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OfflineTrippinRhino
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Single Strain (spore) Isolation and the Benefits..
    #351573 - 06/29/01 06:28 PM (15 years, 11 months ago)

This is kinda a broad question i know. I will break it down:

When cloning from a carpophore, what does one look for in selecting a specimen? I know a fella who recently cloned a mexi-cube by transfering a piece of carpophore and it is growing nice on agar. The pin chosen was both the first pin to appear on the small casing and the largest overall specimen as observed through the fruiting cycle. It was plucked just prior to breaking it's viel and cloned. It seemed to him to be a suitable candidate for cloning...

Will substrate spawned with this isolated strain, in fact, throw a more evenly maturing flush than with mulitispore innoculations? Is there more to know on the matter?....

What is the practical longevity of an isolated strain when it is maintained on agar (continual transfer) and used for grain innoculation?...

Edited by TrippinRhino on 06/29/01 06:38 PM.



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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: TrippinRhino]
    #351585 - 06/29/01 07:04 PM (15 years, 11 months ago)

Don't have any experience with the spore mass injection technique, spore syringe, so no comparison. I only use single strain isolates to make casings. They tend to be very UNIFORM, in fruiting. Very good pin sets. All the mushrooms tend to look similar in size and appereance. Exceptions do occur with uneven casing layers. As far as strain vitality, you can keep a cubensis going for quit a while. Make duplicates for cold storage, revive them at least every four months or so. You don't want to go more than ten transfers or so, but it depends on the strain. Some species can't go more than four transfers without serious degeneration. Cubensis is pretty hardy. I tend to lose my strains to contamination from storage, longest time frame for me was about five years, for a wild Micanopy strain cloned to agar. It eventually got contaminated, and the clean sectors were not as vigorous. There are many techniques that have been discussed here about long term storage. All work to some degree. It really depends on the expense you are willing to spend to maintain the strain.
I still recomend isolation of pure strains as a means to cultivate, if not for any other benefit than to gain experience at the Aeseptic technique. There are definite advantages, but it is definetly not necessary.
As far as characteristics to select for that is personal. In the gourmet mushroom industry they select for characteristics useful to the industry, short stems, large caps, less spores, thicker and more density to the flesh, storage ability, etc..

Edited by Teonan on 06/29/01 07:12 PM.



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OfflineTrippinRhino
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #351602 - 06/29/01 07:38 PM (15 years, 11 months ago)

Your single spores are isolated by means of agar transfer -or- do you clone? This same fella is also in the process of agar transfer isolation using EQs and Thais. 2 series of transfers have been made since the multi-spore innoculation of the dishes. The newest dishes seem to be all the same strain, based solely on uniformity. Is there a way of determining? Is it not better to clone? Cloning ensures a 'fruiting' strain versus agar transfer isolation which leaves you in the blind until fruiting is induced (or not). This is my understanding. Is it lacking?



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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: TrippinRhino] * 1
    #351675 - 06/29/01 10:14 PM (15 years, 11 months ago)

Yes, you are absolutely right. Clones ensure that you have a fruiting strain. Alot of people start with a syringe, inject substrate, fruit, and clone. This is the easiest, most efficient way to get a fruiting strain. I don't mind taking the extra time, because I like to see alot of the strains from any given spore race. I then test these for fruiting capabilities and characteristics. Then I clone what appears to be best to me. When you start with a syringe and inject directly, you tend to get several strains growing in that one jar, there is no gaurantee you are getting the best the spore race has to offer. If you just want a fruiting strain, then syringes are better. If you read through the posts on say PF strain, alot of the complaints about aborting, and degeneration are a result ofnot getting the best it has to offer with . They are usually not lucking out when they roll the dice.
People who develope mushroom strains don't usually start with a syringe, unless they are inoculating agar. Agar enables you to actually isolate individual monokaryons to the point of being able to actually breed mushrooms, for specific traits. You can imagine how time consuming and costly that would be. So unless you work in Research, funded, that is probably a waste of time. But you at least get to see all the spore race has to offer, with isolation of individual strains from multispore germination on agar. But yes you have to test them all for fruitability. So it really depends on what your goals are.
Cloning a fruiting strain, and trying to maintain it is what your after. So syringe to substrate to clone ( pure strain). Strains won't last for ever, so you always go back to spores.

