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Invisibleshymanta
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Strain isolation?
    #5080444 - 12/18/05 11:55 PM (18 years, 5 months ago)

When isolating on agar, how big or small a piece should one cut from the colonized agar and be sure to have only one strain?

Can you tell easily which parts of the plate are each strain?

Can anyone point to a picture which clearly illustrates this?

Thanks all! :smile:

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OfflineRogerRabbitM
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Re: Strain isolation? [Re: shymanta]
    #5080860 - 12/19/05 05:53 AM (18 years, 5 months ago)

Cut a piece no larger than a grain of rice when making isolation transfers. When you swipe spores onto agar, chances are the dish will grow out hundreds of substrains. You likely won't see any sectoring until after the second or third transfer for that reason. The first picture below is a dish after two or three transfers. By holding the dish up to the light, you can clearly see the sectors by looking at the dish from the back(agar) side. The dish on the left side of the second picture is after an additional transfer or two. You can see the rhizomorphic growth developing from the ten and two o'clock positions. By transfering only the rhizomorphic growth, you eventually end up with a totally homogenous, single sector rhizomorphic isolate as seen in the dish on the right of the second picture.
RR


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Invisibleshymanta
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Re: Strain isolation? [Re: RogerRabbit]
    #5081913 - 12/19/05 01:29 PM (18 years, 5 months ago)

Very helpful! Thanks.

I was wondering how many transfers on average does it take to isolate only one strain?

I ask because to begin with, everything I read suggested only one transfer, but later on I began finding reference to more than one transfer for a single isolation.

One more question.  What parameters should agar cultures be incubated? 

I would imagine the temperature would be the same, around 85F.  But What on humidity and air exchange?  (my thinking is that the agar should hold enough water)  This I ask because my first try at agar was in a small Tupperware dish.  While it did yield mycelium, the agar looked slimy and gave off a sour smell that I assumed was bacterial. :frown:

Edited by shymanta (12/19/05 01:33 PM)

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OfflineRogerRabbitM
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Re: Strain isolation? [Re: shymanta]
    #5083684 - 12/19/05 08:42 PM (18 years, 5 months ago)

It will take at least three to four transfers to get to single sector isolates. Five transfers is not unusual. More if you get carried away and use a lot of spores. If using a syringe, use only one drop in the middle of the dish. If using an inoculating loop, take a very small swipe of spores off the print. Continue making transfers until there is no more sectoring. Fruit out each single sector isolate to determine the best fruiter.

I incubate petri dishes at room temperature in a normal room. Dishes are wrapped with parafilm for protection from contaminants and gas exchange.
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."
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OfflineMobius_Strip
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Re: Strain isolation? [Re: RogerRabbit]
    #5087448 - 12/20/05 09:20 PM (18 years, 4 months ago)

So, I guess it's safe to assume that the most prolific mycelium in any particular sector will not be the most prolific fruiter? Have you ever had any cases where a sparse mycelium culture was a prolific fruiter?


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OfflineRogerRabbitM
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Re: Strain isolation? [Re: Mobius_Strip]
    #5110201 - 12/27/05 04:16 AM (18 years, 4 months ago)

No. I actually isolated some fuzzy mycelium once for a fruiting trial. It colonized slowly, then never produced even one single mushroom. When isolating strains on agar, always select fast growing, rhizomorphic mycelium to transfer.
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."
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Invisibleagar
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Re: Strain isolation? [Re: RogerRabbit]
    #5110270 - 12/27/05 05:36 AM (18 years, 4 months ago)

R/R,

ODDLY, I have had some differing results.

I have strains that demonstrated fluffy myc on plates, in WBS jars & on compost.

But, TORE through a casing layer like rhizomorphic bulldozers.

Then, fruited like gang busters.





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OfflineRogerRabbitM
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Re: Strain isolation? [Re: agar]
    #5110305 - 12/27/05 06:08 AM (18 years, 4 months ago)

But, was it from multi-spore, or an isolated, single sector strain? What happens with multispore inoculation is thousands of sub-strains are all growing at the same time, crowding each other and giving the visual impression of fluffy mycelium. When transfered to bulk substrates, sometimes the more aggressive rhizomorphic strains take over.

