[3dem] Vitrobot and BSL 2 virus plunging

Wed Apr 22 09:29:39 PDT 2015

Hi Dez,

Some years ago I used to work with adenovirus. Our protocol was to, at the end of our plunging run, move everything to one side of the BSL hood. Have a couple of biohazard trash bags available and some bleach and ethanol. We used to have one of these surgical mini containers, filled with ethanol and soak everything that was metal or plastic in that for at least 15 mins. Then have a 20% bleach solution (I may be wrong on the %, this was a few years ago), and with paper towels clean the "empty" side of the hood first, where you moved stuff from. The strategy was to wipe clean everything as far as the eye could see and as far as the arm could reach. We'd wipe the sides, the cables, the glass. Everything. And you kept doing this until everything was wiped clean. Dunk the tweezers, Vitrobot plastic caps (for the blotting pads), the tool to close the grid boxes, etc in the surgical ethanol tray. For the Vitrobot, wipe as far as you could reach with ethanol (I think I used to do it twice, just in case). When I got to the side where I'd moved things to, I'd one by one wipe them clean (this could be the pipettor, tip box, etc) and move it to the clean side and continue cleaning until all the hood had been wiped clean.

When I was done, I'd tape the red bad twice and double bag it. Then, I'd take a new bag and take my lab coat off (disposable, of course) and gloves off, and double bag it and tape it. Then we called EHS to schedule a pick up and left the bio hazard trash in a designated place for them pick up. The ethane cup used to be plastic way back when, so that got wiped too (with ethanol).

As far as I remember we never had any contamination issues with adeno, and no user ever got sick. But of course, if you have something more serious than adeno you'll want to be even more careful. Our hood here also has a UV light, so if I remember correctly, we also used to leave it on for a bit (15-30mins?).

When I came back to Vandy I also found that Phoebe's lab had purchased some glutaraldehyde wipes, so that's another option. Strong stuff like bleach, ethanol and cross-linkers or fixatives work well, in my experience.

I wish I'd kept the protocol we wrote. I remember having EHS visit once and Phoebe asking me to write our protocol so it would be approved, but alas, I kept no copies of that with me.

Good luck!

Mariena

Mariena Silvestry Ramos, PhD
Senior Research Specialist - CryoEM Facility
Center for Structural Biology
Vanderbilt University
Medical Center North, Suite RR1207
Nashville, TN  37232-8325
ph. 615-322-4671
________________________________
From: 3dem [3dem-bounces at ncmir.ucsd.edu] on behalf of Benefield, Desiree [DBenefield at morgridge.org]
Sent: Wednesday, April 22, 2015 11:09 AM
To: 3dem at ncmir.ucsd.edu
Subject: [3dem] Vitrobot and BSL 2 virus plunging

Dear colleagues,

I am a post-doc at the University of Wisconsin at Madison. I am also assisting with all things related to biological cryo-EM for our EM facility.

We have some users who would like to use our Vitrobot to freeze cells that are infected with a BSL 2 virus. I am aware that we should do this plunging in a biosafety cabinet/hood but I have yet to find any advice for cleaning the Virobot cabinet post plunge. I was wondering if you might be able to offer any advice? Also, do you know what would be an appropriate way to decontaminate the plunging forceps and ethane cup?

Any advice would be sincerely appreciated.

With many thanks,
Desirée
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