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Offlinetokingnome
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Registered: 09/25/08
Posts: 1,174
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Last seen: 6 years, 9 months
Glove Box Dilemma
    #10171402 - 04/15/09 12:56 PM (14 years, 11 months ago)

So after innoculating some jars a few days ago and losing half of them to yeast contamination, I've been reviewing my sterilization procedures.

I wiped the inside of my glovebox down with iso alcohol, then gave it a spray of air sanitizer. I let it sit for about 10 minutes. After wiping down the inoculation ports of my jars with iso alcohol, I loaded them in to my glovebox. I then took my spore syringe, flame sterilized the needle, wiped it down with iso alcohol, then laoded that in to my glovebox as well. I then inoculated my jars.

I obviously did something wrong as I got some contamination (Hoping and praying it was just half of the jars as the other ones show signs of mycelium and I don't want to lose them to yeast as well)

After reviewing my procedures, this is what I have come up with. Somewhere in between innoculating the firs half of my jars and the second half, the needle got contaminated somehow. I think it may have been because I did not flame sterilize and wipe down the needle with iso alcohol in between each inoculation.

The problem is though, I don't know how I am going to pull off a flame sterilization inside my glovebox as I insert my hands in to rubber gloves that are sealed off inside the glovebox and I fear using any kind of flame inside there would possibly burn the rubber or any iso alcohol inside the glovebox would be ignited. Would I be safe with just wiping down the needle with iso alcohol in between each inoculation or does somebody have a better method for how I should pull this off?


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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Offlinedead
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Re: Glove Box Dilemma [Re: tokingnome]
    #10171453 - 04/15/09 01:03 PM (14 years, 11 months ago)

Quote:

Would I be safe with just wiping down the needle with iso alcohol in between each inoculation




No, because the needle needs to be sterilized from the inside as well. Flame sterilization is needed and you'll just have to figure out a way.


--------------------
"The third eye. You spend years doing everything you can to open it and then the damn thing opens and your friends laugh at you when you tell them you can see their souls behind their eyes burning like rainbows."

Links:
:regularshroom:Nibin's Guide for Noobs
:regularshroom:some easy teks on bulk & grain prep. (my journal)

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Offlinetokingnome
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Re: Glove Box Dilemma [Re: dead]
    #10171527 - 04/15/09 01:10 PM (14 years, 11 months ago)

Fair enough. Do you think a homemade alcohol lamp with a very small flame would be the solution? That way I can place it inside the glovebox but well away from the gloves.


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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InvisibleFractalXplora
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Re: Glove Box Dilemma [Re: tokingnome]
    #10171613 - 04/15/09 01:23 PM (14 years, 11 months ago)

i just use a lighter in the glove box like a renagade. just let the box sit for 5 mins after dousing with alchol / oust. Never had a problem, even using the lighter "dirty" flame, smeems safer than using a A.lamp in a box i rekon.


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Offlinetokingnome
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Re: Glove Box Dilemma [Re: FractalXplora]
    #10171633 - 04/15/09 01:27 PM (14 years, 11 months ago)

Quote:

FractalXplora said:
i just use a lighter in the glove box like a renagade. just let the box sit for 5 mins after dousing with alchol / oust. Never had a problem, even using the lighter "dirty" flame, smeems safer than using a A.lamp in a box i rekon.




The thing is that I have to light the lighter with rubber gloves on, and the flame will be EXTREMELY close to the gloves and I'm kinda uncomfortable with that. You don't think a low flame alcohol lamp would be a safer method?


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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OfflineNineInchNails
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Re: Glove Box Dilemma [Re: tokingnome]
    #10171761 - 04/15/09 01:52 PM (14 years, 11 months ago)

Rubber gloves will not ignite and set you on fire or anything like that. If you use thin tight gloves then you should have no problem.

Personally I flame sterilize ouside the GB using an alcohol lamp. I then quickly move the needle inside the GB where I wipe it with alcohol. Haven't had a problem yet.

My GB is huge and In don't want a lamp in there. I'll never put a lamp in there. I just don't see the need.