By looking at the petri dish pictures in my post above, look at the one labled 0629 Poteet. That dish is after several transfers and I see five distinct substrains: 2 rhizo substrains at the 10 o'clock position, 1 rhizo strain at the 2 o'clock position, and two fluffy strains at 12 and 6 o'clock. If one were to grow out those isolated cottony or fluffy strains, results would be poor. You can see how the rhizomorphic strains are already gaining speed, and if that dish were left for another two weeks, the rhizo strains would cover the entire dish.
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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Invisibleagar
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Re: Strain isolation? [Re: RogerRabbit]
    #5110590 - 12/27/05 08:56 AM (18 years, 4 months ago)

R/R, I don?t disagree with you about rhizo?s & isolating them.

The function of rhizomorphs is related to exploration, and exploitation of nutrients. That is, the strands are actually bundles of hundreds or thousands of hyphae encase in a pseudoparenchymatous sheath that functions to explore for, and when found transport large amounts of water and nutrients over relatively large distances (microbiologically).

In optimal nutrients, cotton like hyphae can/will remain hyphae and effectively colonize those nutrients. In less than optimal nutrients, good strains will develop rhizo?s that start searching, by extending themselves out, exploring for more, or better nutrients.

One can change into the other, and visa versa. Certainly, you isolate rhizo type growth. Because it demonstrates that survival tactic.

If it did not, in less than optimal nutrients, or when nutrients are deleted, it would stand far less of a chance of survival than stains that do not demonstrate that tactic.


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OfflineRogerRabbitM
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Re: Strain isolation? [Re: agar]
    #5110609 - 12/27/05 09:04 AM (18 years, 4 months ago)

Agreed. (I think)

What's up with the black background and blue lettering? I can't read a damn thing in blue over the black background. Admins?
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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InvisibleEonTan
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Re: Strain isolation? [Re: RogerRabbit]
    #5112609 - 12/27/05 07:23 PM (18 years, 4 months ago)

I had a B+ isolate that is always fluffy and rhizo. It looks like a cheese pizza with a single slice of a topping  wedged in with it.  No matter what I do, where I transfer from, it always comes back the same.  You can transfer the fluff or the rhizo sectors and they grow back exactly the same. A 3/4 rhizo, 1/4 cottony plate.
:confused:

If the strain you are working with produces rhizos on agar, transfer the rhizos.

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OfflineRogerRabbitM
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Re: Strain isolation? [Re: EonTan]
    #5112799 - 12/27/05 08:11 PM (18 years, 4 months ago)

Take smaller pieces when you transfer. Try no larger than a grain of rice. Smaller is even better if you have a sharp scalpel. It will take several of those transfers to get a single sector isolate.
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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InvisibleTippinthru
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Re: Strain isolation? [Re: agar]
    #5114376 - 12/28/05 06:07 AM (18 years, 4 months ago)

Good Golly, Agar! Can you actually pronounce "pseudoparenchymatous", My tongue and lips are getting a work-out just trying to pronounce it! You must have a talented tongue! LOL! :grin:


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Invisibleagar
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Re: Strain isolation? [Re: Tippinthru]
    #5114673 - 12/28/05 09:49 AM (18 years, 4 months ago)

:rolleyes: :grin:


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InvisibleTippinthru
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Re: Strain isolation? [Re: agar]
    #5116455 - 12/28/05 07:14 PM (18 years, 4 months ago)

LOL! Ooohhhhh, baby!


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Invisibleshymanta
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Re: Strain isolation? [Re: RogerRabbit]
    #5129659 - 01/01/06 10:01 PM (18 years, 4 months ago)

RogerRabbit,

I had some mycelium growing on agar without peroxide. I transfered it to agar with peroxide. No growth after a week except on the original piece or agar (got fluffier). After thinking back on it, I realized that the peroxide concentration was too high. I poured some more plates with only 3ml H2O2 in 500ml of agar. The mycelium began to grow but with discoloration around the mycelium. Like piss. Is this normal? Maybe the mycelium metabolizing the H2O2?

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