EternalPeace
Rising Star
Introduction
------------
Per request, here are my personal notes and experiences with this procedure. I'll try to keep this really simple, and avoid going into too much detail.
I have found that many people balk at the thought of doing this, which I assume is due to the expected complexity of the procedure. I balked myself during my first round of experimentation, and then gave it a shot for the second. Let me assure you, it is not at all difficult, or any more difficult than the other sterilization procedures that cultivating mycelium requires. As a matter of fact, it appears to be very beneficial to the process. As I have noted in other posts, it speeds it up quite noticeably.
I think that the most difficult part of all this is acquiring the materials. Note that there are a few poor-man's methods of working around the official materials, in the event that ordering online is not possible, or if there are financial limitations, but I'll just write about the official ones that I have used so far.
Stuff you will need:
--------------------
1. Plastic petri dishes (*)
2. Parafilm
3. Agar (potato dextrose or malt extract) (**)
4. Agar pouring device (***)
5. Agar cutting device, such as x-acto scalpel.
6. All the rest of the usual sterilization procedures, materials, and devices
*Buy a plastic wrapped sleeve of 20, because (1) they are easy to dispose of, (2) are pre-sterilized, unlike the glass ones, and (3) the plastic wrapping is actually useful in the procedure.
**I don't think it really matters which type you use. If anything, it is more of a preference related to how easy it is to see through it, which is useful to selecting sectors to transfer. I personally have been using the potato dextrose agar for both Mex-A, Mex-ATL7, and Ps PE without any issue.
***Start collecting short glass bottles that will withstand being PCed. 2-3 of them should do for 20 dishes. You will need these for sterilizing the agar, and they'll need to fit in the PC. An easy measurement comparison when shopping is to keep it around the height of your mason jars, if you are using them. Check out the sauces area of your grocery store, you should find something viable there. The lid won't be needed, so it can be plastic.
Procedure
---------
1. Mix agar (unless already premixed), and pour into glass agar containers. Don't fill more than 2/3 full to prevent boil-over.
2. Cover agar containers loosely with tin foil and put into PC.
3. Use a bit more water than usual in the PC to help prevent agar boil-over.
4. PC at 15psi for 30 minutes.
5. Let cool down in PC (about two hours), but not enough to get cold and harden.
6. While agar is cooling, prep work area.
7. Move agar out of PC into SAB as quickly as possible.
8. Uncover each dish, pour thin layer of agar, and recover.
9. Stack up the prepped dishes back in the plastic sleeve. Seal the sleeve and put in the fridge. No sealing with parafilm yet, just leave them loose. The plastic sleeve will act as the contamination barrier.
10. Let sit in the fridge for a few days to see if anything grows. Toss the contaminated ones.
11. Prep work area again, this time including the spores, the first round's quantity of prepped dishes, and the sealing materials. I went with three dishes per culture for the first round, which worked just fine.
12a. (If syringe) Sterilize tip until glowing red hot (although not too far down towards the liquid!), move inside SAB, let cool, shake to redistribute spores. Quickly uncover a dish, try to drop one single drop right in the center, and recover.
12b. (If print) Sterilize inoculation loop until glowing red hot, move inside SAB, shake to cool, rub on print, then quickly uncover dish, rub off spores onto center of agar, and recover dish. If the print is too tough, drop one small drop of sterilized water onto the print and rub around with loop, it should become easy to work with.
13. Repeat until all dishes are inoculated.
14. Seal all dishes with parafilm, and then they can leave the SAB.
15. Mark each one with what culture it is, that it is round 1.
16. Put in a warm, dark place that mycelium likes to grow in. This is the fun part. Check back each week, or as desired, until the growth has gotten near the edge of the container, or contamination is threatening to ruin the dish. (The better your sterilization procedure, the less of this will occur. It is actually a useful test to visibly see how good your procedure is.)
17. At this point, it is ready to be transferred to a new dish. Prepare your work area again, this time with the previous round of dishes, more prepped dishes, and a scalpel. I transferred the best of the first round's three dishes onto a single dish for the remaining rounds. (If you have higher contamination risk, you might want to go with more dishes per round, but I found it surprisingly unnecessary.)
18. Find the best sector of growth of the first three dishes. Cut a small piece out with a sterilized, cooled scalpel, doesn't need to be big, and drop it in the middle of a new prepped dish. Seal it and mark it as round 2. Put the dishes back in the warm dark place and let them grow out again.
19. Repeat until you have a perfectly uniform culture on the dish, which is referred to as a monoculture. This should be the fastest growing, most aggressive performing culture out of all the rounds that you have completed.
20. Take a piece of that and drop it into your prepped master grain jar, or into your master culture slant.
Additional Notes
----------------
For some pictures of what to expect, see Stro's nice guide on the Shroomery. That's mostly where I learned all this from. Credit and thanks go to Stro for his guide. It will show you what I mean by "sectoring" and "monoculture". Most of it is common sense though--when you're looking at it in your dish, you'll see right away what you need to do.
You will most likely see the yellow blobs that are in some of his pictures--as long as they aren't really bad (like going all over the place, ruining the whole dish), don't freak out, just work around them and keep cleaning. Your final dish should eventually look like the one at the very top of his page. Kind of like a medieval drawing of the sun.
Other credit goes to Roger Rabbit for the plastic sleeve trick.
