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Clarifying questions about agar and strain isolation
    #25814719 -

Hi all, I'm a molecular biologist trying to get started with agar. I'm posting this in the advanced forum because I have some questions about the details of monokaryotic and dikaryotic growth on plates; let me know if I should post elsewhere. Questions are in bold and numbered, feel free to address even just one of the questions. Thanks in advance for your help, I really appreciate it. I plan on writing a protocol and sharing it with the community once I come to a fuller understanding.

I just bought a few spore prints (not syringes). I have two goals: grow a monotub in the near term, and learn how to perform strain isolation to identify a strain that can consistently produce dense, large, potent fruits. Please correct me at any point if my understanding is incorrect.

==============================

From what I've read, you can go two routes with agar:

Process 1. Produce mushrooms fairly quickly: streak multi-spore on agar > transfer clean colony to new plate > transfer piece of rhizomorphic sector to new grain.

Q1: What is the name of this process? If I'm going agar to grain, should I streak to dilute, as I describe in my basic plan below? Do I want single colonies that may or may not be good fruiters, or should I attempt to maintain the multi-spore status by dumping spores in the middle of the plate?

Process 2. Strain isolation: streak multi-spore on agar > transfer many clean colonies to many new plates > transfer pieces of rhizomorphic sectors to new plates > repeat until you have single-sector growth (monoculture?) > transfer monocultures to grain > identify the monoculture with the most desired fruiting qualities > preserve the corresponding mycelium and grow from that each time to ensure consistent, desirable fruits.

My understanding is that #2 can take many months and if you want some mushrooms in a timely manner, you should do #1 and #2 simultaneously.

==============================

My basic plan with questions:
  • Streak spores from print onto agar plates ("Plates 1") to dilute into single colonies that are free from contamination and isolate single "colonies" (is this the correct term?)

    Q2: Are the single colonies monokaryons, dikaryons, or some of both (if both, how do you identify the dikaryons, assuming you want to propagate those)? Please explain what is happening on a cellular/genetic level.

  • Transfer multiple individual colonies to new plates ("Plates 2")

    Q3: At what point should I transfer the colonies? Should I transfer the whole colony at the first sign of growth, or should I wait until the colonies have grown out some and formed rhizomorphic sectors, at that point transfering a piece of the rhizomorphic growth?

    Q4: In Stro's Cleaning and Isolating on Agar instructions, do his Plates A, B, and C single colonies correspond to "Plates 1" or "Plates 2"?

  • Let those new plates grow, then split the process into Process 1 and Process 2 above:
    Process 1. Transfer pieces of rhizomorphic sectors to grain, to get some mushrooms growing in the short term, and
    Process 2. Transfer pieces of rhizomorphic sectors to new plates ("Plates 3"). Repeat agar to agar transfer until there is only one rhizomorphic sector ("Plate N")

    Q5: I'm a little confused about this step with regards to Process 2 -- is there an advantage of achieving a plate with only one rhizomorphic sector, as opposed to transferring a piece of rhizomorphic growth from "Plates 2" to grain and testing the qualities of the resulting fruit of each? If so, what is the advantage?

  • Process 2 final step: transfer a section of rhizomorphic growth from each plate to grain jars, grow each and determine which produces desirable fruits, go back and make slants from that culture's "Plate N"

Edited by replyom (02/15/19 02:24 PM)

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Re: Clarifying questions about agar and strain isolation [Re: replyom]
    #25814763 -

Q1: You'll never get a true mono-culture, unless that molecular biology comes with a lab for you that is above and beyond most amateur access.

Q2: Unfamiliar with the term colony in relation to this. Do you mean culture?

Q3: Most people say when it's about the size of a dime/nickle you should transfer from one plate to the next. No one will be able to tell you if rhizo or tomentose is better because no one knows. Depending on conditions you can transfer rhizo and it'll convert to tomen. :shrug: There is no preference these days as far as I know. Take from the fastest growing (clean) leading edge. You're good.

