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Is this a 'non-sectoring' agar isolate?
    #4133437 -

Strain: Cambodian
Agar: Oatmeal supplemented with kanamycin (200 ?g/ml)

A while back I scraped some spores onto a plate and after some growth, I cut out a more rhizomorphic-looking section and placed it on this new plate.

After 6 days of incubation at 75-80F:



This is the plate it was cut out from (visible at the top):



And just for fun, another multi-spore inoculated plate I'm trying to fruit off of:



So is the mycelial growth in the first picture non-sectoring or does further isolation need to be done?

Also any bets on when (if ever) the plates are going to fruit? I'm giving them a few air exchanges a day and have already cold-shocked overnight in the fridge.

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Re: Is this a 'non-sectoring' agar isolate? [Re: Aeolus1369]
    #4134022 -

I see what appear to be dozens of sectors in that top plate. If you'll hold it up to the light and look at the dish from the back side, you'll see them easier. The middle picture has thousands of sectors. If you want to do an isolation, you need to isolate as many substrains(sectors) as you can, as most will be poor performers. It's that one isolate in a hundred or so that is going to kick ass. I'd take that top plate, and transfer 20 sectors from around the perimeter to a full sleeve of petri dishes. Take specimens to transfer that are no larger than a grain of rice. Grow out each of those twenty dishes(there will still be sectoring) and transfer each one to grains for fruiting, after saving a sample from each dish to a second sleeve of twenty dishes. Be sure to label them. When your grains fruit, you will see which is the best substrain. Go back to the petri dish that corresponds to your best fruiter, and grow that one in bulk. Be sure to save some mycelium in the refrigerator for future use.
RR


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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Is this a 'non-sectoring' agar isolate? [Re: RogerRabbit]
    #4136523 -

Thanks for the detailed response. I've got some follow up questions though...

Since I don't really have the resources (in terms of time and equipment) to do the sort of isolation work you're talking about, I was thinking of an alternative route. If I were to wait until one of the plates in the latter two pictures fruited, then took a tissue sample and set it on a new plate, this would be an isolate right? Granted it may not be a very good isolate since the only trait I'm really selecting for is tendency to fruit on oatmeal agar, but there are other benefits to having an isolate correct (more even pinsets, less aborts etc)?

I guess my question to you is whether it would even be worthwhile to get an isolate through this method knowing that it may have weak genetics in terms of potency, substrate preference, size etc. vs just starting a multi-spore grow and cloning a good specimen later.

Lastly, I just wanted to get a better handle on exactly what 'sectoring' refers to. Here's a more close-up picture:


So are the blue regions sectoring, the red regions, or does sectoring just refer to any lack of uniformity in the mycelium?

Thanks again.

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Re: Is this a 'non-sectoring' agar isolate? [Re: Aeolus1369]
    #4136618 -

Isnt that from the temperature changes?


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Re: Is this a 'non-sectoring' agar isolate? [Re: Aeolus1369]
    #4136703 -

Here's a picture of mycelium that has already been transferred two or three times. You can see about ten sectors or so. Each of these sectors was transferred to new dishes to be grown out and fruited. It helps to see the sectors to hold the dish to a light as shown and look from the back side. The second picture is after the transfers from each sector were made. Each sector(substrain) should be grown out and fruited to find the best one. If you're not going to take it that far, I would recommend using a larger section of the original dish to get the diversity of multi-spore inoculation.
RR




--------------------
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semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Is this a 'non-sectoring' agar isolate? [Re: RogerRabbit]
    #4137033 -

Forgive me if I'm bustin a goove here but I thought I'd ask a few questions myself for further clarification. I'll butt out if I'm intruding.

So basicly, you are taking the healthiest, most prolific (thickest) areas of the mycelium and transfering them to seperate plates to isolate that healthy section of mycelium. Does this mean that every thick streak of mycelium on the plate is stronger than the thin streaks of mycelium?

Since you are transfering them seperately I would assume that each healthy streak has it's own particular strengths. I would also imagine, if the above assumption is correct, that mycelium that appears to be healthy on one type of agar medium may be weak on another type of agar medium.
is this correct?


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The smart way to keep people passive and obedient is to strictly limit the spectrum of acceptable opinion, but allow very lively debate within that spectrum - even encourage the more critical and dissident views. That gives people the sense that there's free thinking going on, while all the time the presuppositions of the system are being reinforced by the limits put on the range of the debate
-Noam Chomsky

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Help Identifying Sectoring in Clone Tissue Petri's [Re: RogerRabbit]
    #6779049 -

I went from Multispore Syringe to PF/BRF Cakes crumbled to HPoo Bulk Substrate in a Monotub and took live tissue from the core of the stipe and of the cap of the most vigorously growing fruit body.

All Agar work/cloning was done inside a glovebox.

I noticed later that the Parafilm under the petri lids had torn on most of the dishes so removed the old tape and double wrapped the lids with new tape folding in half this time to make it thicker.

Are you supposed to stretch the parafilm slightly while wrapping it? Will the double thickness impede FAE?

I'm using Black Agar and it's difficult to hold the dishes up to the light to see any sectoring.

I understand I have a mut since this is a clone from a multispore grow but can anyone even identify any sectoring here? If so how?

I have searched the forums for hours and cannot find any good pics of what a single isolate should look like. I can't even find pics that positively identify exactly what sectoring should look like.