I spray and wipe everything down, place my shit in there, then spray again. Wait for a while then alcohol wipe my hands, utensils, then my hands again. I flame sterilize, bring it inside, then wipe with alcohol.

My GB has holes that I stick my arms in. I use a Tyvek Jacket with rubber gloves on my hands. The gloves go over my sleeves a bit. I even spray Lysol on my sleeves.

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InvisibleBmac420
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Registered: 04/11/09
Posts: 123
Loc: SC
Re: Glove Box Dilemma [Re: NineInchNails]
    #10171771 - 04/15/09 01:55 PM (14 years, 11 months ago)

tyvek jacket where you find them? what did you do cut the arms out?


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

----cledus-----Call me B b mac----

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Offlinehemppy
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Re: Glove Box Dilemma [Re: tokingnome]
    #10171786 - 04/15/09 01:57 PM (14 years, 11 months ago)

You need to pull the gloves out of the holes so you can have freedom of movement. All you are doing is noccing up jars and you don't need a completely sealed box for that. You just need a box with two holes big enough for your arms to fit inside. That way you can take a shot glass and turn it upside down pour in some alcohol and light it. You will be fine going from flame to inside of the box.

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InvisibleBmac420
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Registered: 04/11/09
Posts: 123
Loc: SC
Re: Glove Box Dilemma [Re: hemppy]
    #10171800 - 04/15/09 01:58 PM (14 years, 11 months ago)

it could catch on fire


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

----cledus-----Call me B b mac----

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OfflineBrennus
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Re: Glove Box Dilemma [Re: tokingnome]
    #10171806 - 04/15/09 01:59 PM (14 years, 11 months ago)

Quote:

tokingnome said:
So after innoculating some jars a few days ago and losing half of them to yeast contamination, I've been reviewing my sterilization procedures.

I wiped the inside of my glovebox down with iso alcohol, then gave it a spray of air sanitizer. I let it sit for about 10 minutes. After wiping down the inoculation ports of my jars with iso alcohol, I loaded them in to my glovebox. I then took my spore syringe, flame sterilized the needle, wiped it down with iso alcohol, then laoded that in to my glovebox as well. I then inoculated my jars.

I obviously did something wrong as I got some contamination (Hoping and praying it was just half of the jars as the other ones show signs of mycelium and I don't want to lose them to yeast as well)

After reviewing my procedures, this is what I have come up with. Somewhere in between innoculating the firs half of my jars and the second half, the needle got contaminated somehow. I think it may have been because I did not flame sterilize and wipe down the needle with iso alcohol in between each inoculation.

The problem is though, I don't know how I am going to pull off a flame sterilization inside my glovebox as I insert my hands in to rubber gloves that are sealed off inside the glovebox and I fear using any kind of flame inside there would possibly burn the rubber or any iso alcohol inside the glovebox would be ignited. Would I be safe with just wiping down the needle with iso alcohol in between each inoculation or does somebody have a better method for how I should pull this off?




Never keep a lit alcohol lamp inside a glove box. It's a recipe for disaster.

You need to be keeping your needle over the flame until it burns red-hot and then immediately inoculate. The first drop of solution will cool the needle and allow the rest of your solution to pass without problems.

Are you using latex gloves and tyvek wrist sleeves and wiping those down with alcohol? The excessive cleaning of your glove box is actually unnecessary - glove boxes are NOT intended to be a completely sterile environment; they operate purely on the principal of still air.

Honestly, if you're having problems with yeast, you probably need to review sterilization of your spawn. Are you using grain and sterilizing @ 15 PSI for 90 minutes or are you using PF-style jars?

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Offlinehemppy
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Re: Glove Box Dilemma [Re: hemppy]
    #10171811 - 04/15/09 02:00 PM (14 years, 11 months ago)

You can buy a tyvek paint suit at any big box store that sells paint supplies. I get mine at lowes for about $10

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InvisibleBmac420
B "mac"


Registered: 04/11/09
Posts: 123
Loc: SC
Re: Glove Box Dilemma [Re: hemppy]
    #10171820 - 04/15/09 02:02 PM (14 years, 11 months ago)

ill be damn paint suit do you ware the hole thing lol cause you know i am confined to a wheel chair?


