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OfflinePsilygirl
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Re: multispore agar innoculation [Re: ragadinks]
    #2395759 - 03/02/04 03:25 PM (19 years, 10 months ago)

as long as you sterilize the water, the water itself wont bring in any contams. however, whenever you open up the lid to the jar you of course risk bringing in new contams. this is where a flowhood/glovebox may help reduce airborne contams getting in while the jar lid is open while you pour in the water.


--------------------
"Love says 'I am everything.' Wisdom says 'I am nothing.' Between the two, my life flows."


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Offlineragadinks
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Re: multispore agar innoculation [Re: Psilygirl]
    #2395834 - 03/02/04 03:42 PM (19 years, 10 months ago)

I have got some bottles with a self healing lid and I wonder if I could probably get the water inside with the help of a syringe under totally unsterile conditions ?

That is what the bottles look like:



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Offlinemycohomme
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Re: multispore agar innoculation [Re: Psilygirl]
    #2395844 - 03/02/04 03:44 PM (19 years, 10 months ago)

i still dont think i've heard anything regarding the necessity (ot not) of rehydating the spores before going to agar? Do you all just use an innoc. loop and transfer a bit of spores to the agar? or do you maybe make up a syringe, and add a small amount to the agar?

how many transfers does it generally take to get a pure strain?


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Offlinemycohomme
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Re: multispore agar innoculation [Re: ragadinks]
    #2395880 - 03/02/04 03:52 PM (19 years, 10 months ago)

raga,

I'm assuming you meant "sterile conditions" not "unsterile"
it seems they would do the trick. however, you cant ever be totally sterile. you have to flame the needle to redhottedness, and let it cool of course, so as not to damage those bottles. The needle could still have some contaminants. I dont think its very possible to be completely sterile, if you read one of the previous posts, they something about working cleaner and working faster.

And there is a point where you are trying to hard, you want to do the best you can to keep things sterile, but you can never be perfect. So it just depends on what its worth to you.


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Invisible@cro
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Re: multispore agar innoculation [Re: ragadinks]
    #2395946 - 03/02/04 04:06 PM (19 years, 10 months ago)

The only problem I would see would be doctors are usually only pulling out a couple cc's, not much of a problem. You'd be pulling out at 10-12 cc's which would create a vacuum, I'm not sure if it would let you pull out that much. I suppose you could suck air in to the syringe while it's in the presence of a flame then just shoot that into the jar and suck up the liquid afterwards, of course you would have to do this in a couple steps otherwise you'd still be creating a pressure gradient just in the opposite direction.

Peace - @cro


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Anonymous

Re: multispore agar innoculation [Re: Blue Helix]
    #2396022 - 03/02/04 04:27 PM (19 years, 10 months ago)

There is a very big difference between rhizo in a casing layer, and rhizo on the substrate.

Yes multispore inoculations of substrate guarentees, minus contamination, that you get a fruiting strain. The question is is it the best you can get from any given print. Unless it is by chance, no.

Every population looks like a bell curve, you have the majority right in the middle, you have some that are bad, and some that are above average. If you isolate and test alot of dikaryons, you can find those above average isolates, without rolling the dice.

Is it practical for those just trying to get a fruiting strain, NO. Is it a waste of time for those who want to do the work, NO.

I have never isolated and tried to fruit a non-fruiter either. By selecting vigourous growth, I avoid this. But I have isolated dikaryons from a single print, that showed variability. Bad fruiters, good frutiers, and great fruiters. To me it is worth the effort.

I have never fruited a single PF TEK Jar. I never even tried.

If your are looking for ease and good results. Multispore Jars of substrate, and fruit them.

If you are looking to select something nice, isolation of individual dikaryons is a must, and even better is isolating and breeding monokaryons.


