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OfflineBjJiggles
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Waiting on agar!! What to do?
    #16177439 - 05/03/12 04:20 PM (12 years, 8 months ago)

Ok, so i have a fruit i really want to clone, but agar and dishes wont be here until monday or tuesday. The fruit is big, but it's just beyond the pin stage, probably 2 1/2 inches long, it will probably break its veil, i'm guessing saturday, maybe Sunday.... Is there anyway i could save this fruit for cloning? I was thinking either putting the whole fruit in a ziploc bag and refrigerating or taking a sample from the base of the fruit and placing in a sterile ziploc bag and refrigerating... Do you think either of these ideas will work?


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Alan Rockefeller said:No!  Do not feed the type collection of a new species to animals!

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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177466 - 05/03/12 04:28 PM (12 years, 8 months ago)

check your local grocery store in the asian food isle, don't ask anyone you'll get told they don't have it but its a common food additive.
I just ordered 4 oz a few days ago and saw it in my local grocery store 2 days later.


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Re: Waiting on agar!! What to do? [Re: bishlap]
    #16177479 - 05/03/12 04:33 PM (12 years, 8 months ago)

Local non-chain health food stores will have it too. Not Whole Foods, a local place.

You can also try dropping a piece of tissue into a jar with some grain in it.
Not as reliable as agar but it's been know to work. People have use brf for the same thing.


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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177506 - 05/03/12 04:42 PM (12 years, 8 months ago)

1 tsp brf to 8cc water. put this in jar and sterilize. this will work as a agar, takes about two weeks to fully colonize though. the tek is on here somewhere, ive tried it and it works fine, add a knife tip of wondra flour if you have any


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OfflineBjJiggles
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Re: Waiting on agar!! What to do? [Re: Doc_T]
    #16177519 - 05/03/12 04:45 PM (12 years, 8 months ago)

Well the this i dont have a pc yet, everything this far, including trays and monotubs has been spawned from BRF cakes(sucks i know) but i have my eye on one i plan on getting in a month or 2 (wife willing).... Anyway, i have some presterilized MEA and plates on the way. I have the fruit going at the moment and it's the last project til i get the pc so i can do some grains.. I am wanting to get the clone culture going(from that fruit if possible) and use the prints that i have to get an isolate going in the few months til i get a pc, that way i have some master cultures.... But anyway, i just dont know how i can keep this fruit viable til Tuesday, i guess i could let it over mature, u think?


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Alan Rockefeller said:No!  Do not feed the type collection of a new species to animals!

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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177551 - 05/03/12 04:54 PM (12 years, 8 months ago)

Enter Key dude. Learn to use it.
You might be able to stretch that fruit out until your agar arrives... but how can you do agar without a PC?

Make some cakes, but just like 1/2" in the bottom of the jar.
Can't use regular cake lids because of the holes, but you can put an undrilled lid on upside down.
No dry verm layer, just brf/verm mix. Sterilize as usual.

That's now your petri dish. In a glovebox, add a sterile piece of tissue from inside the stem of the mushroom.
Sterilize your tools in the steamer when you do the jars. Once your minicake colonizes, you can use it like a colonized grain jar.
You can make 'grain lc', do 'g2g', even take a piece out onto a real agar plate if you are ready.

It's not ideal, but it might work. Next time, plan ahead.


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OfflineDazed Belief
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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177562 - 05/03/12 04:56 PM (12 years, 8 months ago)

http://www.shroomery.org/8514/Agar-substitute
heres the link
and no need for a pressure cooker
just up you time
brf doesnt need pc'ed


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Re: Waiting on agar!! What to do? [Re: Dazed Belief]
    #16177582 - 05/03/12 05:02 PM (12 years, 8 months ago)

ive used agar in double broiler with no cantams, not ideal and a P.C. should be your next investment fairly shortly but dont sweat it.


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Re: Waiting on agar!! What to do? [Re: spornication]
    #16177747 - 05/03/12 05:41 PM (12 years, 8 months ago)

Sry doc, iphone typing sux anyway...

Everything mushrooms sells pre-sterilized MEA. You leave the lid sealed and place in some boiling water about 1/2 up the jar, and get to water to a boil. This re-liquifies the MEA then you move the jar of MEA to your glove-box where you'll pour up your plates as usual....

See any problems? Sound ok to me, play around with agar for a while til i can pc some grains, im just so sick of using brf cakes. You have no idea how shitty it is with that being your only source of spawn. I've done so many it hurts.

Anyway, thanks for the help..


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Alan Rockefeller said:No!  Do not feed the type collection of a new species to animals!