Also, I see some petris have concentric rings. I don't see rings in these photos but what do they represent exactly when present?

Do I need multiple transfers here?

Should I grow these out on unsectioned, 3 sectioned or 4 sectioned dishes?

This time I will use regular transparent agar.

-Thanks

Any thoughts?








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Re: Help Identifying Sectoring in Clone Tissue Petri's [Re: eltrkbrd]
    #6780625 -

Quote:
I can't even find pics that positively identify exactly what sectoring should look like.



Look at the picture two posts up from this one.  There's several sectors on that plate that are easily identified.

Most of your pictures look to be non-sectoring, but that's just because through the process of anastamosis, most strains from multispore inoculation combine into a single strain by the time full colonization is complete.  This occurs whether from multispore from a single print/syringe, or if you used spores from multiple 'strains' and mixed them into the same substrate.

Your second dish from the top seems to have a distinct sector in the upper left section at the three o'clock position.  It would be easier to see from the back side with a light, but agar with charcoal in it makes that kinda tough.  Your bottom picture for sure seems to be a single sector.

The idea with isolation is to use multispore inoculation right onto the agar so that the strains can differentiate on the plates and be transferred to new dishes as isolates become distinct.  You then want to grow out each isolate to the fruiting stage to determine which ones have the characteristics you're looking for.

Nice job getting your plates to that point without contamination.  It looks like you have your glovebox sterile procedures working. :thumbup:

All of these pictures below are of non-sectoring isolates.  Sometimes it helps to see different species to make a clearer picture.
RR



--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Help Identifying Sectoring in Clone Tissue Petri's [Re: RogerRabbit]
    #11554574 -

Quote:
Most of your pictures look to be non-sectoring, but that's just because through the process of anastamosis, most strains from multispore inoculation combine into a single strain by the time full colonization is complete.  This occurs whether from multispore from a single print/syringe, or if you used spores from multiple 'strains' and mixed them into the same substrate.




I am confused by this. If multi spore started agar or grain combines into a single strain then why bother cloning and then isolating an individual fruit?

Also, I know that you said to transfer myc to new petris right after germination to avoid anastamosis. What if you didn't and you let the plates grow for a few days? Should you start over?

Also, what are the pros and cons of cloning vs starting with spores on agar and isolating. Cloning seems to be much less work.

I would think there is a better chance of a great colonizer when starting on agar and a better chance of a great fruiter when cloning.


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A Good Substrate: Poo With Extras
Good Liquid Culture, Step by Step
Timer Modification
PM me if you are interested in buying 140ml syringes. $6 each + $7 shipping

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Re: Help Identifying Sectoring in Clone Tissue Petri's [Re: MasFina]
    #11554710 -

I guess this answers my question:

RR said:
Quote:
Most strains of a single species are compatible.  That means over time, the various sectors(strains) will merge into a common whole via the process of anastomosis. By cloning, you get a 'composite' of the original multispore inoculation.  By isolating dozens of strains, you'll get a few good producers, a few poor producers, a zero producer or two, and a stellar kick-ass producer or two.  The latter are what you save for future use and toss the rest out.  It's more work, but way more productive.
RR



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A Good Substrate: Poo With Extras
Good Liquid Culture, Step by Step
Timer Modification
PM me if you are interested in buying 140ml syringes. $6 each + $7 shipping

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Re: Help Identifying Sectoring in Clone Tissue Petri's [Re: MasFina] * 1
    #11556626 -

Once spores germinate, the monokaryotic hyphae seek out compatible hyphae and 'mate' in a fungal version of sex.  After this pairing, cells generally have two or three nuclei per cell, thus we call it dikaryotic mycelium.  Dikaryotic mycelium then 'mates' with other compatible dikaryotic mycelium until there's a coherent whole.  The strains that don't join the network seem to occupy their own space and often fruit independently of the other strain(s).  That's why with multispore inoculation, you often get a substrate that has two or more distinct strains fruiting, and each strain presents a completely different appearance, size, quality, etc.  By cloning, you generally get the exact strain that was fruiting.

The kicker is that it sometimes occurs that more than one strain is active in a single fruitbody, so you may find yourself back to square one by cloning.  I generally only clone outdoor fruits, and if the clone sectors, I fruit each one to see what I like best.
RR


--------------------
Download Let's Grow Mushrooms



semper in excretia sumus solim profundum variat

"I've never had a failed experiment.  I've only discovered 10,000 methods which do not work."
Thomas Edison

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Re: Help Identifying Sectoring in Clone Tissue Petri's [Re: RogerRabbit]
    #11559010 -

Ok, this helps even more.

Although anastomosis combines strains into mutts there are still some strains in a substrate that don't get along and will not intermingle.

Also, the whole anastomosis concept is new to me. I used to think that there was a much wider variety of strains in a multispore grow, but the fact that many strains are just getting merged together makes isolation much more interesting and important to me.

If each multispore grow results in bell curve-like result then you are never experiencing the excitement of the profound differences from one truly unique strain to the next.

Selecting the genetics you prefer from a vast catalog of possibilities sounds like a lot more fun than getting generic, average grows every time.

I wonder if some day humans will be selecting their childrens genetics from catalogs. And would that be wrong. That is a discussion for another forum though


--------------------
A Good Substrate: Poo With Extras
Good Liquid Culture, Step by Step
Timer Modification
PM me if you are interested in buying 140ml syringes. $6 each + $7 shipping

Extras: Filter Print Post Top
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