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OfflineLevster
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Grr.. what the hell is this?
    #23838594 - 11/16/16 11:54 AM (7 years, 2 months ago)

So this is my second batch of inoc'd agar plates and they are about 7 days old so they were sizeable enough for a transfer so this pic I took inside the SAB during the transfer. I transferred it anyway since it was already open but this sure isn't myc, so what the hell? I've done everything right up to this point, all 8 plates basically have this on it, I transferred 3 wedges from 3 different plates to 3 new PC'd plates and then said wtf is the point and didn't bother with the other 5 since that isn't even myc but whatever.. (Forgot to mention, I used  P. cube syringes to drop 2 drops in the center of each agar plate when they were inoc'd)

please help, losing faith.



Edited by Levster (11/16/16 11:56 AM)


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Invisibleazur
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Re: Grr.. what the hell is this? [Re: Levster]
    #23838610 - 11/16/16 11:59 AM (7 years, 2 months ago)

Bacteria. Try antibiotic agar


--------------------


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OfflineLevster
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Re: Grr.. what the hell is this? [Re: azur]
    #23838634 - 11/16/16 12:05 PM (7 years, 2 months ago)

But I don't understand.. There is no Myc anywhere on this plate, or any other plate.. just bacteria. How is that possible, have my syringes gone completely bad? Can I reuse the 16 wasted mini rounds?


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OfflineThe Mycologist
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Re: Grr.. what the hell is this? [Re: Levster] * 1
    #23838641 - 11/16/16 12:07 PM (7 years, 2 months ago)

Spores can take awhile to germinate, bacteria is pretty quick to grow.


--------------------
"That you are here—that life exists, and identity;
That the powerful play goes on, and you will contribute a verse.”
― Walt Whitman, Leaves of Grass
:acidfire::tmckenna:


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OfflineLevster
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Re: Grr.. what the hell is this? [Re: The Mycologist]
    #23838646 - 11/16/16 12:09 PM (7 years, 2 months ago)

and how come this is the first time I've heard of antibiotic agar? Am I that bad at this that I have to use a failsafe method? lol. I followed every instruction to a tee


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OfflineLevster
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839008 - 11/16/16 02:36 PM (7 years, 2 months ago)

If It's cool with you guys, I'm going to need some more information. I don't want to do the antibiotic agar. Should I let these other 5 plates continue to try and colonize or

a) they might as well all be garbage?
b) My sterilization and inoculation is by the book, so that can't be the issue, I feel it's my storage. I'm storing them on a shelf with just the poretape and wad of papertowel on the lid, but it is NOT enclosed in a ziplock bag or covered in cling wrap, is that why this is happening?

I feel like doing another PF tek, that was more fun and successful, but used up way too much of my syringes... I really want to get this one done right so I can transfer to grain


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OfflineThe Mycologist
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839036 - 11/16/16 02:45 PM (7 years, 2 months ago)

I put mine in sandwich ziplocs.

I put spore solution in 10 different petris and only had one colonize in a timely manner.

They might not be garbage, just put them in ziplocs/plastic wrap.

Let us see some pics


--------------------
"That you are here—that life exists, and identity;
That the powerful play goes on, and you will contribute a verse.”
― Walt Whitman, Leaves of Grass
:acidfire::tmckenna:


Edited by The Mycologist (11/16/16 02:59 PM)


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OfflineLevster
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Re: Grr.. what the hell is this? [Re: The Mycologist]
    #23839168 - 11/16/16 03:25 PM (7 years, 2 months ago)

Hey thanks for your reply Mycologist. The pic on the OP was basically what they all looked like. I have already wrapped them all in cling wrap and put them back onto the shelf. Did you want me to do anything further?


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OfflineThe Mycologist
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839176 - 11/16/16 03:27 PM (7 years, 2 months ago)

Well they all are bacterial it sounds like. Try one drop of solution next time. How long did you pressure cook these?