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: ragadinks]
    #2396068 - 03/02/04 04:42 PM (19 years, 10 months ago)

The point of using the H2O2 is so that I don't need a glovebox. Any spores that get in the H2O2 water will be killed. Also by rinsing the agar pieces with pure H2O2, you assure they are clean too, including the sides of the jar which could carry contamination even if the surface does not. I never use a glovebox, but I'm not a slob either. Once you have mycellium growing, you can use H2O2 to do just about anything without need for a glovebox. And contrary to popular belief, H2O2 doesn't kill mycellium very well. It doesn't even stun it in the setting of a mycellium syringe. How do I know? My jars are about 75% complete in 4 days usually. They typically are done is 7. If that's stunned mycellium, then I don't care; it's fast enough for me.


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: ]
    #2396086 - 03/02/04 04:47 PM (19 years, 10 months ago)

When one gets about 11 grams per cake, does it really matter? In my experience, most germinations can yield around 10-12 grams per cake in the first three flushes given proper conditions, yet I often hear of much lower fruitings. I often see trays shown around here that look to be in pretty bad shape, even though they are fruiting. The point is, I think trying to isolate a strain that yields that tiny bit extra is really a moot point when it looks like the biggest concern for me, and probably others, has nothing to do with the strain. Just getting the conditions and timings of the whole procedure right is the real trick in this whole game, not the strain. When you can say that EVERY tray you are laying is getting 10+ rock hard dried (as in they snap like a cracker) grams per cake, then we can talk about strains. Until then I think we're talking above our heads here.


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Offlineragadinks
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Re: multispore agar innoculation [Re: @cro]
    #2396210 - 03/02/04 05:32 PM (19 years, 10 months ago)

You are right with the vacuum. But when they give an infusion to someone they must have some special thing that let's (sterile?) air into the bottle so that no vacuum is created in there. I have to ask a doc about that ...


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Offlineragadinks
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Re: multispore agar innoculation [Re: mycohomme]
    #2396222 - 03/02/04 05:36 PM (19 years, 10 months ago)

Quote:

I'm assuming you meant "sterile conditions" not "unsterile"


Sorry, I meant "sterile conditions". But I think it is important to be as sterile as possible. Especially when you are living in an very unsterile environment as I do  :smirk:


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Anonymous

Re: multispore agar innoculation [Re: Blue Helix]
    #2396254 - 03/02/04 05:44 PM (19 years, 10 months ago)

Well I have higher standards then you, and I actually enjoy looking at a larger sample of the populations of each strain.

My yields are consistently higher then those you are stating, so I guess I am allowed to talk Isolation. Thanks for your permission.

Shrooms don't get rock hard. Not even sclerotia gets rock hard.

The EASIEST WAY TO IMPROVE YIELDS in a stable environmnet is Isolation and testing of MANY INDIVIDUALS within a population.

Yes it is EVERY TRAY, because I am fruiting single dikaryons not rolling the dice and settling for PAR. I play for birdies in golf, and I look for ABOVE AVERAGE YIELD in cubensis.

So back to the original point.

Yes you can multispore on agar, isolate multiple individuals from a single petri dish, and colonize substrate with a single dikaryon, and keep cultures of the best one you find for your particular environmnet. You can do so in as little as one transfer from the original multispore plate, or as many as #, depending on the concentration of spores you inoculate with, and the amount that germinate, and the size of the transfer wedge. You will also be able to determine the cleanliness of the spores before inoculating jars of substrate. You will get a real appreciation for the quantity of individuals within a single print from a single strain, and there variability.

If you just want shrooooms dude, then use a syringe and shoot up pftek jars, and get your doses.


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: ]
    #2396402 - 03/02/04 06:26 PM (19 years, 10 months ago)

Teonan, I don't have a lab. In my opinion, the kind of work you are talking about requires a HEPA filtered lab and thousands of dollars worth of equipment to maintain a very precise environment and thus eliminate other variables that would otherwise grossly influence the outcome. Maybe in your estimation spending that kind of money and setting up such a lab is "the easiest way to improve yields" but in mine, it's not. Aside from that, I'd be curious what sort of yield increase you are getting and all about this variability you say you are finding.

