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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 926
Registered: 02-2003
Posted on Monday, October 20, 2003 - 06:53 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I've been trying to get an agar/petri dish tek written for a long while, but it's really hard to work sterile and take pictures at the same time. Bear with me and I'll get it out gradually. I see more interest in agar work lately, so I thought I'd show you guys and girls one of the tricks of the trade. Strain Isolation. I've said it before, and it's no exaggeration, that a single swipe of spores onto a petri dish can generate hundreds, if not thousands of substrains. Some will be very fast colonizers, some will fruit prolifically, some will not fruit at all. Some will build a few huge mushrooms, others will build small mushrooms, or none at all. A few strains will build lots of huge fruits, and these are the strains we want to seek out. All you need is agar, sterile petri dishes, and lots of patience. The first picture is three transfers away from the original petri dish that was inoculated by the sporeprint. The first transfer should be made within one week of germination. Simply transfer a few pieces of the 'best' looking mycelia at the farthest point away from the point of germination. Transfer these to new petri dishes of antibiotic agar. Wait about a week, and repeat that process. At this point, there are still so many substrains growing all over on top of each other, it's hard to see anything but a mass of white. By the third transfer, you should have something that looks like this. The edges are cloudy because you're looking through the parafilm, which won't be removed until just before the transfers begin.
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You can see some 'sectoring' in the above picture. Each sector is a separate strain. The easiest way to see the sectors if they're grown over with mycelium on top, is to turn the dish over and hold it up to a light. If you look from the back side of the dish, you can easily see each individual sector.
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You will see from the above picture, that the bottom half of the petri dish has been taken over by one substrain. This is the most aggressive mycelium in the dish, and if left unchecked it would take over and devour the other strains. This is what happens when you use multi-spore injection. However, the fastest growing, most dominant mycelium might not always be the 'best' for other qualities. It might not even be a fruiting strain.
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The solution is to transfer a small piece of each strain into separate petri dishes. As you can see, I took two samples of the largest growth, and one each of the best looking strains in that dish. There were more strains I could have isolated, but these were the best. Looking at the above picture, the right half of the dish was 'funky' so I didn't want any of that. There were two more substrains I could have taken from the 9:00 o'clock position, but didn't. The original petri dish was then 'sacrificed' to look at under the microscope, to make sure no bacteria was hitch hiking on the mycelium. You can set the whole petri dish right under the objective, and shine a flashlight on it from above. I don't go over 100X when checking for contamination. That's enough. I don't like to look at it before a transfer, because it's too hard to clean the microscope enough to make it completely sterile, and contams might blow from the microscope down to the agar. It's better to look at it after the transfer is complete, and if you find contams, you have to simply throw all your work away! lol Don't forget to wrap each petri dish with parafilm while still in front of the flow hood, or in the glove box.
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faht (Fahtphish)
Senior Member
Username: Fahtphish

Post Number: 414
Registered: 01-2003
Posted on Monday, October 20, 2003 - 09:16 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

awesome!! :-) great work as usual rodger!!

fahtster
so a man walks into a bar with a chicken in one hand and his cock in the other...
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alligator (Cooscoos)
Senior Member
Username: Cooscoos

Post Number: 111
Registered: 08-2003
Posted on Tuesday, October 21, 2003 - 12:15 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

thats fantastic mr rabbit
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ron (Leveler)
Senior Member
Username: Leveler

Post Number: 132
Registered: 06-2003
Posted on Tuesday, October 21, 2003 - 04:52 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Wow I had a kit before that had agar and that was the only part I got right then years later found the PF tec and I was so leary to try it and I did then was left with three great looking cakes then put them in a grow chamber then knothing showed then I broke them up and put them outside and then it got humid as hell and then I cried when I seen my first ever little Babys then I birth the rest then waited and waited and then when there wherew down and dry I was so scared to eat them,lol lol. I was scared for knothing lol. I triped my balls off and was happer then shit,lol. I thought they would kill me b/c I thought they where not Shrooms but ate them b/c they where blue and at first I thought they where bad b/c i was so damn scared lol.
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Highflyer (Highflyer)
Senior Member
Username: Highflyer

Post Number: 520
Registered: 12-2002
Posted on Tuesday, October 21, 2003 - 06:33 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

When I do plates, I usually use colonized grains to innoculate. It usually ends up cutting back the number of transfers that are needed before a good isolate is attained, thus making the whole process faster. It also will usually give nice rhizzo's with the first plate. Here is a pic of a plate before any transfers are made.