--------------------
"That you are here—that life exists, and identity;
That the powerful play goes on, and you will contribute a verse.”
― Walt Whitman, Leaves of Grass
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OfflineLobi
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839179 - 11/16/16 03:28 PM (7 years, 2 months ago)

It's just bacteria. Excess water in the cup, contam inoculant, improper pc cycle or bad conditions are probably the vectors. Toss them, wash them, try again. Might be your stringe tbh


--------------------
The bonds and ties of the life we know break easily. But through eternity one bond remains; the bond of fellowship.
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of star dust in its endless flight, of suns and worlds,  of gods and men.
The clasped hands of comradeship unite in a bond eternal; the fellowship of spirit.

:mushroom2:My High Quality Lo-Fi Beats  -:mushroom2:MushroomCultivation Compendium  -:mushroom2:- Doing Bulk w/ No PC -:mushroom2:

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Re: Grr.. what the hell is this? [Re: Levster]
    #23839187 - 11/16/16 03:30 PM (7 years, 2 months ago)

I try to use plates asap. If I do store its in the fridge.

You say your inoculation is on point. We hear that a lot.

Elaborate just in case you are inoculating in a 'sterile' bathroom:crazy2:


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InvisibleMad Season
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Re: Grr.. what the hell is this? [Re: Lobi] * 2
    #23839211 - 11/16/16 03:36 PM (7 years, 2 months ago)

Quote:

Lobi said:
It's just bacteria. Excess water in the cup, contam inoculant, improper pc cycle or bad conditions are probably the vectors. Toss them, wash them, try again. Might be your stringe tbh



I've heard this said a lot, and I can't help but disagree. Excess water doesn't cause bacteria. Bacterial CFUs cause bacteria, same with conditions. If there's no CFUs you can have a flooded plate in 50°C. Nothing will grow on it.

The vectors have to be bacteria got in somehow. That could be inoculant, Pc cycle, or techniques. I highly doubt it's the PC cycle, I pc for 15 minutes at 15 psi and have never had bacteria pop up in any cobtrol dishes.


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OfflineLobi
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Re: Grr.. what the hell is this? [Re: Mad Season]
    #23839251 - 11/16/16 03:47 PM (7 years, 2 months ago)

Quote:

Mad Season said:
Quote:

Lobi said:
It's just bacteria. Excess water in the cup, contam inoculant, improper pc cycle or bad conditions are probably the vectors. Toss them, wash them, try again. Might be your stringe tbh



I've heard this said a lot, and I can't help but disagree. Excess water doesn't cause bacteria. Bacterial CFUs cause bacteria, same with conditions. If there's no CFUs you can have a flooded plate in 50°C. Nothing will grow on it.

The vectors have to be bacteria got in somehow. That could be inoculant, Pc cycle, or techniques. I highly doubt it's the PC cycle, I pc for 15 minutes at 15 psi and have never had bacteria pop up in any cobtrol dishes.




Yeah I would agree about the bacterial cfu being the cause and having to exist some where to cause growth.
I have had excess water appear to be the vector for contamination. Water drowning the myc out causing poor colonization and eventually will contam.

Just trying to give some possible pointers to reason for contam, sorry if I posted any misinformation.


--------------------
The bonds and ties of the life we know break easily. But through eternity one bond remains; the bond of fellowship.
The fellowship of atoms,
of star dust in its endless flight, of suns and worlds,  of gods and men.
The clasped hands of comradeship unite in a bond eternal; the fellowship of spirit.

:mushroom2:My High Quality Lo-Fi Beats  -:mushroom2:MushroomCultivation Compendium  -:mushroom2:- Doing Bulk w/ No PC -:mushroom2:

:chillpill:more about my music :chillpill:


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Re: Grr.. what the hell is this? [Re: Lobi]
    #23839341 - 11/16/16 04:12 PM (7 years, 2 months ago)

Excess water moves contams around n makes it hard to clean but that's about it


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OfflineLevster
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Re: Grr.. what the hell is this? [Re: mushboy]
    #23839395 - 11/16/16 04:28 PM (7 years, 2 months ago)

Quote:

mushboy said:
I try to use plates asap. If I do store its in the fridge.

You say your inoculation is on point. We hear that a lot.

Elaborate just in case you are inoculating in a 'sterile' bathroom:crazy2:





Heh, no worries I get it.

