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Tissue Streaking on Peroxidated AgarAdminAdmin1 1 05-06-04  08:43 pm

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hive_bee (Hiveb)
Junior Member
Username: Hiveb

Post Number: 11
Registered: 07-2003
Posted on Thursday, October 23, 2003 - 08:17 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

in working with agar, a first clone was made from PFCs. one chunk put into antibacterial agar, the other in the same but with a much (too much) H2O2. the result was that the non peroxidated agar chunk took off fast. however, that non perox chunk had contam problems in another section of the dish.

here is the kicker. the peroxidated agar did not grow out, but grew brilliant white. perhaps the peroxide layer (an actual several millimeter thick layer of fluid) prevented expansion, but kept the possible contam from the other clone plate out. if so, then a plate like this could be a staging ground for allowing myc to grow on a peice of tissue then transfer to another plate. sort of a precleaning...

comments.

hiveb
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Highflyer (Highflyer)
Senior Member
Username: Highflyer

Post Number: 527
Registered: 12-2002
Posted on Thursday, October 23, 2003 - 10:29 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

The tissue should be taken from the inside of the stem. If this is done, then it should be sterile, and not need a precleaning.

"I hold it to be the inalienable right of anybody to go to hell in his own way." - Robert Frost
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Fitch F. Fitch (Fitch)
Intermediate Member
Username: Fitch

Post Number: 53
Registered: 11-2002
Posted on Friday, October 24, 2003 - 03:13 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Heres a basic rundown of the role of pH in the cultivation of mushrooms.

Higher (more basic) pH is good for killing things on already colonized media, because the mycellium generally holds up better than the contam, so the fungus can recover; but for media you are hoping to grow whatever on, a low pH (acidic) environment is better, as bacteria wont grow but the mycelium will (molds will too, but this is generally not a problem)

Moral: Base is good for fighting off contams, Acidic media are good for culturing as they select against bacteria.

Hope this helps.
And I think it's gonna be a long, long, time 'Til touchdown brings me 'round again to find I'm not the man they think I am at home. I'm a rocket man
burnin' out his fuse, up here alone.

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hive_bee (Hiveb)
Junior Member
Username: Hiveb

Post Number: 12
Registered: 07-2003
Posted on Friday, October 24, 2003 - 03:21 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

well, if sterile conditions (no flow hood, lack of iodine, non sterile scalpel (sp?), etc) are not met, perhaps the precleaning is necessary. these particular fleshy chunks were taken with sterile knife, under flow hood, with an iodine wiped outer skin peeled back from the base of the mushroom. probably sterile conditions.

example of application: glovebox (very primitive) with no positive pressure and questionable air supply. take the chunk of tissue, possibly large chunk, and perform iodine peel back. once chunk is cut, it can be dropped onto the agar plate with a good layer of H2O2. this allows the H2O2 to cleanse while also allowing the chunk of mushroom to grow over with mycelium. this chunk could then be transfered into a karo jar, clean already and with already recovered myc.

hiveb
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RUSTDOG (Rustdog)
New member
Username: Rustdog

Post Number: 1
Registered: 10-2003
Posted on Friday, October 24, 2003 - 03:37 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Can you elaborate on some formulas for starting spores,and on tissue cloning?
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Fitch F. Fitch (Fitch)
Intermediate Member
Username: Fitch

Post Number: 57
Registered: 11-2002
Posted on Friday, October 24, 2003 - 04:29 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Heres a rundown of what I would consider an ideal way to do agar innoculation without any equipment.

THE ROOM:
Innoculate in a still air room. Most of the contams are a foot off the ground or so, not agitating them means they are likely to stay there. Wipe your work area down with: Bleach/Alcohol/Lysol/etc. Wash hands with antibacterial soap, alcohol, bleach, whatever.

THE MEDIA:
For growing fungi, molds, whathaveyou, use agar with a pH of 5.6 or so. This means bacteria will not grow, and unless you live in an old/damp dwelling, you should not have an abundance of spores in the air. Also, spores will tend to fall, as they dont colonize in the air. Spores drop, its what they do, so if you combine THE MEDIA with THE ROOM you do a lot to reduce contams. If you do have lots of spores in the air, this can lead to a wide array of health problems; it is not a good sign.

THE INNOCULATION:
Keep your plate upside down when you innoculate. In THE ROOM, the air is still, so there is no updraft to blow the contaminates up into the plate, and any in the air that settle will do so on the plastic bottom, not your culture media.
When you innoculate, flame sterilize your innoculateing loop/needle/instrument in (preferably) a gas flame. An alcohol flame is good too. (Fill a glass with rubbing alcohol, light it on fire...it burns for awhile, and leaves the glass black) The instrument is sterile when it glows. Allow the instrument to cool for 15-25s or so. Do not wave it about while it is cooling, keep it still. Use the loop to transfer spores to your upside down plate. A T streak should be used if you want to ISOLATE. Store plates upside down, so that water collecting on the lid does not drip into the culture.

