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OfflineBlue Helix
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Agar Strain Isolation - Is it worth it?
    #3770614 - 02/12/05 01:01 PM (12 years, 5 months ago)

I have done quite a lot of searching here at the Shroomery about agar techniques and strain isolation. In principle strain isolation sounds like a good idea just to make sure that the run doesn't suffer from significant competition from several substrains, and to make sure the strain is well cleaned of competing spores which I believe mycelium can carry on it until a later time when they germinate in your casing or such (although I have never read this in a reputable mycological text) But the investment of time is significant, up to two months. True strain isolation seems to take between 2 to 4 transfers from what I have read. Additionally, yields of those not isolating strains are often pretty good if the original spore print was real clean (which is often is not).

So the real question is, is it worth it? Those of you who have used agar and did strain isolation (or at least 2 transfers), did you notice a real improvement in casing penetration, spawn run speed, uniformity of pins, etc. when you went from a multi-strain inoculation to an isolated one? Does anyone out there have pictures to support claims of enhanced yield characteristics? I want to hear from those who know how to grow well enough that they can comment on the effect of multi-strain versus isolated strain without having a dozen other factors cloud the result. I mean if you can't consistently produce a crop, your data is clouded.

My last question is does it really matter what the agar plate substrain looks like? For cubensis, does linear or cottony really produce less than rhizomorphic growth patterns? Does anyone have real yield data to support their claims?


Edited by Blue Helix (02/12/05 01:02 PM)


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Invisibleagar
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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3772058 - 02/12/05 09:31 PM (12 years, 5 months ago)

I primarily use agar PDA petri's to clean up cultures from prints that came from differing sources. Back when, I found that myc changes from one type to the other, dependent of what it's attempting to colonize. It gets Rhizo like when it encounters something like a container wall & attempts to climb it, or when it encounters something tougher to digest, than what it's used to.

To each his own, as all seem to have differing experiances & opinions on the subjct.

But, same spores on plates with differing nutes, gets you differing type myc.


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OfflineBlue Helix
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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3772425 - 02/13/05 01:00 AM (12 years, 5 months ago)

agar, seldom when I make a multispore culture do I see contaminants, but I think the prospect of isolating a strain might be a good idea none the less since I sometimes have casing run issues. If you did not see contaminants in germination petri dish, do you think it'd still worth the time to isolate a substrain?


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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3772578 - 02/13/05 01:54 AM (12 years, 5 months ago)

If all else seems good, if you are working with a strain that for some reason (of the many) has rather feeble or stalled casing runs. It may well be helpful to isolate aggressive rhyzo & try again.

At the same time, it may be that a change in the substrate make-up is needed. Sometimes myc will become so accustomed to the same old - same old substrate, it just gets feedle (IMHO) & needs some change in both the make up & consistency of the substrate.

Like on plates, 1st plate on same PDA you isolate some aggressive myc, then transfer to second plate, so on & so forth (all same PDA recipe) & the myc over that extended time gets cottony. I believe that is simply because it learned (over that time) to emit enzymes to suit & digest the same old nutes, easier & easier.


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OfflineFluxburn
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Re: Agar Strain Isolation - Is it worth it? [Re: agar]
    #3773798 - 02/13/05 12:25 PM (12 years, 5 months ago)

Honestly I must add to this discussion. Why do you grow mushrooms, is it for the love? It's so addictive to grow, the process of learning each and every method is even more pleasureful as the knowledge increases. I have had a great time reading each and every bit of information from this site, still more knowledge is to grasp. The whole dimension of agar adds a fun and exciting twist to the basic mushroom cultivator. Obviously you can bypass agar all together with a karo or honeywater tech as the only difference between these and agar is agar is somewhat solidified(i.e. what agar actually is) with nutrients vs karo and honeywater are just nutrients in a non solid form.

Most of us have done the basics of growing mushrooms from jars or lots of newbies start out with just the myco bags. Many of us venture farther to explore a increased level of satisfaction in our grow, a more complex methodology, as the pleasure threshold only increases to a point that you can only see yourself as enjoying this hobbie for the rest of your life.


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InvisibleATWAR
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Re: Agar Strain Isolation - Is it worth it? [Re: Fluxburn]
    #3779355 - 02/14/05 06:04 PM (12 years, 5 months ago)

Quote:

Fluxburn said:
Obviously you can bypass agar all together with a karo or honeywater tech as the only difference between these and agar is agar is somewhat solidified(i.e. what agar actually is) with nutrients vs karo and honeywater are just nutrients in a non solid form.




