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Glovebox Sterilization Routine and Procedures

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#1 Guest_fuzzymonkey_*

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Posted 23 May 2005 - 02:57 PM

Hello all! SWIM has constructed a glovebox and is wondering how the majority of gb users go about their process.

SWIM sprays with a 1:50 bleach spray inside and underneath the lid..
SWIM wipes it all down with paper towels.

Jars, etc. are placed on the inside and sprayed again.
Obviously swim can not spray everything and the rubbermaid lid
does not make for an absolute air-tight seal.

SWIM has had contamination issues before and figures it must be from his workflow.

Pressure cook everything wrapped in foil (spoons, scalpels, etc.)
*shrug* swim is lost.. Any tips would be greatly appreciated.

ps- swim quit using lysol, it just isn't healthy at all..

#2 insight



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Posted 23 May 2005 - 03:08 PM

You mention having contam issues in the past. Was SMIW using BRF or grains? Was this multispore or culture?

#3 sandman


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Posted 23 May 2005 - 03:20 PM

kick it up to 1/10 bleach

#4 Guest_fuzzymonkey_*

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Posted 23 May 2005 - 03:25 PM

SWIM was using grains, 15psi for 90 to 120 minutes.. Multispore generally.
WBS, several brands even. Pre-soaked for 6 to 8 hours, sometimes 24 hours.
Washed the grain heavily to remove scum, sediment.. Drained fairly well swim thinks..
Use polyfill filters, etc.

Tried adding a wetting agent (kodak photo-flo/jet dry, one drop) to the soaking grain..

SWIM's rate of succcess is closer to 40% as of lately..
In the past, SWIM has had 90% using the oven tek and no glove box. (*scratches head)

Possible only difference is that swim and friends lysoled/bleached EVERYTHING, closed windows.. Used the 'ramsey vermiculite seal' for lids..

Some jars don't grow anything for weeks and then they grow black/brown mold.
Some get wet spot, etc. SWIM must live in the filthiest home ever.

#5 Guest_fuzzymonkey_*

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Posted 23 May 2005 - 03:38 PM

Sandman420, I shall try that. In the past, Swim would use a bleach spray that was about as strong as that, if not a little weaker. It also contained a povidone iodine agent which was messy but helped ease Hectors fears.

10:1, 10 parts water to one part bleach, right?

Also, a closed air environment such as a glovebox only really need be sanitized on the surfaces? If not, how would SWIM sanitize the air? (seeming tough without an ionizer)

sorry for the babble, just want to see swim well on his way and not turn out jar after jar of unsafe bacteria/mold

#6 Guest_Peter Cottontail_*

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Posted 23 May 2005 - 03:52 PM

It isn't dirtier than my house, I assure you. I live in a VERY rainy/wet climate, and have black mold growing around the windows and on the sheetrock near the doors and windows. The carpet near the sliding glass door to the balcony(right next to the grow area) constantly grows mold, yet I can get 99.99% success with grain jars, so don't lose hope. You just have to use proper sterile procedure when called for, and get the moisture content of your grains right. I almost never use lysol, and only use bleach after a major trich outbreak in the greenhouse area, and that's just to kill the trich spores.

If nothing grows in your jars, I'd put the first guess on the grains being too wet, which inhibits mycelium and favors bacteria. Mushroom spores should germinate within a week at normal room temperature. If not, something is stopping them.

If your polyfil gets wet, it's a guarantee of bacterial contamination to come. Bacteria is constantly in the air, and billions of them exude from your breath every time you exhale. If the filter material is wet, bacteria will colonize from the outside of the filter through to the inside where it contaminates your grains. I'm not a big fan of wbs, so maybe somebody who uses it successfully will pipe in to help you get your moisture content right. It sounds too wet for sure. Be sure to have something that will block the grains from getting against the polyfil when you shake the jar. If wet grain juice gets on the polyfil, you're screwed.

Your glovebox sounds ok, so I really doubt that's the problem. There is no reason to use lysol or even bleach in a glovebox. A glovebox is not, and needs not be sterile. A glovebox is simply an environment where no air flow exists. The lack of airflow is what keeps the contaminants out of your jars. Don't try to use a flame in your glovebox as the heat produced causes turbulence, and the flame consumes the oxygen, requiring more air to be drawn in, thus bringing in contaminants with it. If the lid doesn't seal perfectly, that's ok too because without a pressure or temperature differential between outside and inside the glovebox, no air will flow through the cracks in the lid. Be sure to wear a surgical mask so you're not breathing through the cracks and getting bacteria in that way, which I doubt is happening.