Edited by Teonan on 06/29/01 10:17 PM.



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Offlineauto59009
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #351782 - 06/30/01 02:50 AM (15 years, 11 months ago)

hey cool teonan, thanks a lot for ur cool info!!! :smile:
i have been wanting to get attempt isolation for a while now, and i was uncertain of the fruitaility situation...
i do not have a totally aseptic area (at least 99% clean tho) so i ordered some peroxidated mea plates. any idea how long these will last? i was originally going to take substrate sections but i think (after reading ur post) i will wait it out and clone individual carpophores.

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Offlineauto59009
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #351808 - 06/30/01 03:37 AM (15 years, 11 months ago)

hey teonan,
is there any difference in what part of an individual carpophore is cloned? i mean as in would it affect growth on agar or contamination chances or anything...
what part do you usually use?

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OfflineDarK_SavioR
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #351917 - 06/30/01 12:41 PM (15 years, 11 months ago)

This is what I was wanting to know about isolating strains.... First thing, the spores from the shrooms carry the characteristics and traits of the isolated strain, right? Well, after taking spores from a strain you isolated, could you grow those spores out on agar, isolate the best sections of mycelium, grow it out, and repeat over time to get nice and strong genetics or whatever? Or would the strain degenerate from that, its not cloning but growing your spores out on agar and isolating the best strains over and over... guess you could say isolating the best of the best, heh.

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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #352144 - 06/30/01 10:20 PM (15 years, 11 months ago)

I just use a wood box I built about eight years ago, with three walls and a bottom. The top is just clear plastic tacked on. I spray the shit out of the inside with lysol, wait about fifteen minutes and go to work. I get about 10% contamination on average. No matter what stage of the process. It tends to be from mental mistakes during transfers, not the the box. I use MEA with peptone and yeast as a medium. If the print is wild, I use antibotic MEA. Never tried peroxide.




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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #352149 - 06/30/01 10:29 PM (15 years, 11 months ago)

Usually I clone from the cap. Will wipe the upper surface clean with a moist paper towel, then repeat the process with iodine. Do all this in the clean area. Then gently split open the caps top surface and get out a small piece of mycelium. Transfer to agar. Do duplicates. They will get fuzzy white and start to grow out across plates. Transfer away from any contaminants to fresh plates.
Any flesh from the mushroom will work, AVOID SPORES!!! Stem or cap. Just try and get a clean piece to transfer. Thats why I go inside the cap. SURFACE STERILIZE FIRST!!!



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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: DarK_SavioR]
    #352159 - 06/30/01 10:50 PM (15 years, 11 months ago)

If you keep inbreeding like that, you will get more uniformity in active genes. Environment plays a large role in gene initiation. Continuously inbreeding a spore race will not neccesarily make it stronger or weaker, it will just make it more uniform. Degeneration is not gonna happen that easy if at all with spores. You mainly get degeneration from strains, individual dikaryons. To many transfers from initial isolation. Mutation occurs. Your strain on agar will start to sector towards the worst. You loss vigor. The same thing could happen if you keep growing a strain on the same medium or substrate, it could lose it's vigor.
Inbreeding of a spore race to domesticate!!! When you keep inbreeding a spore race, environment is really gonna determine what you get in the end. If you keep selecting great shrooms to be donors, your gonna get more uniformity in your spore race for the specific environment they were grown in. Temperature, substrate, light, air quality, etc.. are gonna be the determining factors. The mushroom contains all the genes, the environment activates them. If you grow on rye grain at cooler temperatures with less light. Over generations of inbreeding, your spore race will become more uniform in that environment. It will still grow outside that range, but will perform best in those environments. That is the best you can hope for. Selecting a clone canidate that grows out of a multispore innoculated substrate, in a specific environment, and cloning the best specimen is essentially the same thing. Now you have a single strain that performs well at your specific environment. When you multispore isolate on agar first, you are just increasing your chances of finding the very best the spore race has to offer, BUT YOU HAVE TO WORK FOR IT!!!!



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Offlineauto59009
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #352177 - 07/01/01 12:02 AM (15 years, 11 months ago)

haha! damn man u sure fuckin know ur shit! thanks so much for the detailed reply...
i will probably be using peroxidated agar. bacteria cannot BEGIN to form a colony on it, ut pre-formed colonies that are transferred will grow... mould spores cannot germinate on it, but neither can mushroom spores. mycellium, however, has an enzyme that can break down the peroxide into hydrogen and oxygen :smile: perfect!
i will do only one transfer from the initial clone. i basically want to store some selected mycellium in a syringe in staesis for preservation. so is this ok?



