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Offlinewikedanjel
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transferring mycelium
    #1200246 - 01/08/03 10:45 AM (13 years, 10 months ago)

A few questions i want to ask.
1. When you transferring mycelium from a PF tek jar to a pint bird seed jar, what size does the chunk need to be?
2. does anything need to be done to the piece before its transferred. I heard you have to dip it in a solution of H202 and water. 20:1?
3. how many pint jars can I inoculate with one 1/2 pint jar?
4. Will it work just as well as if I put a chunk in honey water and let it grow? if so then how much in a 1/2 pint jar of honey water (1/2 tablespoon of honey, PC @15pci for 15 min)
5. if I put a sterile piece of mushroom in honey water will it grow mycelium? Is this and transferring mycelium considered cloning and will I have to start from spores again if I were to keep letting it grow from jars to jars. (although I will start from spores again but its nice to know)

I know some of these questions have been asked before but the responses haven't been very clear to me and I would like to re state the questions again.

:grin: Ok thanks in advance for any useful answers


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OfflineignuF
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Re: transferring mycelium [Re: wikedanjel]
    #1200379 - 01/08/03 11:46 AM (13 years, 10 months ago)

1.- Most people do this using agar in petri plates but it can be done with PF cakes also.  You would need only a small piece, maybe 1/4" X 1/4" about the size of a hit of acid :tongue: Keep in mind all of this needs to be done inside of a sterile glovebox.

2.- I would dip in 100% H2O2 (Live myc. will not be hurt by H2O2) Let it sit submerged in this for about 20 min. Then transfer to your grain jar. 

3.-  :confused: 25-50?  Not sure, it just depends on the size of the wedges you take out.

4.- Yes, reffer to answer #1.  You might not want to transfer pieces of cake to honey water as the vermiculite will make it harder to spot contams :crazy:  Not to mention it will probably clog your syringe when extracting the myc. for use later.

5.- Yes.  Again soak a piece of tissue from inside the stem of the mushroom in 100% H2O2 for 20 min. Then transfer to your honey water.  This is cloning.  Just transfering the myc. is not.  However you could keep transfering a cloned culture (mycelium) from your original clone.  And yes grain to grain transfers can keep you from going back to spores :wink:  Hope this clears things up a bit.  And remember to use a sterile glovebox during all these procedures. 


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Offlinedebianlinux
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Re: transferring mycelium [Re: wikedanjel]
    #1200940 - 01/08/03 02:19 PM (13 years, 10 months ago)

ignuf did a great job of answering your questions. I can give you some pointers based on personal experience with this method. using PF jars as quasi-agar plates is a good way to go because if your already familiar with the tek you don't have to learn the whole agar route and invest in any new equipment. the glovebox is essential tho (or flowhood). personally I do 4 jars of grain to 1 PF cake. I'm using 1/4 pint jelly jars to do my cakes since these colonize unbelievably fast. you could theoretically do over a hundred grain jars with one little PF cake since all it takes is just a little piece (microscopic really) of mycelium to make it go. hell, 2 spores would do the trick. but throwing a sizebale chunk in there will dramatically increase the colonization time of that jar (I should hope your doing quart jars of grain here). the reason being that when you shake it up that PF cake chunk breaks into a shitload of pieces and practically inoculates the whole jar (all kernels) at once. another advantage of only doing a relatively small # of jars per cake is the contamination factor. let's say that one of your cakes was invisibly contaminated or you just mistook some cobweb for mycelium. if you inoculated a crapload of jars with that 1 cake then you just screwed up a lot of grain and hard work. however,  just doing a few jars per cake insures that the larger majority of the jars will make it. keep up the good work and the good thinking. i see our methods are very similar. :smile:

edit: I consider the h202 dunking a bit unnecessary. do it if it makes you fell better. my exact method involves lysol bombing the glove box and some small square containers with lids. I open the PF jars and plop the cake in the container and close it ASAP. lysol the outside of the container again for good measure. lysol your hands/arms and the the 1st grain jar. insert all in the glove box. open the container and break the cake up by hand. open the 1st jar and dump an approximate desired fraction (1/4, 1/8, whatever) of the crumbled cake into the grain. close the grain jar and remove it. lysol hands and next grain jar and insert into glove box and dump the same fraction of the crumbled cake into this jar, repeat until finished. shake the hell out of the jars to distribute mycelium and you should know the rest. 12 jelly jars = 48 quarrt jars of inoculated grain which will colonize in about 1.5 weeks (in my experience). have fun!


Edited by debianlinux (01/08/03 02:25 PM)


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Offlinewikedanjel
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Re: transferring mycelium [Re: debianlinux]
    #1200973 - 01/08/03 02:28 PM (13 years, 10 months ago)

To reply to both of you thanks a lot for the help! Good job guys and good luck with your own projects.  :grin: 


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OfflineMAIA
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Re: transferring mycelium [Re: wikedanjel]
    #1201026 - 01/08/03 02:41 PM (13 years, 10 months ago)

1- I usually use 4x1 cm3 in each jar.
2- This is done just as precaution and it's not needed if you work in a sterile enviroment, ie glovebox.
3- Depends on question 1.
4- I'm not saying you shouldn't do the honey tek but i advice about how contam prone it is, i'd skip honey water and stick with just direct transfers. Anyway, do you want even more spawm material ?
5- Yes, both could be considered cloning techniques. Honey water is just an extra step, although it's a distinct process what matters is the spawn material nature and it's objective, wich at the end it's the same, mycelium reproduction.