Q4: dunno, haven't read it.

Q5: Yes and no. You will continue to isolate further and further if you just keep transferring. You're unlikely to hit mono-culture this for a few years (and if you do, you won't even know if it's got good genetics or traits until you grow it).

If you take transfer two and put it to grain you can grow and then clone from there. Many people find this the better way to go for hobbyists. Read up on the Rusty Whyte thread to see how Pasty isolated specific traits to get that variety.

https://www.shroomery.org/forums/showflat.php/Number/20228521
https://www.shroomery.org/forums/showflat.php/Number/25284782

Good luck!


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My Shoebox Factory Beginner's Tek | Shoebox Assembly Tek | Methodology/teks I use | How I Store Bulk Items

Edited by ShaperDreaming (02/15/19 02:34 PM)

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Re: Clarifying questions about agar and strain isolation [Re: ShaperDreaming]
    #25814852 -

Thanks for your reply.

Q1: You'll never get a true mono-culture, unless that molecular biology comes with a lab for you that is above and beyond most amateur access.

So you're saying it doesn't matter whether I do a diluting streak or simply spread some spores in the middle of the plate, because any and all growth I see will still be "multi-spore"? When you go from a spore print to agar, how do you apply the spores? Do you do a diluting streak as described in this post?

Q2: Unfamiliar with the term colony in relation to this. Do you mean culture?

The picture below is bacteria on agar, the liquid bacterial culture was poured onto the plate at 5 o'clock and was streaked to dilute and thus generate colonies of bacteria at the top of the plate. Each colony is generated from a single cell of bacteria that contains an antibiotic resistance gene and was therefore able to survive and divide. I'm assuming that streaking spores in this manner will result in "colonies" (what is the correct term?) at least somewhat similar to this, but I'm not sure. If so, are these "colonies" dikaryons?



If you take transfer two and put it to grain you can grow and then clone from there. Many people find this the better way to go for hobbyists. Read up on the Rusty Whyte thread to see how Pasty isolated specific traits to get that variety.

Thanks, I will look into this. So just to be clear, this is what you're recommending?
  • Transfer 1 = apply spores from print to agar (simple application to middle of plate, or streak?), wait for dime sized growth
  • Transfer 2 = transfer a small piece of the fast edge of growth to new plate, allow to grow substantially (half of the plate or something)
  • Then transfer six different pieces from fast/rhizomorphic edges to six grain jars, fruit
  • Clone a nice fruit onto agar, then transfer that mycelium to grain, your fruits will be clones of that fruit

Edited by replyom (02/15/19 03:07 PM)

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Re: Clarifying questions about agar and strain isolation [Re: replyom]
    #25814880 -

Cloning is simple. I'm going to assume you have basic agar skills and can clean a culture up grown from spores, produce clean grain spawn and grow fruits.

1. Pick a fruit with desired traits

2. Tear fruit in half with hands (don't cut- it can push contams from the outside in).

3. Drag a piece of inside tissue with a sterile scalpel and place on agar.

4. Let it grow out some, transfer clean growth to a new plate and continue until it's clean.

5. Carefully label and store a plate with clone culture. Grow out and test your clone to see how it produces, test the potency etc.

6. If it's worth saving, store/Slant your clone.


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Amateur Mycologists United
AMU Q&A

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Re: Clarifying questions about agar and strain isolation [Re: replyom]
    #25814884 -

replyom said:
Q1: You'll never get a true mono-culture, unless that molecular biology comes with a lab for you that is above and beyond most amateur access.

So you're saying it doesn't matter whether I do a diluting streak or simply spread some spores in the middle of the plate, because any and all growth I see will still be "multi-spore"? When you go from a spore print to agar, how do you apply the spores?