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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #352180 - 07/01/01 12:21 AM (15 years, 11 months ago)

I've never tried to store mycelium that way, so I don't know!! I just put in slants to store, but I revive them every three to four months. I had a few last about a year without reviving. Never tried to store one for longer than that.



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Offlineauto59009
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #352193 - 07/01/01 12:47 AM (15 years, 11 months ago)

yeh cool...
i just want to store them for about 3 months max... that way i can cycle my fav strains without having to have them all growing at once :smile:

thanks for all ur help teonan! much appreciated...
last q, i have read a bit about slants but cold you please explain the process to me?

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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #352204 - 07/01/01 01:07 AM (15 years, 11 months ago)

A slant is a test tube with medium in it, that was cooled laying at an angle. This angle lets the Agar cool down and gel with a large surface area. Like a petri dish, but using a test tube. You transfer mycelium of known purity and cleanliness into it to store in the refrigerator. All of this is covered in the Mushroom cultivator, and the Growing Gourmet and Medicinal Mushrooms, both by Paul Stametes, and the former co-authored by Chilton.



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OfflineDarK_SavioR
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #352206 - 07/01/01 01:09 AM (15 years, 11 months ago)

Ya, thanks for the info... I had thaught about that and had my own little theories that where close to what you said but I wasn't sure about them. I'm gonna experiment with some stuff now, should have some fun heh. Gonna try inbreeding pan tropicalis and see how well I can get those going, also wanna do the same thing with the puerto rican strain of cubensis I just got in. To revive a slant do you just let it sit out for 24 hours or something and then put it back in the fridge or what? I'd like to do that with some of the original puerto rican agar I grow and then compare it with what I've got it to do over time. Also be nice to have for backup and try and get it use to some other substrates and stuff. Dunno, it'll be fun though. Thanks for the info, agar is definately gonna be my thing from now on : ) well, starting from when I get some more cash anyway, hehe.

Cool, calm, just like my mom. With a couple of valium inside her palm.
Long Live The Free Spore Ring!
All thats stated above is for humor and a lie!!


--------------------
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Ralphster44 & The FSR!
All thats stated above is for humor and a lie!!


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Anonymous

Re: Single Strain (spore) Isolation and the Benefits.. [Re: DarK_SavioR]
    #352278 - 07/01/01 03:35 AM (15 years, 11 months ago)

Remove it from storage, and transfer to fresh plates, and if you don't use it all up, return it to storage. I really don't know how long it will keep in storage. Never tried more than a year.



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Offlineauto59009
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: ]
    #352282 - 07/01/01 03:48 AM (15 years, 11 months ago)

hey dark saviour,

u able to send me a puerto print as soon as they are up? i would appreciate it A LOT :smile: do i owe you anything btw?

thanks!

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OfflineTrippinRhino
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: auto59009]
    #353928 - 07/12/01 11:34 PM (15 years, 11 months ago)

WOW! I have been away for a few weeks...sorry I have not been around to follow up on this thread. I have since cloned a few different spore races. I am about to get down a dirty with the Hawk Tasmanias. I like 'em so far.....NICE RAINBOWS EVERYWHERE!....gotta nice happy feelin' too but all that aside, they seem to outpin just about anything I have seen yet. I'm talking beaucoup pins! I am gonna post some pics of my 2nd attempt with them. It is a couple of multispore casings that provided clone candidates. Some of the clones are being grown out on agar currently. After making rye jars from the agar, they will be subjected to the same substrates and conditions and will be fruited side by side for some comparison. This work could be endless, but fun indeed....Thanks for all the input fellas! Teonan, I am humbled!



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OfflineAnnoA
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Re: Single Strain (spore) Isolation and the Benefits.. [Re: TrippinRhino]
    #359749 - 07/24/01 02:02 AM (15 years, 10 months ago)

Info on long term strain preservation:
http://fungifun.50megs.com/hepa/docs/text/culture-storage-technique.txt


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Mushrooms, Mycology and Psychedelics >> Advanced Mycology

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