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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177789 - 05/03/12 05:51 PM (12 years, 8 months ago)

you kinda want to clone with in the first three days of picking, sooner the better, id go ahead and put a piece in lc, one in agar agarless, agar agar and if you still want save the rest for your Agar Agar, you can alway transport from latter mediums back to Agar when it comes in why not get them going all the while


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Re: Waiting on agar!! What to do? [Re: spornication]
    #16177813 - 05/03/12 05:56 PM (12 years, 8 months ago)

Good point, but isn't the probablity of contam pretty high, just dropping a piece of tissue into LC water? I've just heard it's very hard to get a sterile sample from a fruit growing in an unsterile environment... Thought this was the point of agar and cloning to confirm no contams and transfer away from any contams if they appear.


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Alan Rockefeller said:No!  Do not feed the type collection of a new species to animals!

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Re: Waiting on agar!! What to do? [Re: BjJiggles]
    #16177827 - 05/03/12 05:58 PM (12 years, 8 months ago)

3cc peroxide in 50 ml water put sample in for three sec. then into your lc you can also trya bit of corbon in LC as well to clean it a bit




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Edited by spornication (05/03/12 06:06 PM)

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Re: Waiting on agar!! What to do? [Re: spornication]
    #16177864 - 05/03/12 06:08 PM (12 years, 8 months ago)

I wouldn't add tissue to a lc, its hard for me to imagine that would work well.

as doc t said you can take a tissue sample straight to grain or brf/verm, preferably from inside the stump.

flame sterilize an exacto knife and let cool
grab the top of the stem right where you cut off the cap then gently,precisely and quickly split the stem pulling in 2 pieces exopsing the inside, cut a sample place it in a jar and give it a few days.
I would do at least 3 jars, flame sterilizing each time.

also don't wipe with alcohol or h2o2, just do this in a glovebox or in front of a flowhood


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Re: Waiting on agar!! What to do? [Re: bishlap]
    #16177917 - 05/03/12 06:19 PM (12 years, 8 months ago)

Cloning to lc works extremely well,for me it take one day for them to break down and by day three i have good growth in Karo LC. have not tried to add carbon to LC, i use it on my agar was speculating on carbon in LC though


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Re: Waiting on agar!! What to do? [Re: bishlap]
    #16177927 - 05/03/12 06:23 PM (12 years, 8 months ago)

LC cloning is stupid.
LC without a pressure cooker is somewhere between there and completely fucking stupid.

OP, do it the way I said. Or do it some other way.
But if you want the best chance of success, do it the way I said.
Worst case, you might be able to keep half the mushroom in a bag until you get agar.

Spornication, you need to do some research and some learning.
"Corbon" isn't going to clean an LC. Neither is peroxide. Don't just spout off any stupid idea that comes into your head.
Is it 4:20 where you are? Or are you always like that?


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Re: Waiting on agar!! What to do? [Re: Doc_T]
    #16177987 - 05/03/12 06:36 PM (12 years, 8 months ago)

Doc you should mellow and do your homework, i will find the university files that contain method for peroxide and post when i do, have you heard of black agar? you seem to be stuck on this catholic do as yer told and there is only one way kinda kick you know. and any one who can not sterlize a jar of water with a tiny bit of surgar in just a water bath is pretty silly stupid is a kinda harsh word, play nice my love


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Edited by spornication (05/03/12 06:47 PM)

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Re: Waiting on agar!! What to do? [Re: spornication]
    #16178013 - 05/03/12 06:40 PM (12 years, 8 months ago)

Doc are you saying  by Professor Fanaticus was stupid when he wrote this tek



PF PEROXIDE AND BROWN RICE CLONING TEK
by Professor Fanaticus

The original idea comes from Rush Wayne's cultivation manual "GROWING MUSHROOMS WITH PEROXIDE". The following is a simplification of the idea down to the smallest degree, using brown rice powder, peroxidated water dilutions and small jars.

MATERIALS

1. Small jars with lids
2. Brown rice powder
3. Regular store bought Hydrogen Peroxide (3%) antiseptic solution (usually in a brown bottle)
4. Dissection knife and long needle (exacto etc)
5. Living mushroom or fungus, or culture of fungus

As a preliminary, start reasonable clean, but the following is to be done in the open air, without sterile implements or containers.

Mix the peroxide antiseptic and water at a 20% ratio. Example - 2cc peroxide and 8cc water. Place an amount of brown rice powder into the jar and add some of the peroxidated water to get a slurry, or very wet condition. The diluted peroxidated water sterilizes the medium and the jar. For extra experiments, you should try more potent peroxide contents in the brown rice peroxide slurry medium (30% - 40% - 50%). It is so easy, it can't hurt to experiment and you can benefit from it.

Tear the shroom apart and with an exacto knife, excise a small fragment about the size of a match head. With a long needle, knock or scrape the shroom fragment off the exacto blade and place the fragment on the surface of the peroxidated brown rice slurry. If you are working with previously made plate or slurry cultures that have mold growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi excised out of the culture. If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.

Screw the lid tightly onto the peroxide slurry jar and put the jar in a warm place for growth. The culture can be opened and exposed for observation or experimentation without danger to the growing medium and culture.