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

----cledus-----Call me B b mac----

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Offlinehemppy
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Re: Glove Box Dilemma [Re: hemppy]
    #10171826 - 04/15/09 02:03 PM (14 years, 11 months ago)

I should clarify. I don't mean put the shot glass inside the box. Leave it outside and flame sterelize there. It will be fine going from flame to inside of the box

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Offlinehemppy
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Re: Glove Box Dilemma [Re: hemppy]
    #10171841 - 04/15/09 02:06 PM (14 years, 11 months ago)

I cut the sleeves off the suit. Then I use a rubber band to hold it in place near my elbow and another rubber band near my wrist then I put on vinyl exam gloves and they cover a bit of the sleeve

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OfflineBrennus
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Re: Glove Box Dilemma [Re: Bmac420]
    #10171854 - 04/15/09 02:08 PM (14 years, 11 months ago)

Quote:

Bmac420 said:
ill be damn paint suit do you ware the hole thing lol cause you know i am confined to a wheel chair?




You don't need an entire tyvek suit - you can buy bundles of tyvek wrist sleeves online (I got a box of 10 pairs off eBay for about $5 last year). You want all exposed skin in the glove box to be covered with tyvek or latex to prevent dead skin cells (we discard thousands and thousands of these daily) from flaking off into your jars. Tyvek is more easily wiped down and sanitized with alcohol compared to shirt sleeves.

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OfflineNineInchNails
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Registered: 03/01/09
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Re: Glove Box Dilemma [Re: Bmac420]
    #10171865 - 04/15/09 02:10 PM (14 years, 11 months ago)

Quote:

Bmac420 said:
tyvek jacket where you find them? what did you do cut the arms out?




Sorry ... I don't know for a fact if it is actually constructed of tyvek, but it is a 'painters suit'. It's actually one of those white semi-disposable coveralls.

I cut the legs off and leave the whole upper half intact (including the zipper). That's why I call it a jacket. My wife laughs at me and calls me a dork every time she sees me put it on :crazy2:

I've been looking all over for a suit with elastic wrists, but I can't find one. The only problem with these suits it that the sleeves keep pulling out from under my damn gloves. I have to tuck it back under there and re-wipe my hands with alcohol. I'm on the search for something like a wrist band to hold the sleeves tight around my wrists.

I get these suits for free so I'll be using them forever (I got the hookup), but the wrists are giving me fits. No contams yet ... knock on wood!

Edited by NineInchNails (04/15/09 02:11 PM)

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Invisibleazshroomer
no1

Registered: 01/07/09
Posts: 282
Re: Glove Box Dilemma [Re: Brennus]
    #10171867 - 04/15/09 02:10 PM (14 years, 11 months ago)

Sounds like your not taking proper measures in preparing your spawn.
Ive done 12 G2G transfers in open air and not a single contam.

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Offlinetokingnome
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Registered: 09/25/08
Posts: 1,174
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Re: Glove Box Dilemma [Re: Brennus]
    #10172703 - 04/15/09 04:52 PM (14 years, 11 months ago)

Quote:

Brennus said:
Quote:

tokingnome said:
So after innoculating some jars a few days ago and losing half of them to yeast contamination, I've been reviewing my sterilization procedures.

I wiped the inside of my glovebox down with iso alcohol, then gave it a spray of air sanitizer. I let it sit for about 10 minutes. After wiping down the inoculation ports of my jars with iso alcohol, I loaded them in to my glovebox. I then took my spore syringe, flame sterilized the needle, wiped it down with iso alcohol, then laoded that in to my glovebox as well. I then inoculated my jars.

I obviously did something wrong as I got some contamination (Hoping and praying it was just half of the jars as the other ones show signs of mycelium and I don't want to lose them to yeast as well)

After reviewing my procedures, this is what I have come up with. Somewhere in between innoculating the firs half of my jars and the second half, the needle got contaminated somehow. I think it may have been because I did not flame sterilize and wipe down the needle with iso alcohol in between each inoculation.

