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Offlinemycohomme
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Registered: 02/25/04
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multispore agar innoculation
    #2387060 - 02/28/04 06:57 PM (13 years, 9 months ago)

Does anyone have any experience, tips, or suggestions for a multispore agar innoculation? I have spore prints, and I have read some of the documents regarding techniques. Any advice though would be greatly appreciated.


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Anonymous

Re: multispore agar innoculation [Re: mycohomme]
    #2387303 - 02/28/04 09:31 PM (13 years, 9 months ago)

It helps to hold the syringe needle down to allow the spores to collect at the tip...only one drop in say 3 or 4 well spaced places on the plate. Avoid using large drops - minimize the amount of water on the plate...that's just my 2 cents worth. There was a thread on this just a few days ago...use the search function. Someone with more expertise may have better input....


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Offlinellamaboy
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Re: multispore agar innoculation [Re: mycohomme]
    #2387845 - 02/29/04 01:23 AM (13 years, 9 months ago)

since you say you have a spore PRINT...i'd suggest using an innoculation loop...or if you don't have that maybe a needle of some sort...just scrape the print with the loop or the needle...and then drag it across the agar in a sorta zigzaggy pattern. if you don't see any spores on the loop or the needle, don't worry, there are some there...then you can do your whole transfering of best growing mycelium or whatever.


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Offlinethe man
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Re: multispore agar innoculation [Re: llamaboy]
    #2388044 - 02/29/04 03:25 AM (13 years, 9 months ago)

i think it would be very interesting to put various strains of cubies on one agar dish. all on different parts. then see what one grows faster, fluffier, or if it takes over other myc. (if you could tell)

peace


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Offlinellamaboy
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Re: multispore agar innoculation [Re: the man]
    #2388069 - 02/29/04 03:46 AM (13 years, 9 months ago)

it may be, but i doubt that's what he means by multispore :P


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Offlinethe man
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Re: multispore agar innoculation [Re: llamaboy]
    #2388093 - 02/29/04 04:03 AM (13 years, 9 months ago)

i know just a thought and doesnt need its own thread


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Anonymous

Re: multispore agar innoculation [Re: mycohomme]
    #2388469 - 02/29/04 10:42 AM (13 years, 9 months ago)

My bad...disregard my idiocy. I'm going to learn how to read soon, but I guess I'm hooked on chronic...


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Invisibletripndicular
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Re: multispore agar innoculation [Re: mycohomme]
    #2388620 - 02/29/04 12:18 PM (13 years, 9 months ago)

When you first streak a petri filled with agar ...it is a multi spore germination at first , you in time will harvest particular strains by "issolating" the best myc produced i.e. fastest , most rhizo development , etc ....
You are trying to grow myc that does not sector , meaning two different types of myc
Get copy of TMC , best investment you could make .
Good Luck


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: mycohomme] * 1
    #2389672 - 02/29/04 11:49 PM (13 years, 9 months ago)

I have TMC and GGMM. Forget about sectoring with cubes because you'll just be wasting your time. Cubes ALL grow exceptionally well. I have NEVER seen a germination that didn't support vigorous fruiting. Maybe if we were growing some hard-to-fruit species of mushroom, it's make sense to sector, but with cubes anything that isn't contaminated will produce TONS of mushrooms if you grow them out.

Another thing all those "cottony" strains that appear on agar--and ALL multi-culture spores innoculations start off that way--will turn rhizomorphic given a good growing environment. I challenge anyone to show me a cube culture that is not contaminated that doesn't fruit like gangbusters. So stop worrying about cottny stuff, sectoring, and all the crap. That's stuff you need to worry about if you are growing something else, not cubes.

Anyway, if you have a decent spore print--and forget about spore solutions with agar--you have two options that will prove successful when innculating an agar surface:

1) Work faster
2) Work cleaner

Within reason you need both, but you can substitute a little speed for a HEPA filter room. Likewise, you can substitue a little cleanliness if you can innoculate real fast, say the surface is exposed for less than 5 seconds.