I pc the mini rounds for about ~45 minutes.. The PC has no adjustments, I believe 15 psi is just the standard for it. I just gradually bring up the heat, until it starts hissing and releasing pressure, and I leave it at that state for like 40 minutes then turn the heat off, and let it cool overnight. I then use those mini rounds right away or store in the fridge with cling wrap around it. If I do use them right away, they go into my SAB (just a big tub with 2 arm holes.. I use a damp towel and wet the walls slightly with soapy water to attract any floating bacteria. (I let the box sit for 10 minutes before I work in there to let the air settle). I then inoc them one at a time. I flame sterilize, wipe around the container with alcohol before I open it. Then when I open it, I quickly do the inoc, and close it back up.

Anyways, maybe the bacteria has grown, but if I give it another while, perhaps the myc will start growing, and I can take a piece of out it.. here's to hoping


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InvisiblemushboyMDiscord
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839420 - 11/16/16 04:36 PM (7 years, 2 months ago)

ah. good job on doing it the right way:thumbup:

it could just be the syringe. i recently got a syringe from a sponsored vendor
that was horrifingly dirty. the first batch of plates looked like i nutted into it.
took 6 transfers for clean. ill also point out in maybe 10ish lifetime orders from
the vendor i never had anything like that one syringe. thank god i got into agar.

those spores into jars would of shattered my cult confidence.


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OfflineKenetic
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Re: Grr.. what the hell is this? [Re: mushboy]
    #23839697 - 11/16/16 06:24 PM (7 years, 2 months ago)

I've had a bad syringe before.  I made my agar a little dry and put a drop on the center of the plate, and over the next two days or so the excess water disappeared.  It had about 30 cfu's or so but they didn't merge together like yours.  I just transferred as soon as I saw myc.


--------------------
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OfflineTaizzen
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Re: Grr.. what the hell is this? [Re: Levster]
    #23839960 - 11/16/16 08:12 PM (7 years, 2 months ago)

Quote:

Levster said:
and how come this is the first time I've heard of antibiotic agar? Am I that bad at this that I have to use a failsafe method? lol. I followed every instruction to a tee




Here, take a look at this :wink:
Agar sandwich isolation TEK

Keep faith man :yinyangtrip:


--------------------
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OfflineLevster
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Re: Grr.. what the hell is this? [Re: Taizzen]
    #23840000 - 11/16/16 08:35 PM (7 years, 2 months ago)

Can it really be a syringe though? I got 4 from a vendor, and the first 2 I used on a bunch of cakes, now im using another syringe. But I'll let these sit for awhile,

Do you think Myc will appear? even a little eency weency bit, I will extract


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OfflineTaizzen
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Re: Grr.. what the hell is this? [Re: Levster]
    #23840546 - 11/17/16 01:34 AM (7 years, 2 months ago)

Quote:

Levster said:
Can it really be a syringe though? I got 4 from a vendor, and the first 2 I used on a bunch of cakes, now im using another syringe. But I'll let these sit for awhile,

Do you think Myc will appear? even a little eency weency bit, I will extract




My personals petri dishes observations showed me that the quicker you see the mycelium developping the more you can suspect a contamination : mycelium trying to compete with the quicker conta !
In you case you I think you should already would have seen it !
"Do you think Myc will appear ?" <== no :p sorry.

Now, about your pink bacteria :
I remember getting that a lot few years ago when using homemade glovebox.
Without knowing exactly each stage of you preparation and operation, it would be near impossible to determine/pinpoint the exact cause.
I understand you want "just" suggestions of what could have gone wrong and I don't come with much for the moment.
When I was operating inside my glovebox, I remember over-spraying products inside it : bleach...alcool....
So everything in there was so wet that I ususally sterilizate some pièces of cloth at same time than the jars.... so i can wipe a maximum!
I repeat : This was in this period that I saw thoses pinks colors in agar-agar.

No more since I use a laminar flowhood.

Last advice : practice and practice and try to take a walk in a park and force you to think about it : then you should success !

