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OfflineCyber
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Glove box contamination question
    #3792123 - 02/17/05 09:05 AM (12 years, 1 month ago)

I built a glove box some time ago. It has had a few mods done to it over time but I still seem to get contaminates in 1 out of 6 agar plates. So how can I improve the box or my technique to reduce contamination?

Lets start with the box.





A standard sterlite container. I used some toilet flanges, cow birthing gloves, and Plexiglas to make it. I have it attached to a HEPA filter that is 1micron @ 99% but that seem to make the agar contamination worse. More recently I have used it with no air flow and have the contamination down to 1 in 6.

Before I use the box I clean it out with 91% alcohol, turn off the air in the room and let it set for a couple of hours. I spray the room, air, and box with Lysol. I wash my hands with 10% Povidone-iodine solution, up to the elbows. Once my hands are in the gloves I put on a second set of rubber gloves (Inside the box) and wash them with alcohol. All tools used are autoclaved in self sealing autoclave bags and not opened until I am ready to use them. (Autoclave, 40min@ 15PSI and 15min dry cycle @180F)



The most common contamination seems to be Green Mold. I know it is airborne.

Does anyone have any suggestions that would bring down the contamination rate?

(Just a NOTE, It just seems to be agar plates that give me the trouble. I have made a bunch of spore syringes in it and NEVER had one contaminate. When an agar plate contaminates it is 1 or 2 dots of green mold on the whole plate!)


Edited by Cyber (02/17/05 09:06 AM)


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Offlinebrewwhaha1
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Re: Glove box question [Re: Cyber]
    #3792135 - 02/17/05 09:11 AM (12 years, 1 month ago)

cant help you on the contamination but a great looking box. toilet flanges are a fantastic idea. not sure if i want to build a box is it really necassary? or can you get away with the warmed open oven door tek? or is that a good idea at all???


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Invisibledobinky
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Re: Glove box contamination question [Re: Cyber]
    #3792563 - 02/17/05 11:27 AM (12 years, 1 month ago)

maybe lysol the room...come back 10 min later...then wipe it with alky....seal...put on the gloves and work from there.....?

the waiting an hour seems to long imo.


--------------------
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OfflineCyber
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Re: Glove box question [Re: brewwhaha1]
    #3797295 - 02/18/05 08:11 AM (12 years, 1 month ago)

Quote:

brewwhaha1 said:
cant help you on the contamination but a great looking box. toilet flanges are a fantastic idea. not sure if i want to build a box is it really necassary? or can you get away with the warmed open oven door tek? or is that a good idea at all???




Glove boxes or flow hoods are needed when you work with agar. They are a good thing when you start making your own syringes


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InvisiblePrisoner#1M
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Re: Glove box contamination question [Re: Cyber]
    #3797554 - 02/18/05 10:21 AM (12 years, 1 month ago)

Quote:

Cyber said:
I have it attached to a HEPA filter that is 1micron @ 99% but that seem to make the agar contamination worse.




a true hepa should be filtering down to .3 microns, 1 micron and a fan will pull
lots of spores into your work. when you open your plates try and keep the open
time as brief as possible, lifting the cover only enough to do the work and seal
it as soon as you've inoculated the plate


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OfflineCyber
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Re: Glove box contamination question [Re: Prisoner#1]
    #3797586 - 02/18/05 10:37 AM (12 years, 1 month ago)

Quote:

Prisoner#1 said:
a true hepa should be filtering down to .3 microns, 1 micron and a fan will pull
lots of spores into your work.





I was starting to realize that as I read more on hepa filters. I may have to change the hepa system completely. From the look of it, it may have to be something I make. almost all commercial hepa systems I find are 1 micron (walmart, target, etc)

Quote:


when you open your plates try and keep the open
time as brief as possible, lifting the cover only enough to do the work and seal
it as soon as you've inoculated the plate




Hmmm. I normally remove the cover and take my time laying the wedge down on the plate. Maybe I should try just tilting the cover and not completely removing it and work on getting the slice in as fast as possible.


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InvisiblePrisoner#1M
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Re: Glove box contamination question [Re: Cyber]
    #3797606 - 02/18/05 10:48 AM (12 years, 1 month ago)

Quote:

Cyber said:
almost all commercial hepa systems I find are 1 micron (walmart, target, etc)






those are not commercial hepa filters, they are labeled as hepa to generate sales, they will remove most dust and many spores but they are simply a pleated
paper filter, an automotive air filter is about the same as those, they do make
a good pre filter for a true hepa

Quote:


Hmmm. I normally remove the cover and take my time laying the wedge down on the plate. Maybe I should try just tilting the cover and not completely removing it and work on getting the slice in as fast as possible.




that may be the other source, go through a few practice runs bbefore you do it
in order to get the motions down, I always lift the lid a little, tilt it to
give me soem working area and then place my work on the agar. I work with a flow
hood and generaly have no contams.


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Offlinethenewguy05
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Re: Glove box contamination question [Re: Prisoner#1]
    #3797674 - 02/18/05 11:23 AM (12 years, 1 month ago)

Run a HEPA air cleaner for a few hours in a closed room to clean the air before doing sterile work. ya know to get the room clean even before the box.


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OfflineBlOoMeRsDeLiGhT
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Re: Glove box contamination question [Re: thenewguy05]
    #3946941 - 03/21/05 01:03 AM (12 years, 10 days ago)

you should use hydrogen peroxide 3%.....use 6mls/liter of agar medium...it fights contaminants until the colonizing mycelium safely breaks it down into oxygen


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OfflineUnderhillmaster
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Re: Glove box contamination question [Re: BlOoMeRsDeLiGhT]
    #3953415 - 03/22/05 02:23 PM (12 years, 8 days ago)

Well, first off, I don't work with agar plates hehe, so take my advice for what its worth. I read Mycotopia a lot along with the Shroomery. And Rodger Rabbit over there is a valuable resource for information imo. Anyway, he has some great points over there about the differences between a 'sterlizer' and a 'sanitizer'. If you are willing to put in a little more time cleaning, I would recommend wiping down all surfaces in your work room with a bleach solution. You could also do what I do with my glovebox before I work. About an hr before I am rdy to start, I fill up my bathtub with water and add a couple cups of bleach. Then I dunk my entire glovebox, let it dry, and clean with alcohol like normal before using. Other than that, I don't really have any suggestions. Efficient hand movement while working and minimal air movement are probably key tho hehe. Keep up the good work and good luck.


--------------------
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OfflineArrow
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Re: Glove box contamination question [Re: Cyber]
    #3953613 - 03/22/05 03:02 PM (12 years, 8 days ago)

how often do you change your filter? i make my own one`s. change them every 5th - 10th use. also spray some Lysol on the outside of the filter.. keep a small spray bottle in the g-box and spray your glove in the box just before you getting started... don't worry about the shit in the air outside the box. if your worried about that, then obviously your filter is no good. good luck.


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InvisibleMrMaddHatter
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Re: Glove box contamination question [Re: Underhillmaster]
    #3953858 - 03/22/05 03:43 PM (12 years, 8 days ago)

Quote:

Underhillmaster said:
Efficient hand movement while working and minimal air movement are probably key tho hehe.




:thumbup:


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