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Offlinespooner
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Registered: 03/09/12
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Agar issues
    #18386006 - 06/07/13 11:55 PM (11 years, 7 months ago)

first off I would like to state that I have read through many threads, did a bunch of searches and I just cannot find the answer to my question:confused:

I have decided to branch off into the processes of cloning tissue samples on agar plates, I follow all the proper procedures to get a good sterile sample and I place it on the agar plate and nothing, there is no colonization whatsoever, Iv tried 3 times now. I ordered the agar plates from a trusted vendor, but im just not sure whats going on here. I keep plates in my refrigerator and I leave the plate out a bit before introducing the sample, so it can get to room temp. So if anyone has some suggestions please let me know:laugh:


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Invisiblestonesun
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Re: Agar issues [Re: spooner]
    #18386021 - 06/07/13 11:58 PM (11 years, 7 months ago)

Are you keeping your plates with the tissue
samples in the fridge too?

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Offlinespooner
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Re: Agar issues [Re: stonesun]
    #18386210 - 06/08/13 12:48 AM (11 years, 7 months ago)

no


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Offlinetwistedty
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Re: Agar issues [Re: spooner]
    #18386242 - 06/08/13 12:54 AM (11 years, 7 months ago)

are you getting premixed plates?

make your own just add water and pc



http://www.mycosupply.com/cgi-bin/shopper.cgi?preadd=action&key=PDAA01

pics would help also

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Offlinespooner
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Re: Agar issues [Re: twistedty]
    #18386257 - 06/08/13 01:01 AM (11 years, 7 months ago)

yea they are premixed unfortunately I cant get pics up atm. So when it says PC for 30 mins do you just mix it up and throw it in a jar then pc? what after that? should you only put it in a petri dish?


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OfflineNovanity1
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Re: Agar issues [Re: spooner]
    #18386433 - 06/08/13 03:23 AM (11 years, 7 months ago)

When you collect the tissue sample are you dousing it in alcohol? If so, then you could possibly be killing the tissue and giving it no chance to grow. You can get away without cleaning the stem in iso or h202 if you just rip it open and get a sample of the core.

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Offlinespooner
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Re: Agar issues [Re: Novanity1]
    #18386777 - 06/08/13 07:42 AM (11 years, 7 months ago)

no im only rubbing the outside of the initial fruit


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Offlinecubenpete
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Re: Agar issues [Re: spooner]
    #18386913 - 06/08/13 08:42 AM (11 years, 7 months ago)

spray the stem with 70-90% alcohol (to kill outside contams), then slice open and remove flesh (don't soak or spray this tissue)then add to your project. I have had limited success with this but this method is what is recommended. I think I am right!

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Offlinethe_jerk
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Re: Agar issues [Re: cubenpete]
    #18387923 - 06/08/13 02:00 PM (11 years, 7 months ago)

Quote:

cubenpete said:
spray the stem with 70-90% alcohol (to kill outside contams), then slice open and remove flesh (don't soak or spray this tissue)then add to your project. I have had limited success with this but this method is what is recommended. I think I am right!



Actually, don't "slice into" the stem at all. Any contams that may be left from your spray of alcohol would get forced inside.

Grab it at the base and peel it apart in two much like a monkey does with a banana.
This way there is less chance of forcing a surface contam inside. Then extract center tissue.

Edited by the_jerk (06/08/13 02:01 PM)

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Offlinetwistedty
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Re: Agar issues [Re: spooner]
    #18387956 - 06/08/13 02:06 PM (11 years, 7 months ago)

Quote:

spooner said:
yea they are premixed unfortunately I cant get pics up atm. So when it says PC for 30 mins do you just mix it up and throw it in a jar then pc? what after that? should you only put it in a petri dish?





ya just use water and the pda agar mix in an old whiskey bottle with a hole in the lid like so





if you dont have sfds i think you can get away with stuffing it with polyfil and covering with tinfoil for pc cycle.

as for taking clones dont dip it in alcohol or anything tear it apart with 2 fingers and cut a small wedge out with a heat sterilized blade and drop on agar plate.

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OfflineNovanity1
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Re: Agar issues [Re: spooner]
    #18387981 - 06/08/13 02:13 PM (11 years, 7 months ago)

Quote:

spooner said:
yea they are premixed unfortunately I cant get pics up atm. So when it says PC for 30 mins do you just mix it up and throw it in a jar then pc? what after that? should you only put it in a petri dish?





I'm kinda lost here as to what you are using....

Premixed plates would be sterile petri dishes that have an agar based growing medium already inside them. No further preparation is needed with these.

Or do you have a premixed agar formula such as MEA? In these cases there is some preparation involved.

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Offlinespooner
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Re: Agar issues [Re: Novanity1]
    #18388677 - 06/08/13 05:23 PM (11 years, 7 months ago)

The agar plates that I have are premixed sterile agar plates there is no prep needed.
Step by step this is what I do:
Rub outside of stipe with 70% rubbing alcohol
Break stipe at the very bottom then I split it in half, not cut
Take a small tissue sample with flame sterilized scapel
Drop tissue sample on agar
Seal and wait.

Nothing happens at all there is no mycellial growth. My question is why is nothing colonizing, not how to take a tissue sample and place on agar and not how to prepare agar


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Offlinezpores
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Re: Agar issues [Re: spooner]
    #18388812 - 06/08/13 05:53 PM (11 years, 7 months ago)

What is the longest you have had a sample sitting on the agar? I've done it and it took a week to see growth then it just went nuts and took over the plate.

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Offlinespooner
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Re: Agar issues [Re: zpores]
    #18389207 - 06/08/13 07:13 PM (11 years, 7 months ago)

One has sat for over 2 months...I know the agar plates are good cuz I took one and swabbed the inside of my mouth and bacteria started growing on it, I just can't figure this out :/ is there any specific size of a tissue sample I should do?


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Offlinekinkaku
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Re: Agar issues [Re: spooner]
    #18389216 - 06/08/13 07:14 PM (11 years, 7 months ago)

a really tiny piece closer to the base


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Offlinespooner
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Re: Agar issues [Re: kinkaku]
    #18389240 - 06/08/13 07:18 PM (11 years, 7 months ago)

That's what iv been doing :frown:((, I think I'm gonna try ms to agar should I use spore solution or just go strait from print to agar


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Re: Agar issues [Re: spooner]
    #18389257 - 06/08/13 07:20 PM (11 years, 7 months ago)

print to agar


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Offlinespooner
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Re: Agar issues [Re: cronicr]
    #18389268 - 06/08/13 07:22 PM (11 years, 7 months ago)

Just do a few specs or what?


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Offlinekinkaku
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Re: Agar issues [Re: spooner]
    #18389286 - 06/08/13 07:24 PM (11 years, 7 months ago)

im interested in this thread to because I had the same exact problem lol although I was using nutrient agar and im not sure if that's good


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Re: Agar issues [Re: kinkaku]
    #18389307 - 06/08/13 07:28 PM (11 years, 7 months ago)

swipe it with a sterile inoculation loop then across your agar


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