Yeah, basically it doesn't matter. I'm not going to read the thread you linked (sorry) but if you look up Bod's talks about this you'll see his microscope pictures and they'll explain that no matter how much you think you've diluted this that it's impossible for the amateur to achieve strain isolation.

replyom said:Q2: Unfamiliar with the term colony in relation to this. Do you mean culture?

The picture below is bacteria on agar, the liquid bacterial culture was poured onto the plate at 5 o'clock and was streaked to dilute and thus generate colonies of bacteria at the top of the plate. Each colony is generated from a single cell of bacteria that contains an antibiotic resistance gene and was therefore able to survive and divide. I'm assuming that streaking spores in this manner will result in "colonies" (what is the correct term?) at least somewhat similar to this, but I'm not sure. If so, are these "colonies" dikaryons?




Answering this question is beyond my pay grade. I'd call it mycelium on agar.

Quote:
replyom said:
If you take transfer two and put it to grain you can grow and then clone from there. Many people find this the better way to go for hobbyists. Read up on the Rusty Whyte thread to see how Pasty isolated specific traits to get that variety.

Thanks, I will look into this. So just to be clear, this is what you're recommending?
  • Transfer 1 = apply spores from print to agar (simple application to middle of plate, or streak?), wait for dime sized growth
  • Transfer 2 = transfer a small piece of the fast edge of growth to new plate, allow to grow substantially (half of the plate or something)
  • Then transfer six different pieces from fast/rhizomorphic edges to six grain jars, fruit
  • Clone a nice fruit onto agar, then transfer that mycelium to grain, your fruits will be clones of that fruit



Almost! Things to add: these transfers a dependent on making sure that there's no bacteria or other contam on the plate, and if there is you transfer away from there and try again.

Also, I am not saying anything about rhizomorphic growth. The type of myc is more dependent on things unrelated to good/desirable genetics.

Other than that, yup! If you want to go the most consistent way to get the types of fruits you're looking for you need to grow them at some point. Cloning from the traits you want to isolate is easiest to do from grabbing them out of your tub, not isolation on agar.

Also, do a forum search for the tiger drop :smile:


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My Shoebox Factory Beginner's Tek | Shoebox Assembly Tek | Methodology/teks I use | How I Store Bulk Items

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Re: Clarifying questions about agar and strain isolation [Re: ShaperDreaming]
    #25814902 -

Here's that response about spores by Bod: https://www.shroomery.org/forums/showflat.php/Number/25810284#25810284
bodhisatta said:
denger said:
bodhisatta said:
No that would be the third way. By the time you see mycelium on a dish its long been dikaryotic if you streaked out spores


That depends on the density of the streak. If it shows single colonies with some distance between them they could be monos with some probability.


I purposely diluted spores down quite a bit then streaked them. Looked at with a microscope there's still just too many spores. And spores like to clump. If you could dilute spores and also keep them from grabbing a partner then maybe. But even using jet dry or other surface tension agents wasn't able to get spores broken apart enough to get monokaryotic growth no matter how hard I tried. I think it would take a micromanipulator scope to achieve single spore monokaryotic growth reliably

Even with hefty agitation you'll see clumps like this


Diluted and streaked


If you made a 10cc syringe with the smallest speck of spores you can see its still too many spores


Even at the hardly visible growth phase they're long dikaryotic from streak plates(not super diluted in this case)



Ive played around a bunch with germination and trying to get monokaryotic growth but i think its out of the reach of hobby equipment


Spores clump like madmen despite your best efforts to make them disperse evenly



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My Shoebox Factory Beginner's Tek | Shoebox Assembly Tek | Methodology/teks I use | How I Store Bulk Items

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Re: Clarifying questions about agar and strain isolation [Re: mushpunx]
    #25814916 -

Again I'm going to assume you have rudimentary agar skills.
Here is another basic run down.

