advanced mycology:help with substrain isolation

M

MinnesotaHomegrown303

Active Member
So I've recently gotten into agar work and was wondering some opinions on isolating sub strains from tissue. I have a few clones that I have begun trying to reduce to the substrains contained within. I am very lost in how many transfers to make from each sector. And after you transfer that and there are several sectors do you then transfer each of those? That seems exponential to me and would seem even from one clone that you could isolate hundreds of substrains. Maybe I'm missing something? Please help
 

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The picture above is a clone from an 80g Brazil cluster. It appeared to have 9 rhizomorphic sectors. I transfered each to new dishes and lost 7 and 9. The rest I still have but if they sector out into multiple each do I take the strongest rhizo or all of the different sectors? Seems like if I took them all I would have hundreds of isolates to test just from one clone? That sounds more like ms to me.
 
The first picture in this comment is a clone from a 34 cluster mushroom also Brazil from a different bin, ms. There were no sectors visable until a day before the culture was refrigerated and a wedge was cut to create a master jar for testing. The second and third picture seem to be extremely close to isolates but I'm not sure. I understand homogenisis but they didn't sector to begin with. I would love that last little bit of help understanding exactly how to isolate from tissue down to a t. Any input is greatly appreciated
 

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Take only the most ryzomorphic sector. Be sure your agar is completely homogenous. No corn steep.

Swap PDA with mea at least once. If your original tissue was from a cap it is already likely you have a clamped monoculture.
 
What are you after?

My work with coprinous and plurotus requires I fruit in the conditions I am selecting for, you may not have to do that depending on your goal.
 
canndo said:
What are you after?

My work with coprinous and plurotus requires I fruit in the conditions I am selecting for, you may not have to do that depending on your goal.
Click to expand...
Interesting. So I should try to take my tissue samples from the cap? I'm just after a vigorous sub strain. All my pictures are from tissue some cap some stipe. When they sector do I isolate each rhizo sector or choose one from each clone? Also when transferring to isolate, if I've taken a rhizo sector of a clone and put it on new agar and two or more sectors occur do I again transfer these out or choose just the strongest one? My first picture was a clone with 9 distinct rhizo sector . I am growing those out but if they all have multiple rhizo sectors it seems like within the next transfer or two I will have 30+ kinds to test which seems extremely excessive
 
At
MinnesotaHomegrown303 said:
Interesting. So I should try to take my tissue samples from the cap? I'm just after a vigorous sub strain. All my pictures are from tissue some cap some stipe. When they sector do I isolate each rhizo sector or choose one from each clone? Also when transferring to isolate, if I've taken a rhizo sector of a clone and put it on new agar and two or more sectors occur do I again transfer these out or choose just the strongest one? My first picture was a clone with 9 distinct rhizo sector . I am growing those out but if they all have multiple rhizo sectors it seems like within the next transfer or two I will have 30+ kinds to test which seems extremely excessive
Click to expand...
At least for just one clone and not ms. I didn't think that clones were made up of so many substrains. I have multiple clones and no room for 100+ dishes so I'm just looking for the best way to isolate a few with potential rather than 100s which I do not have the time or resources to test
 
Of you have sectors after an isolation or two, suspect your medium. You likely have a monoculture. Remember that only one sub strain will produce basidium.

Gather as few hyphae as possible, and transfer.

Here is a trick. If you place two identical substrains on the same dish then you will see no deliniation between them, they will merge. If they are at all different, there will be a border formed at the convergance. No border? Same sub strain. Easier than trying out a bunch of sectors that are really the same.

Some strains just sector, that's the way it is. Some detect tiny differences in substrate.
 
Seems odd that you would have to take tissue from the cap since the fruit body is made from the same mycelium, with the same genetic makeup. When collecting tissue I have taken samples from both the stipe as well as the pileus. It mainly was dependent on which tissue was least damaged when collecting. Best to rip the mushroom open, as internal tissue is not contaminated, and you lessen the risk of contaminating your tools, as could be the case when cuttin g into the shroom.
 
GranolaCornhola said:
Seems odd that you would have to take tissue from the cap since the fruit body is made from the same mycelium, with the same genetic makeup. When collecting tissue I have taken samples from both the stipe as well as the pileus. It mainly was dependent on which tissue was least damaged when collecting. Best to rip the mushroom open, as internal tissue is not contaminated, and you lessen the risk of contaminating your tools, as could be the case when cuttin g into the shroom.
Click to expand...
Thanks. I've found that the stipe tends to have the smallest number of sectors and most prolific fruiters. Since this thread I've isolated several sub strains of Brazil Australia and b . All I can say is I love life and thank you cando
 
GranolaCornhola said:
Seems odd that you would have to take tissue from the cap since the fruit body is made from the same mycelium, with the same genetic makeup. When collecting tissue I have taken samples from both the stipe as well as the pileus. It mainly was dependent on which tissue was least damaged when collecting. Best to rip the mushroom open, as internal tissue is not contaminated, and you lessen the risk of contaminating your tools, as could be the case when cuttin g into the shroom.
Click to expand...


I usually take material from where I can get it.
 
MinnesotaHomegrown303 said:
The youngest mycelium is the strongest
Click to expand...


That sort of depends upon substrates. I look at it as a factor of length. The lomger, linearly, the mycelium has grown the more likely it will be weaker, however, it gains strength or delays age by exercising a variety of enzymes so long as it is fed a variety of substances rather serially or all at once. This is why I suggest varying agar types, PDA, NEA, cornsteep, straw steep, dog food, grain flour etc. Eventually the mycelium will begin to fail regardless but I have seen viable growth at ten generations.
 
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