I have searched this site to find a complete guide for isolating mycelium from multi-spore to pure culture, testing, and long term storage. But I did not find anything that had all the info in 1 post. So I decided to put one together for anyone out there who may be interested in agar work and/or isolating and finding consistently good producing cultures.
Cloning is much faster, but I have decided to do isolating from spores instead.
This procedure does take some time, but if one is patient, it will be well worth the wait IMO. Also, one can isolate pretty much whatever characteristics coveted. I personally like to isolate the fastest and most productive cultures. That’s the beauty of cloning and/or isolating, one can pick and choose what works best for them or what they like to grow. And the crap shoot of multi-spore growths are no more.
The type and strain I decided to work with is P. cubensis PES Hawaiian.
Agar streaked from a spore print and allowed to colonize:
I transfer 2-3 very small pieces from the best sectors I can locate into a fresh agar plate. I normally do this for 5-6 more additional plates giving me several different isolates producing many sectors to choose from (the more the better). For the purpose of this post, I decided to keep it simple and only do 2 plates. One can transfer a single wedge as well, I just prefer to prepare lesser amounts of agar for the same results. Or if you like to use Petri’s, you can purchase the plates that have sections with built in barriers and do 3-4 per plate. I prefer to use 4 oz. jelly jars for all of my agar work. I also change agar recipes frequently. This helps the mycelium remain aggressive by not allowing it to get used to the same food source:
Several pictures of the some transfers to show the different mycelial growth. Picking the mycelium that produces the best growth (in the case of P. cubensis, ropey is the best) over time you will be able to isolate the growth you are looking for. One plate became contaminated with Aspergillus on the corner, but had some promising growth. As you can see many of these plates are throwing off rhizo. growth, but are not uniformly rhizomorphic. Some still have linear or tomentose growth as well as the ropey type. If the plate is not uniform, you do not have a pure culture, and just because you isolate a pure culture with the best growth, you cannot be sure it will grow well until you test:
After several transfers, I now have 4 different pure cultures isolated exhibiting uniform growth and the rhizomorphic mycelium I was trying to isolate:
Now comes the fun part of testing each isolate, choosing which one or ones you would like to work with and grow indefinitely if preserved correctly. For this post, I have used rye grain to colonize and fruit in mini grows. I have chosen to fruit from grain because it’s faster than waiting for bulk to colonize. Although, using bulk to test does have its advantages and must be done for certain species that do not grow well on grain alone. One could also use cakes, but I prefer whole grain for cubes. If using cakes you can make a slurry or LC to inoculate (you can also use this method for grain). If making an LC, it will take a little longer due to the fact that you have to wait for it to colonize and then test for cleanliness. But either way will get the job done. For the edible wood lovers I have isolated, I used the cake method with a slurry into saw dust based cakes for testing.
Alternatively, you could skip the mini grow and test each culture one at time using whatever style you prefer or if you have room, you can do all together in larger grows. I prefer doing the mini grows as it does not take up much space and it gives plenty of opportunity for a side by side contrast/comparison without all the space required for larger grows.
I labeled each culture in the exact order of the above picture so I can keep detailed notes and compare which one/s are winners. As stated above, the more pure cultures you have to test, the better you will be:
I place 2 medium size wedges of agar from each culture in a pint jar of grain, shake and allow to colonize, labeling each and taking good notes:
Rhizomorphic growth from one of the agar wedges:
After all are colonized I break up and pour into a small container, case with 50/50+ pasteurized (sterilized casing will work as well) at 160 degrees for 60-90 minutes at a depth of ½-1 inch, and cover with foil or something similar with a couple of small holes for general gas exchange and allow it to colonize. Again they are carefully labeled and I continue to make detailed notes of how fast and even the casing colonizes, pin sets, speed of maturity, size and shape of fruit, etc. Side note: I did not know before I read a recent post by MLBjammer that peat bogs are being depleted. I think I will change my casing recipe after reading that post, but this is what I have always used and did not know about that fact when this was done:
Matured and ready for harvest in exact order of labeled cultures:
Although all produced, culture 3 was the best and most prolific fruiting culture. It wasn’t as rhizomorphic as the other 3 and it was one of the slower one’s to mature, but it is the one I would pick as my culture of chose from this grow. This culture continued to produce well and gave me 4 flushes all together. The other cultures clearly reiterates the point that just because you have the perfect type of mycelial growth does not mean you will have a rock star culture. This is why methodically testing each isolated culture is paramount. If I had isolated 8-10 cultures, I may have had 1-2 more great or greater producers that were perhaps a little faster to mature. So as said before, the more cultures you have to test, the better chances you will have at getting a great producing culture/s.
And now finally to store the culture long term, place a small piece into a slant, let it colonize and refrigerate:
The slants can be stored this way for years. I prefer to take mine out every 1-2 years and re-grow and transfer into new slants for continual storage. It is a good idea to take fresh spore prints periodically as well, just in case anything goes wrong and you lose the culture/s. Using it to grow, you want to colonize at least one agar plate from the original isolated one for further growth and transfers, and ONLY transfer from the slant when needed. This will keep the cell divisions down and will insure you have the same healthy culture you isolated for many years to come. Think of it as your main backup and use the plate/s as a “master”. Continue to transfer and refrigerate from this “master” to new plates until it contaminates or senescence becomes an issue. Then you can use the slant (your main backup) with very few cell divisions to create a new master plate and start the whole procedure again.