Isolating Single Spore Monokaryons

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mind.at.large
Myconerd

Registered: 12/13/16
Posts: 1,230
Loc: Floating in liquid gardens

Isolating Single Spore Monokaryons

23
#28708274 - 03/22/24 10:08 AM (2 years, 4 months ago)

Hey y'all! It seems like forever since I've been around these parts but I miss all of ya and I've got some cool stuff to share. I've always been fascinated with monokaryons but it wasn't till about 6 months ago that I really dove into what it takes to isolate monos. I've tried a couple different techniques and have found single spore isolations using serial dilutions to be the easiest and most consistent way to find monokaryons. So I'm going to detail my process here. Hit me up for any questions.

Isolating monokaryons is useful for breeding and once you have a monokaryon, you can essentially breed it with any variety (and possible intraspecific hybrids). This vastly opens up the possibility for breeding and creating as many varieties as you want. A quick description of what a monokaryon is for anyone who doesn't know. Pretty much all the mushrooms we cultivate are basidiomycetes (except for a few, Cordyceps being the main example). Basidiomycetes need two mated monokaryons to reproduce. A monokaryon is a single germinated spore. Once mated, they share nuclei and become a dikaryon. Monokaryons are, by nature, eager to match with another monokaryon to create a dikaryon and reproduce (produce mushrooms). So, isolating a monokaryon is the first step in creating a cross/hybrid. Technically, crossing can easily happen by placing spores of differing varieties on the same plate and letting them mate. This is for sure a great way to cross, however, it requires a guessing game to find the crossed fruit. Isolating monos takes away this guessing game.

The method I like to use to isolate monos is through serial dilution. Essentially, this means to dilute a spore solution so much that in theory, each drop will have one single spore. Once you get the proper dilution rate, all you have to do is put a drop on a plate and let it grow. If your dilution rate is correct, once that single spore germinates, there's your mono. Now this method works very well but I have found a few tricks to help ensure more monos. The materials/equipment I use are probably not in your average cultivators arsenal, but you can totally repeat this process without any of the "fancy" stuff. Don't have a micropipette? Use a syringe. Don't have centrifuge tubes? Use jars. You get the point.

Materials/Equipment:
- Spores (prints, swabs, syringe, etc)
- Agar plates
- Tween 20 (surfactant used in cosmetics and microbiology) very cheap and easily found - also called Polysorbate 20
- 1.5 ml microcentrifuge tubes and rack
- Micropipette and tips
- A couple mason jars with lids

Steps:
1. Take a jar, fill with water, and add a very small amount of Tween 20 (I added a single drop to this jar)

2. Fill a jar with microcentrifuge tubes

3. Sterilize Tween 20 solution, micro centrifuge tubes, and pipette tips for 30 minutes at 15 psi

4. Once everything is cooled down, set up in aseptic conditions

5. Set up your centrifuge tube rack and load up some tubes like this

Each column of 3 is going to be for one variety

6. Open up the tubes and using the pipette, add 1000 ul of tween solution into the first tube, and 900 ul into the other 2 tubes behind it.

7. Add spores to first tube with 1000 ul of tween solution. Obviously there's many ways to add spores, but I have a feeling y'all can figure that part out. No need to add a lot, a little is just fine.

8. Shake that tube like you're shaking a baby. If you think you've shaken it enough, do it again. This helps break up spore clumps and ensures an even distribution of spores throughout the solution.

9. Add 100 ul of the spore solution to the 2nd tube and once again shake the ever loving shit out of it.

10. Add 100 ul of the 2nd tube to the 3rd tube and once again, get shakin!

11. Now you have a 100x diluted spore solution. Many people like to dilute it one more time to get to 1000x, but I have found 100x to be good enough. However, spore density varies widely so you might have to play around with finding the right dilution rate

12. I then like to add one drop of each tube to separate agar plates. I then use a sterilized tool (scalpel, loop, spreader, etc) to spread the solution throughout the whole plate. Keep track of where you originally dropped the solution, because that is where most of the spores will be. Label all the plates to help know what dilution rate works best for whatever you're growing.

Here's a plate that I found a few monos from but then took this pic a few days after. I'm assuming most of those colonies are now dikaryotic but there's how I like to spread the solution around

13. Pay diligent attention to your plates over the next few days. At the first signs of any germination, gently transfer those over to fresh plates. Be very careful and surgical when transferring as you're trying to remove the smallest piece you can. A dissecting microscope is incredibly helpful for identifying germinated spores

14. Once transferred, let it grow a few days and assess growth patterns. Tomentose and uniform are signs of monokaryotic growth, however, it is impossible to verify monos without a microscope.

Here are some microscopy photos of monokaryons lacking clamp connections

Always verify and reverify your potential monos, but once you feel confident, go ahead and start breeding. I'll be adding some more info about breeding, crossing, and generally what to do with monos once you get them, but here's a start! Now get out there are start grabbing monos!!

(Also, go check out the marketplace right now cause I'm giving away a couple of these monos!!!)

Edited by mind.at.large (03/22/24 10:55 PM)

Extras:

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