MAIA


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OfflineFred Garvin
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Re: transferring mycelium [Re: wikedanjel]
    #1201201 - 01/08/03 03:20 PM (13 years, 10 months ago)

It's really a lot simpler than it seems. I use pf cakes as a lazy man's petri dish.

I grate my pf cakes on a cheese grater, and spoon the juicy bits into quart jars. I usually do about 4 quart jars per pf cake. I like to use more inoculate, because the colonization time is decreased tremendously, cutting down on the time any contams might have a chance to gain a foothold.

In my opinion, a glovebox or flowhood is not absolutely necessary, and is a bit of overkill. Just make sure your work area is clean as can be, you are as clean as can be, and all your apparatus and tools are clean as can be. A liberal spraying of the air in the workspace with lysol or alcohol prior to starting is good, as well as running a HEPA room filter. A face mask and rubber gloves are essential.

I get my quart jars ready first, by loosening the lids and getting them arranged in working order. I then grate my cakes into a sterile stainless steel bowl, and cover with a filter disc. Then I spoon the bits into the jars quickly and replace the lids loosely. After all the transfers are complete, I go back and tighten the lids and shake the jars to distribute the mycellium throughout the birdseed.

I've been doing this for quite a while, and have had very few contam problems, and those I think were due to using rye grain instead of birdseed.

A lot of people tend to overcomplicate things.

Just my two cents.

BTW- I think using pint jars is kinda pointless, why go from a 1/2 pint to a pint, when you can go to a quart instead? Using quarts you get twice as much colonized grain for the same amount of effort. Unless you have space issues, etc.



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Edited by Fred Garvin (01/08/03 03:30 PM)


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OfflineMAIA
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Re: transferring mycelium [Re: Fred Garvin]
    #1201220 - 01/08/03 03:26 PM (13 years, 10 months ago)

Quote:

a glovebox or flowhood is not absolutely necessary, and is a bit of overkill




Overkill ? Yes, perhaps for multispore but not for transfers.

MAIA


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OfflineFred Garvin
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Re: transferring mycelium [Re: MAIA]
    #1201242 - 01/08/03 03:35 PM (13 years, 10 months ago)

Yes, if the transfer procedure is done with proper precautions, it is overkill. I have done many transfers with negligible contam problems, and I don't think it's due to luck.

But again, just my opinion. Peace and happy growing :cool: 


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OfflineMAIA
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Re: transferring mycelium [Re: Fred Garvin]
    #1201251 - 01/08/03 03:39 PM (13 years, 10 months ago)

Sure, what works is the best for us.

MAIA


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OfflineFred Garvin
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Re: transferring mycelium [Re: MAIA]
    #1201260 - 01/08/03 03:43 PM (13 years, 10 months ago)

Thats what I'm sayin'





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Offlinewikedanjel
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Re: transferring mycelium [Re: Fred Garvin]
    #1203524 - 01/09/03 09:07 AM (13 years, 10 months ago)

Well thanks everyone for all your help, maybe i will goto quart jars instead but I stil like the idea that if one contams its not like Ill be loosing so much, but then again if it goes well them I have twice the fun to play with  :wink: OK yeah im going to get some Quart jars. 


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OfflineCurious_George
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Re: transferring mycelium [Re: wikedanjel]
    #1203582 - 01/09/03 09:27 AM (13 years, 10 months ago)

I'd like to do quarts, but can't cuz my PC won't take them,, jars are too tall.

I have a presto 6 Quart i think, and with the plate at the bottom the lid will fit but the jars will be touching the PC lid.

Soooo.. i have to do pints!!

arghh

cg


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InvisibleLanaV
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Re: transferring mycelium [Re: wikedanjel]
    #1204483 - 01/09/03 03:56 PM (13 years, 10 months ago)

You should consider 1/2 gallon jars.  Now they're fun :smile: 

Lana 


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Offlinedebianlinux
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Re: transferring mycelium [Re: Lana]
    #1206031 - 01/10/03 05:54 AM (13 years, 10 months ago)

big problem with 1/2 gals (for me): can only fit 1 in my PC. can fit 7 quarts in there. 7 quarts=1.75gal and if contams hit i don't lose it all in one fell swoop. now if I could fit 7 half gals in a pc....

started thinknig about it and maybe what i thought were half gallons are actually gallons? i dunno... they don't say and I'm not home to check the volume. any1 know how many 1/2 gals fit in an AA921 PC?

even so, my argument still stands because the best I would be able to do is two maybe three 1/2G's and that still doesn't add up to 1.75 and the loss to contams is still a big factor (like 3 times as high). I don't seem to have much of a problem with contams anymore but I'm still scared to death of them.


Edited by debianlinux (01/10/03 05:58 AM)


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