If the moisture content of your wbs is correct, there might be a possibility the distributor puts fungicide on it to increase the shelf life. I know this is done with feed grains which are usually stored in damp barns. The fungicide keeps spores from germinating, but usually doesn't inhibit live mycelium. Personally, I didn't like wbs because of the different size seeds in it, which have different moisture holding capacity. If one group of seeds are the right moisture, others will be either too wet or too dry. I recommend organic rye berries. Don't give up though, and good luck.

#7 paradox00



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Posted 23 May 2005 - 03:53 PM

I've had some serious problems with contams lately also, even when working in my glovebox. I havn't been able to do grain to grain transfer very successfully or make many syringes.

Remember if you are making your own syringes that could be the source of contamination. I had such a big problem making them that I had to just buy one from a sponser even though I have lots of spore prints around. If you know you have clean syringe, even in a very unclean environment innoculation can be done via oven tek with a good success rate. I gave up using my glovebox for now, after getting so many contams while using it. Although, after just reading the tips roger gave I think I may give it another try.

You can sanitize the air with a new product called Oust available at most grocery stores.

#8 sandman


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Posted 23 May 2005 - 04:09 PM

Another thing.... You need to move very slowly and precisely. Dont skimp and start moving quickly, it will suck air in.

#9 Guest_fuzzymonkey_*

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Posted 23 May 2005 - 05:27 PM

Rodger, swim used wbs due to cost and availability. He could get rye but it is pricey (from a health food store) down here. He does admit that his grains could be a little too wet. When shaking the jars, it does make a nice 'ping noise'.. Swim will _definitely_ try another 'cleaner' brand at least, if not switch to something like millet or rye.

Sandman, slowly? Swim works pretty quickly/forcefully :/ Except for last night when swim had taken a clonazepam.. :)

That could make a lot of sense as his lid does not seal well. He has used plastic tarp and a rubber bungee strap around the diameter of the lid to seal but am not sure if it's even worthwhile.

#10 Guest_Peter Cottontail_*

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Posted 23 May 2005 - 07:56 PM

" He has used plastic tarp and a rubber bungee strap around the diameter of the lid to seal."

There's your answer. He cut off the air at the same time as it had too much water. Mycelium drowns and bacteria thrive.

#11 Guest_fuzzymonkey_*

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Posted 23 May 2005 - 11:36 PM

Rodger, I think that swim meant the 'bungee/tarp sealing' on the lid of the glovebox.
Swim just tried a bunch of g2g's, spore prints and a bit of cloning so he shall get back to me on how everything looks.

Thank you all for the help, i love this place.

#12 highflyer



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Posted 23 May 2005 - 11:58 PM

Ive seen WBS to not colonize very well when starting your culture from spores. It is the slowest out of the grains. Id only use WBS for a secondary the substrate you are transfering an established culture into.

Getting the moisture content of WBS right should be a fairly straight forward process. A 24 hour soak followed by a rinse and 30 minute minimum drain.

Either Lysol or a bleach concentrate in the glovebox should work well.

Id suggest another grain to start your cultures. Possibly using popcorn and then transfering to WBS. I bet that your G2G's will colonize very well with the WBS.

#13 morthos



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Posted 24 May 2005 - 12:36 PM

Oh-Tay! I have some comments for this, and some Q's regarding your grain & jar prep.

First, your grain prep: My wbs/popcorn method has yet to fail me;
1. put wbs in pot, cover with water. boil 10~30min
2. dump into colander.(add a fine wire mesh if stuff gets thru the colander!) rinse excessively
3. put back in pot & cover with water, a lot. boil 5~10min to kill bacteria in water
4. let pot cool off some. cover top with saran-wrap so nobody gets in from the air. refigerate overnight
5. bring to boil. dump in colander & strain 45 min. it is important to boil b4 straining as much more moisture will evaporate from a hot material!!
6. load jars & PC 60 min. my lids have 4 holes around rim, then tyvek, band, and foil. my jars are loaded 4/5 full.
7. tighten & shake jars 1 hour after PC'ing- when they are cool enough to manage. let cool overnight, shake again, and inoculate. shake like your life depended on it -the inoculum must me distributed well for victory.

