tinfoil off the first grain jar. 
9. Take one alcohol wipe, and rip it open, and wipe the alcohol wipe over the black sticker port on one jar. Wipe it for several seconds. 
10. Take a second alcohol wipe, and do the same. 
11. Do this on all three jars quickly. 
12. Pick up your first syringe, shake it a couple of times. 
13. Take off the lid to the needle. 
14. Inject the needle into the black port. 
15. When the needle goes through and you can see it go into the jar, you can then start injecting the spores. 
16. Keep track of how much liquid you are injecting. I inject 4 cc (also called ml or milliliters. They are all the same thing). 
17. I will usually inject 1 cc in one spot, then change the angle of the needle and spray another 1-2 cc and change the angle and spray another 1-2 cc. That way the mycelium will start growing is several places. 
    
18. I inject 4 cc into each jar, but I've seen others say as little as 1 cc is enough. 
19. After you inject the 4 cc into one jar, take the needle out of the first jar, quickly wipe the next jar with an alcohol wipe, and immediately inject the needle into the next jar. 
20. Do not touch the needle, do not let anything touch the needle. Do not put the syringe down. 
    
21. Do this for the three jars, until all the fluid in the syringe is gone. 
22. If you have to put your syringe down, put the lid of the needle back on first, very carefully to keep the needle as clean as possible.
    

This video below has all sorts of problems with it, but I want you to see what it looks like to inject the syringe into the jar and spray the spores.

It's a good video to learn what you shouldn't do. He's not wearing a mask. He sanitizes his hands instead of washing them (do both!) He's not wearing gloves. He's not working in a clean room. This is how you get lots of contamination! 

He also rubs the STERILE needle with the disinfected cloth! 🙅 Every part of me shudders when I see this! Don't do that!! The needle is sterile. Don't touch it with anything! 

How he uses the towel with the alcohol is a great way to save money. That would work to use instead of alcohol wipes. So wherever we use alcohol wipes or a paper towel with the alcohol, this method would work well. 

But the video shows you the process of putting the needle in the port, squirting the liquid, and what that looks like.

Start it at 3:14 and go until 3:44

He shakes his jar after the inoculation. I didn't do that, but I'm sure it would be fine. The issue for me, is that it's important for those spores to be next to each other for the mycelium to grow. So that's why I don't mix up the jar until after the mycelium is growing well.

Pat Yourself on the Back!! 

You did it!!! You have done the hardest part of phase one!! CONGRATS!!! Great job! 

Now go put your feet up and get a cold drink. You deserve it!

Next Step: (Step 9) Let it GROW!! Growing Mycelium into Grain Spawn

9 Steps of Growing Psilocybin Mushrooms: Making Grain Spawn

1. (Step 1) Planning - Timing and Schedule Breakdown
2. (Step 2) List of Items You Need
3. (Step 3) Making the Lids
4. (Step 4) Prepare the Rye Berries for the Pressure Cooker
5. (Step 5) Clean Room - Set up and Cleaning 
6. (Step 6) Using the Pressure Cooker for the Grain Jars
7. (Step 7) Last Prep for Clean Room
8. (Step 8) Inoculation of Grain Jars
9. (Step 9) Let it GROW!! Growing Mycelium into Grain Spawn