Courage :wink:


--------------------
:takingnotes: My Trade List :leaving:

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OnlineLogicaL ChaosM
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Re: Grr.. what the hell is this? [Re: azur]
    #23840652 - 11/17/16 03:48 AM (7 years, 2 months ago)

Quote:

azur said:
Bacteria. Try antibiotic agar




This is real?!

:mindblown:


--------------------
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OfflineThe Mycologist
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Re: Grr.. what the hell is this? [Re: LogicaL Chaos]
    #23840794 - 11/17/16 06:14 AM (7 years, 2 months ago)

Yeah they use aquarium antibiotics. But it seems a lot of people don't like it.


--------------------
"That you are here—that life exists, and identity;
That the powerful play goes on, and you will contribute a verse.”
― Walt Whitman, Leaves of Grass
:acidfire::tmckenna:


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OnlineLogicaL ChaosM
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Re: Grr.. what the hell is this? [Re: The Mycologist]
    #23841095 - 11/17/16 09:51 AM (7 years, 2 months ago)

Best to not use antibiotic agar huh? I could see that.

I would argue that being super sterile around and with regular agar to prevent infections.


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Re: Grr.. what the hell is this? [Re: LogicaL Chaos]
    #23841104 - 11/17/16 09:56 AM (7 years, 2 months ago)

Not to disagree with Azur, but my thought is if your agar is so bad you need antibiotic agar, you might as well just start over. Sorry to be blunt, but you obviously weren't trying hard enough.


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InvisibleMad Season
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Re: Grr.. what the hell is this? [Re: Moabfighter] * 3
    #23841129 - 11/17/16 10:07 AM (7 years, 2 months ago)

What azur meant was if his spore source is bacterial, use antibacterial agar, and if op did things as right as he says, the bacteria is undoubtedly because of the syringe.

There's many people I know (kizzle especially swears by this method) who either germs or transfers to antibacterial agar at least once in case there's any hidden bacteria growing with the mycelium.


--------------------
contam and car window art
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Improve your sterile techniques! (A comprehensive guide to agar)
Links upon links of literally EVERYTHING UP TO DATE

AMU Q&A
No trees were killed in the sending of this message. However, a large number of electrons were terribly inconvenienced.


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Re: Grr.. what the hell is this? [Re: Mad Season]
    #23841132 - 11/17/16 10:08 AM (7 years, 2 months ago)

Alright I apologize for bad information then. Disregard. Sorry.


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Re: Grr.. what the hell is this? [Re: Moabfighter]
    #23841158 - 11/17/16 10:17 AM (7 years, 2 months ago)

i've gotten plenty of dud syringes, which is what seems to be the case here.  It took a while racking my brain trying to figure out what i was doing wrong before finally realizing that half the syringes i was getting from a certain vendor were dead.  Once i switched to a reliable vendor it was smooth sailing.  I highly recommend sporeworks.


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Re: Grr.. what the hell is this? [Re: crackbaby]
    #23841176 - 11/17/16 10:24 AM (7 years, 2 months ago)

I had an ape syringe that would not put out anything but bacteria. Finally I put it on antibiotic agar and sandwiched it with antibiotic agar. Clean my grew through and I very easily obtained a pure culture.


--------------------


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FALL IN LOVE WITH LC
FOTTSE!!!
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OnlineLogicaL ChaosM
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Re: Grr.. what the hell is this? [Re: Mad Season]
    #23841294 - 11/17/16 11:05 AM (7 years, 2 months ago)

Quote:

Mad Season said:
What azur meant was if his spore source is bacterial, use antibacterial agar, and if op did things as right as he says, the bacteria is undoubtedly because of the syringe.

There's many people I know (kizzle especially swears by this method) who either germs or transfers to antibacterial agar at least once in case there's any hidden bacteria growing with the mycelium.




Im dumbfounded vendors even sell baterial syringes. blows my mind.


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Re: Grr.. what the hell is this? [Re: azur] * 1
    #23841338 - 11/17/16 11:16 AM (7 years, 2 months ago)

Quote:

azur said:
I had an ape syringe that would not put out anything but bacteria. Finally I put it on antibiotic agar and sandwiched it with antibiotic agar. Clean my grew through and I very easily obtained a pure culture.