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"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 935
Registered: 02-2003
Posted on Tuesday, October 21, 2003 - 07:03 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

But, what do you inoculate the grain with in the first place?
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Highflyer (Highflyer)
Senior Member
Username: Highflyer

Post Number: 521
Registered: 12-2002
Posted on Tuesday, October 21, 2003 - 08:48 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Spores. When I first started doing this, I was figuring out how to get the whole process moving faster. I was using the spores at the time to innoculate the dishes. I found that the grain-agar transfer will shave at least a week off of the total isolating time, and has several other nice benefits. This has really been working well because of the speed at which popcorn colonizes.

Could you please describe what contams, like bacteria, look like under a scope? Also, would you have any suggestions for a prospective microscope buyer?

Thanks for the writeup. I learned several new things. :-)


"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 936
Registered: 02-2003
Posted on Tuesday, October 21, 2003 - 11:02 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

If you're using multi-spore inoculation on grain, then the most dominant strain is what is taking over the jar, and it's the strain you're transfering to the agar. That is why you don't see a hundred strains on your petri dish. If you look at the dish above, it was about three days away from that one strain eating up all the others. Had I made the transfers a few days earlier, I would have picked up three or four additional strains that were already gone when I got around to doing the work. In two weeks, all those new substrains will be grown out, and each dish will inoculate two quarts of rye or popcorn, with a small piece of mycelia going into a new dish for safekeeping. After the grain fruits, I can see which strains I want to keep, and then can grow out those petri dishes to inoculate more grain masters, and the process continues. . .

As for looking for contaminants, the main thing to look for on mycelia is bacteria. It's easy to spot once you've seen it a few times. Look for short 'pencil' looking objects. Sometimes they're referred to as bacterial rods. You want to look for the same thing on sporeprints. I usually crank the objective around to 400X when looking at sporeprints, and also look for trich spores. They look almost like mushroom spores, except they're green. There's an excellent chapter in Paul's book, "The Mushroom Cultivator" on microscopic identification of contaminants. If you go to one of his seminars you'll get hands on work as well.
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Highflyer (Highflyer)
Senior Member
Username: Highflyer

Post Number: 523
Registered: 12-2002
Posted on Tuesday, October 21, 2003 - 11:23 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

What are the benefits of having several substrains, and not just the most dominant one?
"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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faht (Fahtphish)
Senior Member
Username: Fahtphish

Post Number: 448
Registered: 01-2003
Posted on Tuesday, October 21, 2003 - 11:40 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

so is isolation only good until you collect spores again? are you just transfering better genetics through this process?

faht
says: hello :-)
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 938
Registered: 02-2003
Posted on Tuesday, October 21, 2003 - 01:25 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

yes, and no. When you go back to your spore print, you'll be starting over from scratch. However, if you find a killer strain, you can use it 'forever' if you're careful. Allow a petri dish (a test tube would be better) of a great strain to grow out partially, then store it in the refrigerator. Anytime you need to, just take a piece of mycelia out of the dish or test tube the size of the chunks you see above, and put it into a fresh dish. Return the 'mother culture' to the refrigerator. Allow the new dish to grow out for two weeks, then transfer it to grain. If you do this, the mother culture will last for many years.

If you constantly isolate strains for the best qualities, then sporeprint them, you will gradually improve the genetics of your crops.
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 939
Registered: 02-2003
Posted on Tuesday, October 21, 2003 - 01:49 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Highflyer,
To answer your question, the most dominant mycelia in a petri dish may or may not be the best fruiting strain. The most dominant growing mycelia will gobble up all the others in its path. How often have we all seen posts here where someone ate 3 grams and didn't hardly get off, and someone else ate 2 grams and went to Mars? The difference is in the particular strain that was dominant in taking over their jars. By isolating strains and keeping them properly labeled, you can tell when a casing or cake fruits which petri dish it was inoculated with, and if you like what you see, use that strain again. If not, throw it away, and try another. If you'll run several experiments at once, you can make side by side comparisons and speed up the whole process. I would recommend everyone interested in this to go to Sears and buy a brand new $99 'dorm' refrigerator to keep in their lab. You need a very clean fridge if you're going to store cultures.
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Gnome (Gnome)
Senior Member
Username: Gnome

Post Number: 133
Registered: 10-2003
Posted on Tuesday, October 21, 2003 - 02:03 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Excellent Work, Rabbit. I thank you on behalf of all Mycotopians!!!