CLONING:
Take your tissue sample from the middle of the mushroom. Rinse it in dilute H202 (3%). It makes it easier to spread the tissue over the plate if the tissue is liquified. Mix H202 (3%) with the tissue puree. This will kill mold spores, and any bacteria will not be able to grow on THE MEDIA anyways. Some H202 may react with THE MEDIA to form water. If the plates are stored upside down, this water does not collect on your agar.

ISOLATING:
Draw a T on the bottom of the agar plate containing THE MEDIA. The small zone over the top of T is zone one. Streak your culture onto ZONE ONE ONLY. Now, with your instrument, draw some of the innoculant from zone one into ZONE TWO (either of the two remaining zones) ONLY. Draw a single amount from zone one into zone two, and spread this amount over zone two. Only go from zone one to two once. Draw some innoculant from zone 2 into zone three. Only go from two to three once; spread the innoculant in zone three. The instrument is sterilized in between transfers. This is the T streak technique. The advantages are that you can get very isolated colonies from zone 3...that is you will not be using a multispore innoculation. I believe this is better for yields. Also, the spread out colonies can be selected from based on how aggressively they grow. This selection process leads to increased yields. To avoid strain degredation, senescence, alternate between cloning and T-streaking spores. Eventually, it is best to stick to T streaking spores only...cloning will always degrade the race. ALternating cloning and streaking just prolongs the inevitable.

All this stuff is in the archives, but spread over many threads and sections...I believe this is all the info you need to work with agar in one place. I hope you find it useful.

Lost in a Roman...wilderness of pain
And all the children are insane
All the children are insane
Waiting for the summer rain, yeah


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rodger rabbit (Skyypilot)
Senior Member
Username: Skyypilot

Post Number: 975
Registered: 02-2003
Posted on Friday, October 24, 2003 - 04:47 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

One good way to cool the red hot scalpel quickly is to swipe it through the agar on the receiving dish.
"I feel rowdy and I don't know why. . .Excuse me, while I kiss the sky!" jimi hendrix
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Fitch F. Fitch (Fitch)
Intermediate Member
Username: Fitch

Post Number: 60
Registered: 11-2002
Posted on Friday, October 24, 2003 - 06:48 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

In general I would not recomend slicing up the agar, especially if you just use regular agar; opens you up to a whole new level of contams that thrive in the abscense of oxygen. Small colony starts at the bottom of the cut, can spread throughout the underside, where you cant gtt at it. Maybe hasnt happened to you, but is a definite possibility.
Lost in a Roman...wilderness of pain
And all the children are insane
All the children are insane
Waiting for the summer rain, yeah


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FarmerWop (Croppinsloppy)
Advanced Member
Username: Croppinsloppy

Post Number: 94
Registered: 01-2003
Posted on Friday, October 24, 2003 - 01:13 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

I'm not sure I get what this thread is about....

Was there a specific question?
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RUSTDOG (Rustdog)
New member
Username: Rustdog

Post Number: 2
Registered: 10-2003
Posted on Friday, October 24, 2003 - 02:11 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

What do you use to make the agar acidic?
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Fitch F. Fitch (Fitch)
Intermediate Member
Username: Fitch

Post Number: 61
Registered: 11-2002
Posted on Friday, October 24, 2003 - 03:44 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

You buy sabouraud agar, which is already made up to the proper requirements. You can get it offline. You could also probably use regular agar and lower the pH on your own, but youd have to know some chemistry.

The thread was about the use of peroxide in agar, and RUST asked for a rundown of agar and cloning work, so I gave it to him. The info was also there for hive to sort through as well, to get an understanding of the process and purpose of materials and such.
Lost in a Roman...wilderness of pain
And all the children are insane
All the children are insane
Waiting for the summer rain, yeah


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Imok Urok2 (Imok)
Moderator
Username: Imok

Post Number: 452
Registered: 07-2002
Posted on Friday, October 24, 2003 - 06:26 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Archive Material to Cloning
Thanks for helping
Hope this helps :-)
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RUSTDOG (Rustdog)
New member
Username: Rustdog

Post Number: 3
Registered: 10-2003
Posted on Saturday, October 25, 2003 - 05:42 am:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Thanks Fitch
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Fitch F. Fitch (Fitch)
Intermediate Member
Username: Fitch

Post Number: 65
Registered: 11-2002
Posted on Saturday, October 25, 2003 - 05:51 pm:Edit Post Quote Text Delete Post Print Post Move Post (Moderator/Admin Only)

Any time, bud.
Lost in a Roman...wilderness of pain
And all the children are insane
All the children are insane
Waiting for the summer rain, yeah


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