And as for the innoculation loop, it's fine, but in my experience it just isn't practical to use it fast. You end up spending too much time dinking around with it, and touching the agar surface never works better than just getting the spores to fall on the surface because you end up damaging the surface. Here is how to do it:

First, you should be wearing latex or, better yet, those rubber or neoprene dish washing gloves, and the gloves should have been rinsed on the outside with rubbing alcohol or denatured ethanol or the like. Also, keep in mind that you should NEVER breath onto the agar surface. You should never have your hair or bare skin over the exposed agar surface either at any point--remember your whole body is a shower of contamination, even if you are a clean person.

Now get a good metal scrapy thing. A good set of tweesers works pretty well. Just heat the scrapy thing up in a blow torch flame. Wait a minute or so for it to cool. And then in one smooth motion, take a hand with the spore print paper between your index and thumb and open the jar or petri with the ball that hand. During the entire process, you should not lay down the lid or face it upwards. Just keep it clenched in the hand that has the spore pring between the index finger and thumb. Practice this beforehand if you have to. Be smooth.

Turning the spore print sideways or facing downward on your index finger above the agar, take the tweesers or whatever in you other hand and scrape it with the scrapy thing until the tiniest bit of spores fall on the agar. If you can see a speck, you have PLENTY. Under no circumstances touch the agar surface with anything, including the scrapy thing. Rapidly close the jar or petri dish. It's my experience that you want to do all that in less than 10 seconds and in a draft-free room and a sterile hard surface. Remember be smooth and work fast and you won't need even a HEPA filter.


Edited by Blue Helix (02/29/04 11:56 PM)


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Offlinemycohomme
stranger withcandy
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Re: multispore agar innoculation [Re: Blue Helix]
    #2391932 - 03/01/04 06:48 PM (13 years, 9 months ago)

thanks for all the replies! I read though that you should rehydrate the spores in h2o. is this not necessary?


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Anonymous

Re: multispore agar innoculation [Re: Blue Helix]
    #2392151 - 03/01/04 08:07 PM (13 years, 9 months ago)

The site says we are trying to stop misinformation.

Please reread the books you claim to possess.  Becasue I don't think you understand them.

Cubensis is not unique.  It to contains good dikaryons and bad dikaryons, and isolation of individual dikaryons and testing them at different environmnetal parameters will give you a REAL understanding of this.

If you wan't to state that itis not neccesary to get good fruitings, that is one thing, but to imply that cubensis doesn't obey the same rules as every other mushroom with the same reproduction system, or every other living thing on the planet is just  stupid.

Inoculation loop works great.  You can inoculate agar with spore solutions.  And scraping a print directly onto agar is not the cleanest and safest method of getting spores from print to agar.  A loop is far safer, and more controlled.

Not all cottony sectors will become rhizo.  It is very possible to isolate non fruiting sectors from a very diluted multispore inoculation.

I think your heart is in the right place, but you really are simplifying things way to much.

If you wan't to see the full variety within each Strain, you got to do what you consider a waste of time.

Not all Dikaryons are created equal in each and every environment.  Nature doesn't remain 80 F, 95 % humidity all year long.  The seasons change, and life changes with it.

Just like there are short people, tall people, skinny people, fat people, smart people, dumb people, etc...........  There are differences between Strains , and individual dikaryons of cubensis.

If you haven't seen a bad cubensis fruiting, you haven't really seen a good one yet either. 

