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OfflineMycowlogist
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Glove Box Needle Flaming
    #14378505 - 04/30/11 04:21 PM (12 years, 9 months ago)

Do you use a glove box?  Have you ever done G2G transfers in a glove box?

If you can use a glove box to do G2G transfers why do you have to flame sterilize the needle before innoculating each jar inside the GB?  Shouldn't the first sterilization be sufficient; as long as the needle is kept covered in a alcohol soaked pad.

I would like to understand the reason why? 

Thanks for all your help!


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"And in the end, it's not the years in your life that count. It's the life in your years."  Honest Abe

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OfflineSomeGuy
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14378564 - 04/30/11 04:38 PM (12 years, 9 months ago)

I don't re-sterilize between jars. It's strange, because when I number my jars, and one becomes contaminated, it's usually not the last one, so I inoculate good ones after the contaminated one.:crazy2:It's too much work to sterilize between every jar

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OfflineMycowlogist
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Re: Glove Box Needle Flaming [Re: SomeGuy]
    #14378623 - 04/30/11 04:57 PM (12 years, 9 months ago)

Quote:

paducahovoids said:
I don't re-sterilize between jars. It's strange, because when I number my jars, and one becomes contaminated, it's usually not the last one, so I inoculate good ones after the contaminated one.:crazy2:It's too much work to sterilize between every jar





Thanks.  Would you say that the jar it self was not properly sterilized?  What is your contam ratio?

Great Idea about numbering my jars, I will do that to help me track possible cross contamination.


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"And in the end, it's not the years in your life that count. It's the life in your years."  Honest Abe

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InvisibleDoc_T
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14378646 - 04/30/11 05:03 PM (12 years, 9 months ago)

No need to re-flame the needle unless you touch something other than a sterile jar.
It's just extra security to flame between jars, not a requirement.


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OfflineSomeGuy
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Re: Glove Box Needle Flaming [Re: Doc_T]
    #14378690 - 04/30/11 05:18 PM (12 years, 9 months ago)

my contam ratio varies, with how much beer I've drank:shrug:average of maybe 20%(failure) on pf, and 40% on wbs. I'm not very high tek with it. Occasionally I'll have a whole batch(of 5) go bad and that drives up the ratio. If only one of five goes bad, it always develops near an inoculation site, suggesting to me that possibly I damaged the filter too much or the verm. didn't fall back to cover the hole left by the needle.

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InvisibleDoc_T
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Re: Glove Box Needle Flaming [Re: SomeGuy]
    #14378709 - 04/30/11 05:22 PM (12 years, 9 months ago)

Quote:

paducahovoids said: Occasionally I'll have a whole batch(of 5) go bad and that drives up the ratio.




I count that as "one failure".


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InvisibleDoctor_Inoc
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Re: Glove Box Needle Flaming [Re: Doc_T]
    #14378783 - 04/30/11 05:45 PM (12 years, 9 months ago)

Quote:

If you can use a glove box to do G2G transfers why do you have to flame sterilize the needle before innoculating each jar inside the GB?  Shouldn't the first sterilization be sufficient; as long as the needle is kept covered in a alcohol soaked pad.



There's no needle involved in the process of grain to grain transfers.  I prefer to flame sterilize instead of keeping needles covered with an alcohol soaked paper towel or pad. 

Flame sterilizes the insides of needles where contaminates may possibly be harbored and are safe from alcohol soaked paper towels or pads. 

The alcohol from a soaked paper towel or pad is also prone to make it's way up ito the needle.  This opens vectors for contaminate spores to make their way up the needle and into the solution filled syringe barrel, causing infection.

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OfflineMycowlogist
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Re: Glove Box Needle Flaming [Re: Doctor_Inoc]
    #14379380 - 04/30/11 08:20 PM (12 years, 9 months ago)

"There's no needle involved in the process of grain to grain transfers."  Dr._Innoc, of course there is no Needle needed for G2G transfer.  Logically one would think that a G2G transfer would require more exposure than a needle piercing a inoculation port (obvious). 

Contaminants may make their way into the syringe only if the spawn were improperly prepared.  So, if you can properly prepare your spawn jar, it is not necessary to flame sterilize before each inoculation.  IMO.  But thanks for your feed back!


--------------------
"And in the end, it's not the years in your life that count. It's the life in your years."  Honest Abe

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InvisibleDoctor_Inoc
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14387069 - 05/02/11 06:43 AM (12 years, 9 months ago)

Quote:

Logically one would think that a G2G transfer would require more exposure than a needle piercing a inoculation port (obvious).




Obviously.  However, when contaminates inside of a needle are present upon inoculation, level of exposure won't matter and contamination of the medium inoculated will ensue. 

Quote:

Contaminants may make their way into the syringe only if the spawn were improperly prepared.