The problem is though, I don't know how I am going to pull off a flame sterilization inside my glovebox as I insert my hands in to rubber gloves that are sealed off inside the glovebox and I fear using any kind of flame inside there would possibly burn the rubber or any iso alcohol inside the glovebox would be ignited. Would I be safe with just wiping down the needle with iso alcohol in between each inoculation or does somebody have a better method for how I should pull this off?




Never keep a lit alcohol lamp inside a glove box. It's a recipe for disaster.

You need to be keeping your needle over the flame until it burns red-hot and then immediately inoculate. The first drop of solution will cool the needle and allow the rest of your solution to pass without problems.

Are you using latex gloves and tyvek wrist sleeves and wiping those down with alcohol? The excessive cleaning of your glove box is actually unnecessary - glove boxes are NOT intended to be a completely sterile environment; they operate purely on the principal of still air.

Honestly, if you're having problems with yeast, you probably need to review sterilization of your spawn. Are you using grain and sterilizing @ 15 PSI for 90 minutes or are you using PF-style jars?




I would think that if I had a problem with preparing my spawn, I would have the same problem in every jar.

I don't know EXACTLY that it's yeast, but from pics I've seen in the contam forum, it looks like it.

My PC's don't have gauges or anything on them so I have to judge by the steam, but I have them sitting on my stove tops at med-hi heat. Again, if it were a PC problem, I would think I would see the contam in each jar as it would be a problem with the grains themselves, not the solution I put in to them. Mind you where I saw the contam in the other jars is exactly where I had squirted in the spore solution.


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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Offlinetokingnome
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Re: Glove Box Dilemma [Re: tokingnome]
    #10172920 - 04/15/09 05:24 PM (14 years, 11 months ago)

Anyone else wish to share their opinions?

If everyone thinks my grain sterilization procedures are wrong, then I don't wanna waste my spores. I PC them for 60 minutes at what should be 15 psi. Based off the PC guide I looked at, 15 psi will give a steady stream of steam coming out of the top. I made sure that there was a heavy flow of steam shooting out of the top, and as I've used this method of sterilizing before and been able to colonize jars, I would think that I just messed up sterilizing my syringe in between innoculations.


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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Offlinedead
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Re: Glove Box Dilemma [Re: tokingnome]
    #10173031 - 04/15/09 05:41 PM (14 years, 11 months ago)

You should PC longer. 75 minutes for pint jars or 90 minutes for quart jars.


--------------------
"The third eye. You spend years doing everything you can to open it and then the damn thing opens and your friends laugh at you when you tell them you can see their souls behind their eyes burning like rainbows."

Links:
:regularshroom:Nibin's Guide for Noobs
:regularshroom:some easy teks on bulk & grain prep. (my journal)

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Offlinetokingnome
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Re: Glove Box Dilemma [Re: tokingnome]
    #10173044 - 04/15/09 05:44 PM (14 years, 11 months ago)

Would it be beneficial for me to PC my jars again and do it for 90 minutes?


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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Offlinedead
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Re: Glove Box Dilemma [Re: tokingnome]
    #10173070 - 04/15/09 05:50 PM (14 years, 11 months ago)

Why not.


--------------------
"The third eye. You spend years doing everything you can to open it and then the damn thing opens and your friends laugh at you when you tell them you can see their souls behind their eyes burning like rainbows."

Links:
:regularshroom:Nibin's Guide for Noobs
:regularshroom:some easy teks on bulk & grain prep. (my journal)

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Offlinetokingnome
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Registered: 09/25/08
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Re: Glove Box Dilemma [Re: tokingnome]
    #10173094 - 04/15/09 05:53 PM (14 years, 11 months ago)

Well one last question, could anyone explain why half of my jars did fine and the others didn't? I'm just confused and curious.

I will go ahead and PC the jars again.


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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Offlinetokingnome
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Re: Glove Box Dilemma [Re: tokingnome]
    #10173378 - 04/15/09 06:43 PM (14 years, 11 months ago)

Anyone? RR? :P


--------------------
Confucius say, "Man who go to bed with itchy butt, wake up with stinky finger." :strokebeard2:

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