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Isolating/Testing/Storing Cultures


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#1 peacefrog

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Posted 29 July 2015 - 02:33 PM

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: 

1. Multi-spore.jpg

 

Transferring:

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:

2. First transfer.JPG

 

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:  

4. First transfer grown(3).jpg 5. First transfer grown(4).JPG 6. First transfer grown(2).JPG 8. Second transfer grown(1).JPG 9. Second transfer grown(2).JPG 10. Second transfer grown(4).JPG 11. Second transfer grown(3).JPG IMG_0096.JPG IMG_0097.JPG 11. Second transfer grown(3).JPG 12. Third transfer 1.JPG 13. Third transfer 2.JPG 14. Third transfer 3.JPG 15. Third transfer 4.JPG 16. Fourth transfer 1.JPG 17. Fourth transfer 2.JPG 18. Fourth transfer 3.JPG 19. Fourth transfer 4.JPG

 

After several transfers, I now have 4 different pure cultures isolated exhibiting uniform growth and the rhizomorphic mycelium I was trying to isolate:

22. Fifth transer 3.JPG 23. Fifth transer 4.JPG

 

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:

30. Cultures.jpeg

 

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:

25. Grain 1.JPG

 

Rhizomorphic growth from one of the agar wedges:

27. Grain growth.JPG

 

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

31. Tray of grain.JPG 32. Tray of grain cased.JPG 33. Cased grain covered.JPG 35. Colonized casing.JPG

 

Matured and ready for harvest in exact order of labeled cultures:

44. Harvest.JPG 45. Harvest.JPG 43. harvest.jpeg 46. Harvest.JPG

 

Conclusion:

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:

29. Slant.jpeg

 

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.    

 

 

 

 

 


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#2 mushfun

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Posted 29 July 2015 - 04:17 PM

Cool write up, thanks.
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#3 happy4nic8r

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Posted 29 July 2015 - 04:18 PM

Really nice Peacefrog, I have to give it a full erection, and definitely more than just a "like".


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#4 Mycomaniac2007

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Posted 29 July 2015 - 05:52 PM

Awesome post and very noteworthy. Great details and easy to follow! Im glad you put that up and sometimes it hard when someone has to pick a tek or details from a bunch of different thread to get the idea.

Great job with fruiting directly from grain to speed test those cultures, simple and easy :-)
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#5 MLBjammer

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Posted 29 July 2015 - 07:51 PM

That's a wonderful write-up.  It would help if the type was a bit bigger, though--well, I am half-blind anyway.

 

This kind of thing is very helpful for new or intermediate growers just getting into agar work.

 

Awesome work, man.


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#6 TurkeyRanch

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Posted 29 July 2015 - 08:02 PM

Well written! Thanks peacefrog.

Marked for Archive material.
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#7 peacefrog

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Posted 29 July 2015 - 11:30 PM

Thanks for the kind words guys! And sorry about the font. I'm surprised I didn't notice that when I posted it. I started isolating in January and typed everything up as I progressed on Word on my computer. I then just copied and pasted. I haven't put that much effort into a cube grow for some time now, but it was really fun working with them again.
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#8 TurkeyRanch

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Posted 30 July 2015 - 09:42 AM

Yep, they are a great species to play around with because they tend to be very forgiving. Thanks for contributing, this will certainly help lots of beginner cultivators.
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#9 drmcnasty

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Posted 30 July 2015 - 10:18 AM

Very well put together.
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#10 peacefrog

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Posted 30 July 2015 - 06:59 PM

I hope it does help some cultivators out there wanting to explore agar work. This is a wonderful community where we can all share, help and learn. One big happy family! Cheers to all!
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#11 azure7

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Posted 09 August 2015 - 04:40 PM

great info ...


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#12 toadshroom

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Posted 20 August 2015 - 05:26 PM

This should be in the vaults. Clear, concise write-up! Good job peacefrog! :-)
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#13 CatsAndBats

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Posted 28 October 2015 - 04:26 PM

this is such a helpful post @peacefrog! bookmarked. 


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#14 sahtanoj

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Posted 28 October 2015 - 04:37 PM

very good!

those slants you bought it ready or make it yourself the culture?


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#15 peacefrog

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Posted 28 October 2015 - 06:05 PM

Thank you very much!

Sahtanoj,

The slants were purchased from an online source, poured to about 3/4 full of water, a heaping teaspoon of agar and sterilized. Then placed on an angle to cool and solidify. I then placed a piece of the culture in there and allowed to colonize.
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#16 MLBjammer

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Posted 28 October 2015 - 06:05 PM

I agree that this is absolutely vault material--a wonderful write-up.

 

When you have time, you should do a write up on your whole process of making and using agar.


Edited by MLBjammer, 28 October 2015 - 06:06 PM.

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#17 peacefrog

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Posted 28 October 2015 - 06:26 PM

Perhaps I will post a few.

Thank you all!

Edited by peacefrog, 28 October 2015 - 06:55 PM.

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#18 MLBjammer

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Posted 29 October 2015 - 03:31 AM

It would be very helpful to newer growers interested in agar.  You know how tough it is to find good information sometimes.


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#19 peacefrog

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Posted 31 October 2015 - 05:52 AM

I agree. In have posted some recipes and my style on a couple of other people's posts before, but I think it might be easier to find and see if it were on its own thread. I'll do one showing some of my favorite recipes.
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#20 CatsAndBats

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Posted 31 October 2015 - 09:41 AM

I have some agar anomalies (I guess). Should I put the photos here or start a new thread? 


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