The reason I am skeptical of sectoring and isolating strains is because the only folks who seem to beat this drum are those who have a vested interest in selling strains. Folks who sell strains place their entire business viability on fact that their strain is better than most of the ones you'd get by chance. They have a VESTED interest in convincing you of what they are claiming, and indeed, if it's not true, they are selling just a dream.

It's a bit like the so-called traits that are reputed to be associated with spores found in various parts of the world. As far as I know, spores don't carry many unique traits, be them B+ or B or Amazonian or whatever, yet the spore vendors swear they are really very different. Are they? I suppose spores are as different as an Asian man is from a white man (not much), but to get an "Arnold Schwarzenegger" mushroom, you'd need a clone of a strain which proved to have super strength. Can you find such a strain by sectoring? Yeah, you can but it's going to take a lot of work. You have to make it to the fruiting stage time and again without other factors interfering with your observations or else you can't say what is related to the strain and what is just dumb luck. Doing that means a lab and money.

As for "go do some shrooms dude," the experience tends to be a little on the super intense side for me, even given really modest doses, and as a result I don't do it often. But to each his or her own. I certainly don't feel superior to someone else who gets high on shrooms on a monthly basis or even every weekend.


Edited by Blue Helix (03/02/04 06:31 PM)


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Offlinedaywalker
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Re: multispore agar innoculation [Re: mycohomme]
    #2400749 - 03/03/04 10:46 PM (19 years, 10 months ago)

Quote:

mycohomme said:
i still dont think i've heard anything regarding the necessity (ot not) of rehydating the spores before going to agar?  Do you all just use an innoc. loop and transfer a bit of spores to the agar? or do you maybe make up a syringe, and add a small amount to the agar?
...




I am also waiting for that answer. I hope to be working with some agar in the next week and have a print I'd like to try. Taken it upon myself to acquire the tools so is it "spores-to-agar" or "spores-to-h20-to-agar"? If germination will occur with using a loop to streak the agar - no rehydration, then I am all for it! :laugh:


--------------------
"I suggest we ... learn to love our ... selves before it ... becomes illegal."


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OfflinePsilygirl
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Re: multispore agar innoculation [Re: daywalker]
    #2401065 - 03/04/04 12:56 AM (19 years, 10 months ago)

no, you dont need to rehydrate it, it rehydrates itself on the agar.

just sterilize an innoc. loop (use isopropyl, wait for it to dry and the flame dry it with a lighter...be careful, obviously isopropyl is flammable) and collect some spores. then just streak them across agar (once its settled in the petri dish, or whatever you use).

hope this helps.


--------------------
"Love says 'I am everything.' Wisdom says 'I am nothing.' Between the two, my life flows."


Puget Sound Mycological Society


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Anonymous

Re: multispore agar innoculation [Re: Blue Helix]
    #2402425 - 03/04/04 11:30 AM (19 years, 10 months ago)

I don't have that kind of lab, or a hepa, and Isolation and testing works just fine.

I am searching for the best isolates for my particular environmnet, throughout different times of the year. My home temps fluctuate during the seasons by about 10 degrees. Those isolates that perform best during the winter, perform not as well during the summer.

Multispore results in average casings, which is better then poor, but not as good as above average.

10 original multispore plates can yield 50+ vigourous substrains to test. Of those fifty+ you might find 1-2 that are superior to the middle of the road. Instead of yielding 11 grams bone dry, you might be yielding 15 grams bone dry.

an extra 4 grams per cup, is an extra ounce per 7 cups substrate. Now you can process far less substrate to get the same yields. Personally I don't like cleaning jars.

The extra effort at the beggining, results in maintaining your Strains as close as possible to there oringinal state you obtained them in. It also enables you to have clones of known vigor, at known parameters, yielding greater consistency. Instead of having 1 average GT, you now have multiple GT clones that are above average, and varied from each other. You also still have the original print in storage, and prints from each generation you grow out from each clone.