Uh...


Liquid fermentation using a spore-mass (such as what is commonly done with Karo or honey) and isolating a strain on agar are two completely different things. A liquid TEK does not really have a place in strain isolation. The 3-dimensional growth medium does not allow for any "isolation". There just is no possible way to be sure you are contaminant and sector free when using a liquid fermentation technique as there is with an agar plate.

2 dimensional if you want to do isolation.
3 dimensional if you want to grow mycelium.


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OfflineCyber
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Re: Agar Strain Isolation - Is it worth it? [Re: ATWAR]
    #3780260 - 02/14/05 09:17 PM (12 years, 5 months ago)

With the stuff you order, I would say no. It is fun to learn but not needed.

Now dealing with wild prints, YES! You need to clean up the spores and get a good mycelium growth. If you go that far you might as well isolate a good strain!


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OfflineBlue Helix
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Re: Agar Strain Isolation - Is it worth it? [Re: Cyber]
    #3780993 - 02/14/05 11:42 PM (12 years, 5 months ago)

I personally grow for the pleasure of growing and, a far far second, because I and my friends like to trip on special occassions.

The reason I am trying agar is because it looks like fun and agar techniques are essential to growing many non-hallucinogenic mushrooms. But there is more to why I am trying agar...

I believe that sometimes multi-strain germinations on agar yield mycellium that doesn't go through real casings (like 1" casings over 5" substrate, not some see-through casing). I gather this from many failures from a set of prints I took--failures that never happened before. I would say that over half my runs from these prints went great through grain and through manure, yet would not go through the casing. Time and again I would have to vacuum off the casing or else the whole tray wouldn't give me a single flush. And after I did vacuum off the casing, I would get only one decent flush before green mold got me! Keep in mind that this same mycellium raced through 5" deep of manure in about 12 days! And I even did sensitive pH testing of the casing as well as water content analysis! It just didn't make sense since I never had trouble like that before and I've been growing some kind of mushroom for a LONG time (over a decade).

Now that really stressful group of failures are what drove me to trying out agar. I guess I'll know my opinion in a few weeks, but I am excited and wanted to know if anyone else could shed some light on how effective it really is.


Edited by Blue Helix (02/14/05 11:46 PM)


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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3783957 - 02/15/05 04:57 PM (12 years, 5 months ago)

"and to make sure the strain is well cleaned of competing spores which I believe mycelium can carry on it until a later time when they germinate in your casing or such (although I have never read this in a reputable mycological text)"

Search a plant tissue culture text. It's called piggybacking. It does happen in mycelium culture sometimes. though I never read about it in mycological context, I have seen it happen. Example: A agar wedge with a non sectoring mycelium is transfered to fresh plates and put upside down on the new agar. Mold spores that were ontop of the mycelium, non-germinated, now are in contact with usuable nutrients and begin to grow out. What appeared to be a clean culutre now has contmainats growing side by side with the mycelium.


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OfflineMycelium5150
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Re: Agar Strain Isolation - Is it worth it? [Re: EonTan]
    #3785621 - 02/15/05 10:18 PM (12 years, 5 months ago)

here is a good pic of what happens when one doesn't isolate. Note the varity or mushrooms. If you were to isolate a strong strain then you most likely would end up wth healthy mushrooms. Image the top of a casing being the top or a petri dish that hasnt been isolated. You will have different type of mycel in different sectors. Notice the second pic with the different shrooms. Note how most of them look like they are from the same clumps, areas, or sectors of the casing. I hope i explained this clearly



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Edited by Mycelium5150 (02/15/05 10:22 PM)


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OfflineBlue Helix
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Re: Agar Strain Isolation - Is it worth it? [Re: Mycelium5150]
    #3786389 - 02/16/05 12:00 AM (12 years, 5 months ago)

Mycelium5150, that is very interesting. Normally I do not see that kind of variability in a tray of cubes, but I get the point. Thanks.

EonTan, never heard of piggybacking, but I think it happens with mycellium too. Of course, I don't have proof.


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OfflineDig
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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3788699 - 02/16/05 12:01 PM (12 years, 5 months ago)

Would it be possible to transfer some of the mushroom tissue to a liquid culture in order to bypass the agar step? Meaning take a sterile syringe or whatever and take a center piece of the mushroom and transfer it to the liquid. I may have to try this myself soon!