Contaminates "making" their way into the syringe and grains being improperly prepared are 2 completely different vectors for contamination.

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InvisibleSillicybin
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14387563 - 05/02/11 09:23 AM (12 years, 9 months ago)

Quote:

Mycowlogist said:
Shouldn't the first sterilization be sufficient; as long as the needle is kept covered in a alcohol soaked pad.

I would like to understand the reason why?




Alcohol does not kill everything all the time.  Especially in porous surfaces like a pad or a cottonball, contaminants can still hide and escape death.  It also takes 10+ minutes of exposure to the alcohol before many bacteria and spores to even be killed (again, in porous surfaces.)  It only takes 3 minutes on hard surfaces, but do you actually expose your needles to alcohol for 3 mins?

Nothing survives a good flame sterilization.  Not even prions.

Iodine (especially in an alcohol solution) is actually a superior antiseptic.

Quote:

Wikipedia said:
The great advantage of iodine antiseptics is their wide scope of antimicrobial activity, killing all principal pathogens and, given enough time, even spores, which are considered to be the most difficult form of microorganisms to be inactivated by disinfectants and antiseptics.



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OfflineMycowlogist
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Re: Glove Box Needle Flaming [Re: Sillicybin]
    #14402358 - 05/04/11 09:44 PM (12 years, 9 months ago)

Nice feedback! 

So technically the time from when the syringe leaves the sterilized grain jar contaminants can crawl unto the needle through the inoculation port, and possibly up the syringe once the needle breaks through. 

Once you fire the syringe do you still cover it with an alcohol pad to cool it?  Wouldn't that technically be cross contamintantion?  If you use a little of the syringe water to cool it, aren't you exposing the "stirile" syringe to an exposed environment?

I am asking questions to better understand the logic.  If Glove boxes can be used with a good success rate doing agar, G2G, Clonning, why do you need to fire the needle?

If it assurance its nice, but it seems unnecessary if not needed.

I am debating between designing a Syringe port in my G.B for just plain ease of use.


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"And in the end, it's not the years in your life that count. It's the life in your years."  Honest Abe

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Offlinear1es
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Re: Glove Box Needle Flaming [Re: SomeGuy]
    #14402973 - 05/04/11 11:39 PM (12 years, 9 months ago)

Quote:

paducahovoids said:
I don't re-sterilize between jars. It's strange, because when I number my jars, and one becomes contaminated, it's usually not the last one, so I inoculate good ones after the contaminated one.:crazy2:It's too much work to sterilize between every jar





man i thought i was lazy

its really easy to sterilize between jars

only take a minute or so


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InvisibleSillicybin
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14403851 - 05/05/11 06:36 AM (12 years, 9 months ago)

Quote:

Mycowlogist said:
Once you fire the syringe do you still cover it with an alcohol pad to cool it?  Wouldn't that technically be cross contamintantion?  If you use a little of the syringe water to cool it, aren't you exposing the "stirile" syringe to an exposed environment?

I am asking questions to better understand the logic.  If Glove boxes can be used with a good success rate doing agar, G2G, Clonning, why do you need to fire the needle?




Well, it might not be considered cross-contamination, but you are going from total sterility to "mostly clean" by swabbing the needle with alcohol.  The problem that I tend to have is that if I do not cool the needle before injecting, the needle will melt a larger hole than needed in my injection port.

Firing on a needle is helpful not only because it achieves 100% sterility, but also because it can kill nasties that have made their way inside the needle.  The syringe liquid does not always have enough surface tension to not drip out of the needle some over time.  The volume those drips left behind is replaced with air, which could have a contaminant.  An alcohol swab would not be able to reach inside the needle to kill contaminants there.

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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14403865 - 05/05/11 06:40 AM (12 years, 9 months ago)

Quote:

Mycowlogist said:
If Glove boxes can be used with a good success rate doing agar, G2G, Clonning, why do you need to fire the needle?




You don't need to- IF you have perfect sterile technique.
Newbs don't have perfect sterile technique, so we tell them to flame.

...and yes, newb, you are a newb. Flame your needle, expel a drop, then shoot.


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OfflineRogerRabbitM
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Re: Glove Box Needle Flaming [Re: Mycowlogist]
    #14403926 - 05/05/11 07:09 AM (12 years, 9 months ago)

Quote:

Mycowlogist said:


Contaminants may make their way into the syringe only if the spawn were improperly prepared.  So, if you can properly prepare your spawn jar, it is not necessary to flame sterilize before each inoculation.  IMO.  But thanks for your feed back!




This is incorrect.  Contaminant spores can and do make their way into the bore of the needle, which to bacteria or mold spores is like a freeway tunnel to a bird.

Also, flame sterilizing between each jar prevents cross contamination.  You should have no more than a 1% fail rate if you'll simply follow proper sterile procedure.
RR


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