JUST MY TWO CENTS. :tongue:


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: ]
    #2392420 - 03/01/04 09:47 PM (13 years, 9 months ago)

Could be, could be... I am just saying I have never seen a cubensis strain that did not show rhizomorphic character before fruiting in the casing layer, and I have never came across a cubensis strain that did not fruit. I'd say my experience is the norm too considering there are thousands of folks around who are using 1/2 pint grain jars as their petri dish in a little tek called PF Tek. Of the many thousands of jars, each like a petri dish, I can't remember the last time I read about a cake that did not fruit unless contamination took down the cake. Doesn't that seem a little odd to you? Thousands upon thousands of jars and they all seem to fruit well. I think the real challenge would be to find a strain that didn't fruit well. That might be about as uncommon as an albino mushroom or the like, one in tens of thousands probably.


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Invisible@cro
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Re: multispore agar innoculation [Re: Blue Helix]
    #2392499 - 03/01/04 10:16 PM (13 years, 9 months ago)

Quote:

Blue Helix said:
Of the many thousands of jars, each like a petri dish, I can't remember the last time I read about a cake that did not fruit unless contamination took down the cake. Doesn't that seem a little odd to you? Thousands upon thousands of jars and they all seem to fruit well.




Doesn't seem strange at all to me. When you're referring to multispored inoculations in a jar you have many different substrains in that jar, so if there was a substrain which didn't fruit it would just be shadowed by the other substrains which do.
But that leads to the rest of the question, you now have part of a jar completely useless for your purposes. If you were to have isolated a substrain which is known to be productive you wouldn't have that substrate going to waste. Plus the benefit of any other characteristics you isolated it for in the first place.

Peace - @cro


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: @cro]
    #2392728 - 03/01/04 11:10 PM (13 years, 9 months ago)

I personally don't use jars--way too hard compared with agar and mycellium syringes--but I don't believe that a significant part of the cake is warring strains. I'd bet all of that fighting is done on a very small scale. I have no proof, but that's what I'm betting.


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OfflinePsilygirl
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Re: multispore agar innoculation [Re: Blue Helix]
    #2392901 - 03/01/04 11:58 PM (13 years, 9 months ago)

You dont use jars??  So you go straight from agar to a casing???  :wtf:


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OfflineBlue Helix
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Re: multispore agar innoculation [Re: Psilygirl]
    #2393083 - 03/02/04 01:13 AM (13 years, 9 months ago)

Ooops, I do use jars. What I meant is that I don't use spore-injected jars. I use a 1/2 pint agar jar, let it grow out, break up the surface with a scrapy thing, pour in 20 ml or so of h2o2 on the broken pieces, shake up the jar, pour in a couple hundred ml of sterile water, shake up the jar, and then I use the solution to inject birdseed/brown rice/vermiculite jars using a really big syring (60 ml). When those are done colonizing--I give them about 7 days to colonize and another 4 days just for good measure--I apply the casing, incubate the tray for 5 more days covered with syran and in the dark, and eventually fruit out the tray. Fruiting chamber is misted using an ultrasonic on a timer and kept circulating using a cool mist and a PC fan (both on an inverted timer of the ultrasonic).


Edited by Blue Helix (03/02/04 01:14 AM)


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OfflinePsilygirl
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Re: multispore agar innoculation [Re: Blue Helix] * 1
    #2393122 - 03/02/04 01:22 AM (13 years, 9 months ago)

gotcha. just confused on your wording.

good luck


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"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: Blue Helix]
    #2394458 - 03/02/04 11:23 AM (13 years, 9 months ago)

How do you prepare and pour in the H2o2 and sterile water into the agar jars ? Do you use a glovebox ?


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OfflinePsilygirl
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Re: multispore agar innoculation [Re: ragadinks]
    #2395220 - 03/02/04 03:25 PM (13 years, 9 months ago)

i'm not sure a what point you're asking about, just make sure you're not adding H2O2 to anything that doesn't have mycelium


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"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]
    #2395627 - 03/02/04 04:53 PM (13 years, 9 months ago)

I just wonder if I can pour the water into the jar without risking to contaminate the jar again.


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Mushrooms, Mycology and Psychedelics >> Mushroom Cultivation >> Mushroom Cultivation Archive >> Agar and Liquid Culture

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