This...remember to switch to regular agar before going to gran though


--------------------

It doesn't matter what i think of you...all that matters is clean spawn

I'm tired do me a favor


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Re: Grr.. what the hell is this? [Re: cronicr]
    #23841584 - 11/17/16 12:25 PM (7 years, 2 months ago)

Yup. Only use the antibiotic agar to obtain a pure culture. Only use it when battling bacteria, not mold. Once a pure culture is obtained, rocknroll with regular agar


--------------------


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FALL IN LOVE WITH LC
FOTTSE!!!
ALL NOOBS READ THIS!!!



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OnlineLogicaL ChaosM
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Re: Grr.. what the hell is this? [Re: cronicr]
    #23841586 - 11/17/16 12:26 PM (7 years, 2 months ago)

Quote:

cronicr said:
Quote:

azur said:
I had an ape syringe that would not put out anything but bacteria. Finally I put it on antibiotic agar and sandwiched it with antibiotic agar. Clean my grew through and I very easily obtained a pure culture.



This...remember to switch to regular agar before going to gran though




What happens if u dont switch?


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Re: Grr.. what the hell is this? [Re: LogicaL Chaos] * 1
    #23841592 - 11/17/16 12:27 PM (7 years, 2 months ago)

You're wasting money.


--------------------


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Re: Grr.. what the hell is this? [Re: LogicaL Chaos] * 1
    #23841599 - 11/17/16 12:30 PM (7 years, 2 months ago)

Quote:

LogicaL Chaos said:
Quote:

cronicr said:
Quote:

azur said:
I had an ape syringe that would not put out anything but bacteria. Finally I put it on antibiotic agar and sandwiched it with antibiotic agar. Clean my grew through and I very easily obtained a pure culture.



This...remember to switch to regular agar before going to gran though




What happens if u dont switch?



The antibiotis don't kill bacterIA they just stop it so when you go to grain it kicks in again.


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Re: Grr.. what the hell is this? [Re: azur]
    #23841602 - 11/17/16 12:32 PM (7 years, 2 months ago)

and you wont see if your transfers are clean of the bacteria since the bac wouldn't grow on antibac-agar. it would once you put it to grain tho.

edit cron you fuckin type-nerd..  :stoner:


Edited by spacechildo (11/17/16 12:32 PM)


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OnlineLogicaL ChaosM
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Re: Grr.. what the hell is this? [Re: cronicr]
    #23841611 - 11/17/16 12:34 PM (7 years, 2 months ago)

How intriguring.

Interesting....


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Re: Grr.. what the hell is this? [Re: LogicaL Chaos]
    #23841784 - 11/17/16 01:34 PM (7 years, 2 months ago)

Levster:
Pour and sterilize agar, then under your SAB, just open 1 plats like you were going to inoculate but don't !
Close and wait 1 week to see if its agar stays clean.
if you see pink color you'll know that it is not the serynge.
When you new to agar, It is a good idea to have a "witness" agar plate every time you work under your "sterile" setup until you perfect your technic.


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OfflineKenetic
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Re: Grr.. what the hell is this? [Re: Taizzen]
    #23842007 - 11/17/16 02:35 PM (7 years, 2 months ago)

I would also like to add that you can do blank transfers as well to see if you get any contams from your technique.  I'm confident that the agar plate full of bacteria in your post is from the inoculant though.


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OfflineTaizzen
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Re: Grr.. what the hell is this? [Re: Kenetic]
    #23842556 - 11/17/16 04:53 PM (7 years, 2 months ago)

Quote:

kenetic said:
I would also like to add that you can do blank transfers as well to see if you get any contams from your technique.  I'm confident that the agar plate full of bacteria in your post is from the inoculant though.




yeah ! ! blank transfert ! ! :laugh:


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Re: Grr.. what the hell is this? [Re: crackbaby]
    #23870413 - 11/26/16 08:30 PM (7 years, 2 months ago)

edit -- nvm


Edited by Levster (11/26/16 11:33 PM)


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Re: Grr.. what the hell is this? [Re: Levster]
    #23870670 - 11/26/16 10:09 PM (7 years, 2 months ago)

Naming vendors in cultivation threads is against the rules. Please edit your post.


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