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Re: Agar Strain Isolation - Is it worth it? [Re: Dig]
    #3789744 - 02/16/05 05:00 PM (12 years, 5 months ago)

yes its possible to take tissue from a shroom. its called cloning. This doesnt ensure that every shrooms will pin at the same time and be the same size, but it should reduce aborts and grow hardy mushrooms. Best bet if you can be sterlile is to take a piece of a mushroom and put it in some karo/broth and wait a few days then use the broth to inoculate. I did this with a port that pinned on agar even though it wasnt cased. Should grow fast and strong


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Re: Agar Strain Isolation - Is it worth it? [Re: tahoe]
    #3791246 - 02/17/05 02:34 AM (12 years, 5 months ago)

oh my ignorance, hey a good book is the mushroom cultivator from paul stamets... I bet that might explain it.


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OfflineDig
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Re: Agar Strain Isolation - Is it worth it? [Re: Fluxburn]
    #3793804 - 02/17/05 04:52 PM (12 years, 5 months ago)

Yes I bought the book 5 years ago. I was just wondering on a sterile technique that involved liquid culture.


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InvisibleOlgualion
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Re: Agar Strain Isolation - Is it worth it? [Re: Dig]
    #3804502 - 02/19/05 09:54 PM (12 years, 5 months ago)

Yes, This is definitely possible so long as you are very careful!

Lets start from the beginning so I can give my opinion on the first question in the same answer.

Multispore innoc to grain. Spawn to manure, or just friut the grain. Pick:

1: the first shroom,
2: the biggest shroom,
3: the shroom that gives you the best "vibe"

Hopefully they are in different areas of the container.

Then clone them each onto peroxidated agar.

Or, you can take a sterile syringe with some sterile H2O and a 16 gauge needle or so and UNDER STERILE CONDITIONS separate the mushroom stem in half and core the mushroom stem lenghtwise. Immediately shoot it into the sterile Honey Water. You can add a few cc's of newly opened peroxide to the honey water. If done correctly (you don't need a flow hood, just well thought out sure movements), you will soon have an isolate in HW.


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OfflineDig
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Re: Agar Strain Isolation - Is it worth it? [Re: Olgualion]
    #3805258 - 02/20/05 01:21 AM (12 years, 5 months ago)

When you mentioned coring the stem, does that involve just the center of the stem or parts of the cap as well (as shown in stamets)?

Thanks for the reply


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InvisibleOlgualion
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Re: Agar Strain Isolation - Is it worth it? [Re: Dig]
    #3808121 - 02/20/05 08:55 PM (12 years, 5 months ago)

I feel sterility is of the utmost importance. It is just easier IMO to be sterile if you can quickly core the inside stem tissue, and in the next hand movement inject into the hole in the jar lid (covered with masking tape before sterilization).

You dont have the luxury of the 2D petri, so you need to eliminate as many possible contamination vectors as possible. It really sucks when you inoculate your only 6 grain jars with a contam'ed liquid culture...

I would use the cap tissue if cloning onto plates though...

One good jar of an isolate in H.W. can last a good while stored properly in the fridge. It can also inoculate a shitload of jars! The benefits of using an isolate can be huge IMO. The benefits of inoculating with a pure liquid mycelial culture can be even bigger!


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OfflineDig
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Re: Agar Strain Isolation - Is it worth it? [Re: Olgualion]
    #3810852 - 02/21/05 02:13 PM (12 years, 5 months ago)

That is my plan... to inoculate with an isolated liquid strain. Beat the contams to the punch and add some umpff to the harvest wt. !


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Re: Agar Strain Isolation - Is it worth it? [Re: Blue Helix]
    #3816381 - 02/22/05 12:45 PM (12 years, 5 months ago)

Quote:

real improvement in casing penetration, spawn run speed, uniformity of pins, etc. when you went from a multi-strain inoculation to an isolated one?




not necessarily, not necessarily, definitely. Also you get more uniformity, and less aborts IMO.

If you have a stablilized strain, multispore can be just as good if not better than a good isolate. But, it is my opinion that inoculating from very dark spore syringes can be counterproductive. I see it being analagous to shoving 100 people in a room. 20 would be fine though...

Quote:

My last question is does it really matter what the agar plate substrain looks like? For cubensis, does linear or cottony really produce less than rhizomorphic growth patterns? Does anyone have real yield data to support their claims?



IMO, not really... I have seen both varieties produce rather well. Enough data would probably show that the isolates with desired results more often come from the rhizo variety, but there is just as much potential within cottony as well..


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