After inoculation day I don't re-shake my jars. I sit them on a rack and let them have ambient roomlight until fully colonized or time for invitro harvesting. If you skip the boiling before final straining, I can see you having excess moisture problems!

As to your glovebox method: I'm curious how you people use your Lysol exactly. I've built a styrofoam glovebox out of (obviously) a cooler. I cut holes in the front and duct-tape large dishwashing gloves to the holes on both sides. The top has the center cut out, then a pane of glass set in and secured with white glue.

My method is as follows:

1. place 2 layers of paper towel in glovebox to A)make cleaning up easier B)absorb excess moisture/lysol
2. place lysol in corner of box. add in pre-sterilized tools inside sealed lunch baggies including:paper towels, exacto-knives, fork & spoon, rubbing-alcohol soaked paper towel, etc.
3. place jars in box, still covered with original foil. If a bowl in needed, it is placed upside-down in the box. Add in other required items, covered if necessary with foil to protect from Lysol.
4. add lid to box. tape down each corner with electrical tape in case something stupid happens. :) spray lysol on all walls inside box, plus gloves.
5. remove hands, wait 20 min
6. put hands in gloves and get it done. bowls are to be wiped with alcohol-towel. In case of fogging, take dry towels from baggies to absorb it.

The only limitations I have with my glovebox is it's size; I can work with a maximum of 8 500ml jars with an oven bag & tools in there. It is important that no specimens are left exposed to the lysol. You can take your time in there, as no air can get in. Never do wrist-pushups before working in there!:)

While growing all jars must have some method of air exchange, with the debated exeption of the first 2 weeks of PF jars. Failure to breathe can only lead to suffocation. I'm going to toot my own whistle and recommend my double-layer tyvek filter patch design for those that are having problems with contams wicking into the specimens: tyvek layer, metal mesh, tyvek...contams can't reach the other side. Check out my invitro bag threads for more detailed descriptions.

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#14 morthos



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Posted 24 May 2005 - 01:45 PM

Just thought I'd add a few more details:

I don't clean my glovebox; I simply wipe it out. The thing permanently stinks of lysol anyway! I only use bleach for surface sanitizing & bleach dunking. My tools are washed in Sunlight dishsoap water, dried, then wiped with rubbing/iospropyl alcohol before insertion into lunch baggies.

Fuzzymonkey, I'm curious as to your spawning & cloning technique. Please post & we'll see what improvements/suggestions can be made.

#15 vrooota



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Posted 24 May 2005 - 01:54 PM

I've seen GB's with the gloves taped to the holes and not taped. I have a non taped version. Which do those of you who have experience with both prefer? Maybe it makes little difference, I dunno

#16 morthos



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Posted 24 May 2005 - 01:59 PM

I've had to re-tape the gloves once already, after four months use. Can't complain...

#17 wayback



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Posted 24 May 2005 - 08:11 PM

24 hr soak on the seed, but I don't rinse for 30 min like Highflyer. A five minute rinse while constantly moving the seed in a small hole strainer works well. PC 60-75 min, then wipe glove box with alochol, just in case you drop any of the grain while doing transfers on the floor of the box. Also, I spray with a 1/20 bleach solution just prior to closing box for 15-30 before going to work.

Sure, I have some failures this way, but my success rates have gone up with time. Peace.

#18 Guest_fuzzymonkey_*

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Posted 25 May 2005 - 02:20 PM

Morthos, thank you for your well-written reply!
I basically spray the heck out of the glovebox, place everything inside and spray again.. Everything inside gets very wet.

My gloves are hot-glued and silicone-sealed. My g2g's are a success!
And I did minimal gb sterilizing, just a smooth and steady workflow inside the glovebox.

I'm starting to think that you can go completely overboard and hurt your results.

#19 morthos



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Posted 25 May 2005 - 03:36 PM

You're welcome Fuzzymonkey. :)

I have no info on how effective a bleach solution is on sanitizing the glovebox air. I agree with your thoughts on Lysol being unhealthy, but that's only if you let it touch your stuff. If you seal everything in foil or lunch baggies and wait 20 min for the Lysol to settle out of the air, it is no longer a consideration.

Good luck!

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