Under magnification (10x), the fragment of mycelium appears to "melt". It also turns blue, as if it were killed. Within a few days, tiny white hair like tendrils (hyphae) will appear on the "melted blue" fungus. It will grow and fill the the culture jar.

To use the culture for further inoculations, add 20% peroxidated water to the culture, mix the brown rice and fungus, and with a syringe, draw up water and inoculate PF jars, with the standard PF spore syringe technique. This is done nonsterily also because the peroxidated water will kill contaminant spores. But flame sterilize the needle before injecting into the sterile PF substrate. Instead of flaming the needle, an even better way to sterilize the needle (outside of the needle) is to wipe the needle with a tissue soaked with rubbing alcohol. Let the needle briefly dry of the alcohol before injecting. Use the culture well before it grows in. That way, it is much easier to get a usable slurry for the syringe without clogging.

As an addition, try more potent peroxidated water ratios. For instance, a 50% ratio works also. That would be half peroxide antiseptic and half water. Increase peroxide content until the mycelium doesn't survive. Then back off the amount of peroxide and use a near death peroxide load for guaranteed clean results. But a 20% peroxide to water ratio seems to be perfect.

If you are working with previously made plate or slurry cultures that have mold or bacteria slime growing along with the pure white good fungi, follow this tek using a small fragment of the desired fungi excised out of the culture. Make sure you don't have any growing mold or bacteria in the good mycelium because the peroxide won't kill it (as it doesn't kill the good fungi). If the culture grows back clean, that doesn't mean that it really is clean, because there will be contaminant spores in the mycelium (which is there anyway because of the open nature of this tek). When the culture is to be used PF style, when it is reslurried with fresh peroxide water, the contaminant mold spores and bacteria endospores will be killed while the growing fungi mycelium will survive to grow clean - PF style or any style you like.

TEK PROBLEMS

The only problem is syringe needle clogging. As a remedy, do not allow the culture to fully grow in or get thick. Keeping the culture "thin", allows a good breakdown of the mycelial fragments for use in a syringe. At first, doing the tek can be messy, but learning is quick and easy. Finding and using needles as big as possible is important.

But another route is very possible and actually preferable. Using long glass bulb pipettes are very good to use, but here, one must customize and "tweak" the teks a bit (one must be careful when injecting PF style jars with the bigger pipetts and not to breach the top vermiculite contaminant barrier). Also, flaming the glass pipetts can sometimes break the glass. In this case, always sterilize the outside of the pipette with the rubbing alcohol. Bulb pipettes are actually better to use than needles and syringes because they don't have much problem with clogging as compared to needles and syringes with mycelial slurries. But if the Professor's tek is followed closely and the cultures are not allowed to grow in and get thick, the 18 gauge needles with 10cc syringes work OK.

PRINCIPLES OF THE PEROXIDE TEKS

Hydrogen Peroxide is a powerful antiseptic. The solution of Hydrogen Peroxide bought in a drug store is 3% Hydrogen Peroxide and 97% water. Even at this low concentration, and with further dilutions, the germ killing is potent. But that germ killing power only works for micro fungi spores and bacteria endospores. A micro-organism that has germinated into its secondary form (mycelium), is safe from the antiseptic power of diluted Hydrogen Peroxide. But the ungerminated spores, bacteria endospores, and microbes are all susceptible. If there is bacteria that is growing (germinated), it will not succumb to the peroxidated water (just as the fungi mycelium is not succumbing). Also, any mold that is growing will survive. A clean fragment of shroom flesh or mycelium from a mold contaminated culture has germs all over it, but only in the spore or endospore form. They won't germinate in the peroxidated medium and water or on the recovering mycelia.

The tek is like a tightrope act. The mycelium that is cultured in the peroxide enriched medium can survive and grow, but it is not clean. Any spores or bacteria that are "piggy-backing" on the mycelium and not in contact with the peroxidated medium can come to life if given the opportunity.

After the culture of mycelium grows, it can be rehydrated with more peroxidated water. The spores and bacteria endospores that are "piggy-backing" on the mycelium will die. The mycelium in its fully secondary form, will survive the new peroxidated solution, "cleaned".

BY PROFESSOR FANATICUS


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Edited by spornication (05/03/12 06:43 PM)

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OfflineBjJiggles
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Re: Waiting on agar!! What to do? [Re: spornication]
    #16178074 - 05/03/12 06:52 PM (12 years, 8 months ago)

Cool, sounds like a winner doc-t, do a brf clone and save the other half for agar...

Btw so you say just make a pf jar up with 1/2" @ bottom, no dry vern layer and in a glove box drop the sample on top of it?? Also, how will it get it's GE? Maybe the seal being loose from the upside down jar lid? And if i dont have any without holes, could i tape them up or leave and aluminum foil cover over it?


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Alan Rockefeller said:No!  Do not feed the type collection of a new species to animals!

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