------------
Per request, here are my personal notes and experiences with this procedure. I'll try to keep this really simple, and avoid going into too much detail.
I have found that many people balk at the thought of doing this, which I assume is due to the expected complexity of the procedure. I balked myself during my first round of experimentation, and then gave it a shot for the second. Let me assure you, it is not at all difficult, or any more difficult than the other sterilization procedures that cultivating mycelium requires. As a matter of fact, it appears to be very beneficial to the process. As I have noted in other posts, it speeds it up quite noticeably.
I think that the most difficult part of all this is acquiring the materials. Note that there are a few poor-man's methods of working around the official materials, in the event that ordering online is not possible, or if there are financial limitations, but I'll just write about the official ones that I have used so far.
Stuff you will need:
--------------------
1. Plastic petri dishes (*)
2. Parafilm
3. Agar (potato dextrose or malt extract) (**)
4. Agar pouring device (***)
5. Agar cutting device, such as x-acto scalpel.
6. All the rest of the usual sterilization procedures, materials, and devices
*Buy a plastic wrapped sleeve of 20, because (1) they are easy to dispose of, (2) are pre-sterilized, unlike the glass ones, and (3) the plastic wrapping is actually useful in the procedure.
**I don't think it really matters which type you use. If anything, it is more of a preference related to how easy it is to see through it, which is useful to selecting sectors to transfer. I personally have been using the potato dextrose agar for both Mex-A, Mex-ATL7, and Ps PE without any issue.
***Start collecting short glass bottles that will withstand being PCed. 2-3 of them should do for 20 dishes. You will need these for sterilizing the agar, and they'll need to fit in the PC. An easy measurement comparison when shopping is to keep it around the height of your mason jars, if you are using them. Check out the sauces area of your grocery store, you should find something viable there. The lid won't be needed, so it can be plastic.
Procedure
---------
1. Mix agar (unless already premixed), and pour into glass agar containers. Don't fill more than 2/3 full to prevent boil-over.
2. Cover agar containers loosely with tin foil and put into PC.
3. Use a bit more water than usual in the PC to help prevent agar boil-over.
4. PC at 15psi for 30 minutes.
5. Let cool down in PC (about two hours), but not enough to get cold and harden.
6. While agar is cooling, prep work area.
7. Move agar out of PC into SAB as quickly as possible.
8. Uncover each dish, pour thin layer of agar, and recover.
9. Stack up the prepped dishes back in the plastic sleeve. Seal the sleeve and put in the fridge. No sealing with parafilm yet, just leave them loose. The plastic sleeve will act as the contamination barrier.
10. Let sit in the fridge for a few days to see if anything grows. Toss the contaminated ones.
11. Prep work area again, this time including the spores, the first round's quantity of prepped dishes, and the sealing materials. I went with three dishes per culture for the first round, which worked just fine.
12a. (If syringe) Sterilize tip until glowing red hot (although not too far down towards the liquid!), move inside SAB, let cool, shake to redistribute spores. Quickly uncover a dish, try to drop one single drop right in the center, and recover.
12b. (If print) Sterilize inoculation loop until glowing red hot, move inside SAB, shake to cool, rub on print, then quickly uncover dish, rub off spores onto center of agar, and recover dish. If the print is too tough, drop one small drop of sterilized water onto the print and rub around with loop, it should become easy to work with.
13. Repeat until all dishes are inoculated.
14. Seal all dishes with parafilm, and then they can leave the SAB.
15. Mark each one with what culture it is, that it is round 1.
16. Put in a warm, dark place that mycelium likes to grow in. This is the fun part. Check back each week, or as desired, until the growth has gotten near the edge of the container, or contamination is threatening to ruin the dish. (The better your sterilization procedure, the less of this will occur. It is actually a useful test to visibly see how good your procedure is.)
17. At this point, it is ready to be transferred to a new dish. Prepare your work area again, this time with the previous round of dishes, more prepped dishes, and a scalpel. I transferred the best of the first round's three dishes onto a single dish for the remaining rounds. (If you have higher contamination risk, you might want to go with more dishes per round, but I found it surprisingly unnecessary.)
18. Find the best sector of growth of the first three dishes. Cut a small piece out with a sterilized, cooled scalpel, doesn't need to be big, and drop it in the middle of a new prepped dish. Seal it and mark it as round 2. Put the dishes back in the warm dark place and let them grow out again.
19. Repeat until you have a perfectly uniform culture on the dish, which is referred to as a monoculture. This should be the fastest growing, most aggressive performing culture out of all the rounds that you have completed.
20. Take a piece of that and drop it into your prepped master grain jar, or into your master culture slant.
Additional Notes
----------------
For some pictures of what to expect, see Stro's nice guide on the Shroomery. That's mostly where I learned all this from. Credit and thanks go to Stro for his guide. It will show you what I mean by "sectoring" and "monoculture". Most of it is common sense though--when you're looking at it in your dish, you'll see right away what you need to do.
You will most likely see the yellow blobs that are in some of his pictures--as long as they aren't really bad (like going all over the place, ruining the whole dish), don't freak out, just work around them and keep cleaning. Your final dish should eventually look like the one at the very top of his page. Kind of like a medieval drawing of the sun.
Other credit goes to Roger Rabbit for the plastic sleeve trick.