Hard to fruit species are hard to fruit becasue they require very specific conditions to fruit. Isolation won't increase the range of those conditions, but it can help you find those individuals that are skirting the edge of the range. IE, if the species fruits in a raqnge from 60-70 F, you might find the better producers at the 70F side of the range, which makes fruiting easier if you have trouble maintaing 65F temps, but can maintain 70F. This is SELECTION from a populaiton. This is how all breeding programs start, once pedigree is determined. Identifying the best individuals from the population for your specific requirements.

If you are just happy getting fruits every time, AVERAGE is just fine.

EDIT. I too am REALLY sensitive to shrooms. I use them very infrequently also. I don't feel superior to anyone or anything. I just like to see Facts remain facts, and not get obscured.

The vested intrest you are refering to is TO STAY IN BUSINESS. To offer products that do not live up to CLAIMS will not keep you in business forever. I can assume you are refering to STAMETES and Fungi perfecti. The guy is still in business, and still sells CULTURES. He is offering cultures of KNOWN PRODUCERS.

When someone is going to invest money in commercial growing of edibles/medicinals, they usually want to be able to take the Mushroom for granted. Ie. I am using this substrate in this environmnet, so I buy a culture that has been tested, and selected for inTHOSE EXACT ENVIRONMNETAL PARAMETERS. Then you just have to worry about maintaining those variables.

I find it funny that you question the legitamicy of a practice that has been used since man began Agriculture. We selected for desirable traits, from large poluations of individuals. We did not select for PAR.

In a natural poulation of Marijuana, the majority of individuals will be hermaphroditic, and average potency. Is this desirable if you like to smoke pot.


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Anonymous

Re: multispore agar innoculation [Re: Blue Helix]
    #2402496 - 03/04/04 11:57 AM (19 years, 10 months ago)

On a side note if you are interested in STRAIN VARIABILITY. Search a thread on Copelandia cystidia variation. It has alot of pictures that might interest you, as well as measurements. That thread is showing you visually just how variable a single species can be throughout the world.

That is morphological variation of a just pleurocystidia and spores only!!!

In the plant pathogenic fungal world, you can have a single species that has variants that only eat one type of tree, while other variants only eat another type of tree. One species, becoming multiple species becasue of host specificity. Just an Example of variability.

Having clones that colonize at 75 F, and others at 85 F. Ones that fruit at 85F and others that fruit at 65F. Doing so without any difference in time frames. Variability.


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Offlinedaywalker
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Re: multispore agar innoculation [Re: Psilygirl]
    #2404078 - 03/04/04 07:19 PM (19 years, 10 months ago)

works for me :laugh: thanks for the tips!


--------------------
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Offlineazurescens
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Re: multispore agar innoculation [Re: daywalker]
    #2405043 - 03/04/04 11:26 PM (19 years, 10 months ago)

@cro

Is your good friend Billy Baldwin?? Because that is who that quote is from in the movie 'threesome'


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Invisible@cro
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Re: multispore agar innoculation [Re: azurescens]
    #2405259 - 03/05/04 12:15 AM (19 years, 10 months ago)

I'm not sure what you're referring to, I didn't quote anything in my posts.  And I definately don't know any Baldwins I try my best to avoid their movies.  :wink:

But as for sterilizing your utensil I would flame first then wipe with alcohol to cool it down and not kill the spores.

Peace - @cro


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OfflineScourge
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Re: multispore agar innoculation [Re: ragadinks]
    #2407025 - 03/08/04 12:38 PM (19 years, 10 months ago)

while not as good as a glove box IMO, adding h2o2 is a fairly safe method. there is little risk of contamination because h2o2, in proper concentration, will kill any bacteria and mold spores. the only real risk of contamination using proper moisture and h2o2 concentration would be if living mold got onto your substrate. not likely.


--------------------
"Those who walk in Love and Truth shall grow in Honor and Strength."


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