Strain Isolation :

1. Streak spores on plate,let germinate.

2. Transfer clean germination.

3. Keep growing the culture (quarter to half dollar size), choose healthy looking growth (rhizo, tomentose- doesn't matter just clean healthy looking growth) and keep making transfers.

4. Eventually, typically around 8 to 10 transfers you will start to see obvious sectoring. See Stro's guide to see photos of what sectors look like.

5. Make a transfer from each individual transfer you can see. I shy away from anything really weak looking. Transfer from as many as you can see, I shoot for 10 to 15.

6. Grow out each of the 10-15 transfers from sectors you took.
If you are lucky, you will already have some monocultures/isolates- again see Stro's guide for photos. Monocultures won't show any sectoring.
Label and save each isolate.

7. Any cultures that still show sectoring, continue to make transfers until those also show no more sectors and are monocultures.

8. Take your 10-15 isolates, label each very carefully and store a  in the fridge. Fruit out each isolate and look for any desirable traits - speed, yield, clustering, :important: potency!! etc.

9. Compare each isolate and look for the best one/ones .

10. Store and slant the winning isolate.


Note: the way I'm using the term monoculture is just a single sector isolate. At least as close as we can get to one at home yea?


--------------------

Amateur Mycologists United
AMU Q&A

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Re: Clarifying questions about agar and strain isolation [Re: mushpunx]
    #25814923 -

mushpunx said:
Note: the way I'm using the term monoculture is just a single sector isolate. At least as close as we can get to one at home yea?


:whathesaid:
If you're going to use monoculture, this is the understanding we need. Can we call it monoishcultures?


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My Shoebox Factory Beginner's Tek | Shoebox Assembly Tek | Methodology/teks I use | How I Store Bulk Items

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Re: Clarifying questions about agar and strain isolation [Re: mushpunx]
    #25814959 -

Thank you mushpunx, makes sense to me.

I'd be interested to read about the results of strain isolation from a clone. Once you have your 10-15 isolates from that clone and spawned/fruited them, how much variability would you observe between the isolates? This would tell you a lot about the utility of strain isolation vs cloning.

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Re: Clarifying questions about agar and strain isolation [Re: ShaperDreaming]
    #25814981 -

Thanks a lot ShaperDreaming. Btw here's a quick link to the streaking method I'm talking about and the resulting "colonies". With this method the colonies are going to be way less genetically diverse. After reading some more today I see that you are correct, you can never get single-spore colonies, but by spreading the spores out on the agar by streaking, each colony will have arisen from fewer spores. That will speed up strain isolation immensely.

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Re: Clarifying questions about agar and strain isolation [Re: replyom]
    #25815118 -

replyom said:
Thank you mushpunx, makes sense to me.

I'd be interested to read about the results of strain isolation from a clone. Once you have your 10-15 isolates from that clone and spawned/fruited them, how much variability would you observe between the isolates? This would tell you a lot about the utility of strain isolation vs cloning.


The goal of both is to get a great producing culture that will produce consistent results. Strain isolation from spore is a whole lot of work. Cloning can get you those results much faster.

When you isolate on a culture germinated from spore, there are hundreds or more strains packed together. That's why it takes so many transfers before you can even start to see individual sectoring.

With clones there are much less present. Sometimes you will take a clone and it will already be a monoculture from the start.

Sometimes it might have as little as two or three sectors, others can be quite a few.
You will likely end up with much less isolates to test than from spore.

I usually just keep my clones as is if they test well. Sometimes they work better that way. But I have isolated and tested the different sectors in clones and in my experience , there aren't usually huge differences or anything.
But if I have a really great clone, I might want to see if I can get a killer isolate.

Key points are that you want to end up with multiple isolates to test from a culture as that is the best chance of finding a killer producer.
And the other is to always label each isolate very carefully and test them out, and remember to test potency..

If you have lots of isolates to test it can take quite a while because the only way to test potency is to eat them. Keep good notes.


--------------------

Amateur Mycologists United
AMU Q&A

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