You must be the change you wish to see in the world- Gandhi
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Hippie3 (Admin)
Board Administrator
Username: Admin

Post Number: 7233
Registered: 02-2001
Posted on Tuesday, October 21, 2003 - 02:56 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

archive material .
great posts & pix,
a great addition to the knowledge stored here.

http://archives.mycotopia.net/discus/messages/1/29852.gif
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The AOC (Angryorangecat)
Member
Username: Angryorangecat

Post Number: 35
Registered: 09-2003
Posted on Tuesday, October 21, 2003 - 04:56 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Thank you sir Rabbit, I learned a lot today. May the day that I have the resources and knowhow to do this sort of thing come on swift wings! :-)
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faht (Fahtphish)
Senior Member
Username: Fahtphish

Post Number: 451
Registered: 01-2003
Posted on Wednesday, October 22, 2003 - 07:16 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

ok, so lets say i have a popcorn pan... are all the mushies on that pan one strain? if so would it be worth taking a kernal from a popcorn casing if that casing is ubundant in fruits and potency and isolating that? :-)

fahtster

(Message edited by fahtphish on October 22, 2003)
says: hello :-)
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 954
Registered: 02-2003
Posted on Wednesday, October 22, 2003 - 09:06 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

It would be better to clone a fruitbody in that case. A fruit is by definition an isolated strain. I have seen multiple strains make it to fruiting when several are strong enough to survive. That's also why sometimes you see completely different results from first flush to second. Sometimes one strain is dominant for the first flush, and a second strain comes out for the second. Typically however, one strain gobbles up all the others before fruiting.
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jimmy jublanski (Zoomerhead)
Senior Member
Username: Zoomerhead

Post Number: 154
Registered: 05-2003
Posted on Saturday, October 25, 2003 - 01:20 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

first of all props to rodger!! great stuff!!

some questions come to mind after reading all this so here goes:

so after isolating a substrain there is no garantee that it will even fruit. therefore the only way is to isolate say 10 substrains and try fruit them all. then pick the strain with the characteristics you like the most.

once you have found the substrain that fit's you. you can "save" this strain and pull off the master culture to innoculate new cultures. people talk of reduced genetics and such...dosen't that apply here as well? and if so any guesses for reasonable limits to this?

now along the same guide lines. one could take from the master culure and use that too make a new culure. then use this culure to innoculate jars of grain. then one could perform gtg tranfers till he had all the spawn he wanted. is this correct?

sorry if this seems redundant...it's been a long week!! thanks
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 981
Registered: 02-2003
Posted on Saturday, October 25, 2003 - 08:15 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Correct on just about all. If you go back to your original cultures in the refrigerator, then take just a small piece to grow out on agar to transfer to grain, you can keep the master culture for years without degradation. Yes, you have to fruit each strain you isolate, and throw out the poor performers. What you will be left with will give those monster flushes you see in the pictures, if you isolate for maximum flushes. Other strains might produce less but be extremely potent. You have to fruit them all, then the hard part. . .You have to eat them... , then finally

I plan to follow and document in this thread the six isolates above all the way to fruiting. They're now all about the size of a half dollar (after four days) and will be transfered to grain in two or three more days. Stay tuned.
"I feel rowdy and I don't know why. . .Excuse me, while I kiss the sky!" jimi hendrix
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philnweird (Highroller)
Intermediate Member
Username: Highroller

Post Number: 73
Registered: 08-2003
Posted on Saturday, October 25, 2003 - 08:39 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Very respectable work rodger! madd props to ya!
Well, I've been meek, and hard like an oak...
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jimmy jublanski (Zoomerhead)
Senior Member
Username: Zoomerhead

Post Number: 157
Registered: 05-2003
Posted on Saturday, October 25, 2003 - 04:10 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

do you like the disposable ones or the glass ones better? 150mm?
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 994
Registered: 02-2003
Posted on Monday, October 27, 2003 - 01:20 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I hate glass petri dishes. Too much of a pain in the butt to wash, then re sterilize. Plastic ones can be had for less than 20 cents each.
"I feel rowdy and I don't know why. . .Excuse me, while I kiss the sky!" jimi hendrix
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jimmy jublanski (Zoomerhead)
Senior Member
Username: Zoomerhead

Post Number: 165
Registered: 05-2003
Posted on Monday, October 27, 2003 - 01:42 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

k i'm sold...where do you get yours from just out of curiosity?
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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 998
Registered: 02-2003
Posted on Monday, October 27, 2003 - 04:59 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

You can get a box of 500 for $89 at fungi.com
"I feel rowdy and I don't know why. . .Excuse me, while I kiss the sky!" jimi hendrix

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