NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from January 1, 2015 to February 28, 2015. Complete listings and subscription information can be obtained at
http://www.microscopy.com. Postings may have been edited to conserve space or for clarity.
Specimen Preparation: tracking samples
I¹m sure this question has been asked many times before, but I wanted to see if people might share their systems for keeping track of TEM samples. In grad school it wasn’t too bad, since I was focused mainly on my own samples, but now I’ve been deluged with samples from many diff erent projects. I’m considering using Evernote in combination with a naming and tracking scheme, but I’m open to other options as well. Steven Spurgeon
steven.spurgeon@pnnl.gov T u Feb 12 From my experience, having good tracking, a naming method and physical storage for samples is key. I would suggest going beyond a simple text fi le, as this becomes unwieldy quickly. A database can be as simple as a spreadsheet, with columns for all the parameters of interest and links to image storage locations. A spreadsheet can be fi ltered and searched and doesn’t require any programming - unless you need to do a real database for PNNL tracking or billing. Set viewing and editing permissions so that only those you trust to enter data properly can make changes. A physical organizer for grid boxes, with names that relate to your database for getting at them later if desired, is really helpful. Larry Scipioni
les@zsgenetics.com Fri Feb 13 We give each project a unique project number, which is incorpo- rated into sample labels, processing records and image records, as well as a project database (currently Filemaker Pro) so every step can be traced back to the project. Ours is prefi xed by an identifying code, followed by the fi nancial year, followed by the project number - i.e., EMLP 14-15.037 T e database includes client name, department (or company), order number, brief description of work, fi nancial details, etc. Each project has a processing protocol and job summary that is fi led in a folder headed with the corresponding project number. T is way, as long as you’ve got the project number, you can refer back to any sample. We also give each image a unique image number for the same reason. Natalie Allcock
nsa2@leicester.ac.uk Fri Feb 13
Specimen Preparation: fi xation of mitochondria
I am very confused about how to go about fi xing a sample of isolated mitochondria for TEM. All the procedures seem to be very vague about “washing the pellet”. To me, this implies re-suspending the pellet in buff er and then spinning it back down. But the procedures I have looked at never mention the g force for this step, but do specify the time. I would assume just use the g force of the last centrifuge step performed, but this step involves addition of agarose solution to enrobe the pellet, so it seems like this would be a distinct and unrelated step. Also, the addition of agarose is never mentioned again, so would I add it to the buff er for the washing steps, assuming washing involves more centrifu- gation? Alternatively, does washing the pellet just mean inundating the pellet with buff er and then pouring it off ? If that is the case, how do I do this for 20 minutes? Do I just let the buff er sit on the pellet for 20 minutes and then pour it off ? Sam Gonzalez
sameesh@uga.edu Mon Jan 26
64
Liquid agarose is added to make processing easier. Processing some samples such as cell suspensions and organelles can be tricky, especially when it a very small quantity, as the majority of the sample gets lost during all the washing steps—plus it can get very hard to pellet them in the thick embedding resins. Adding liquid agarose in the earlier stages—then leaving it to cool so that it sets hard - embeds the samples in a larger, gel like pellet that can then be processed through as if it were a larger sample—without the need for centrifugation at each step. When I process organelles, I usually fi x in primary fi xative, wash in buff er, centrifuge it down into a good pellet, remove as much supernatant as possible, then add a small drop of warm (not too hot) agarose (3%), making sure there is no air bubbles between it and the pellet, leave in a warm water bath for 10 minutes to allow the agarose to infuse with the pellet, then move to the fridge until the agarose is completely set. I then carefully remove the agarose/sample from the tube, and if necessary, use a razor blade to cut it into 1mm 2 pieces for further processing. You should then have some nice cubes of agarose/sample (or maybe just a small smear of sample in the tip if you’re processing organelles or a diffi cult sample) that you can process through without the need for further spinning. Sometimes, despite all my best eff orts, the sample pellet gets leſt behind in the Eppendorf when I remove the set agarose gel. In this instance, I carefully slip the pellet onto a glass slide, and sandwich it between the agarose that way. If anyone has any helpful hints to combat this, it would be much appreciated! Natalie Allcock
nsa2@leicester.ac.uk Tue Jan 27
Microscopy: recycling old EM negatives
A colleague of mine found several boxes of old EM negatives while cleaning out her basement. She contacted me and asked if we could recycle them. It never crossed my mind that these could be recycled but apparently there are hundreds of them and it does seem kind of a waste to just through them out. Does anyone out there no if old EM negatives (Kodak 4489) can be recycled? Greg Hendricks gregory.hendricks@
umassmed.edu Wed Jan 7
Kodak has a document called KES-60, which has a list of
scrap fi lm buyers. T e most recent version I could fi nd in Google was this:
http://www.kodak.co.uk/ek/uploadedFiles/Content/About_ Kodak/Global_Sustainability/Health,_Safety_and_Environment/ HSE_Support_Center/Product_End_of_Life_Management/KES-60_ Scrap_Film_Buyers.pdf. Ben Micklem
ben.micklem@pharm.ox.ac.uk Wed Jan 7 T ere are people around the world that recover silver from both
unexposed fi lm and negatives. Many have gathered together on the http://goldrefi
ningforum.com/ forum. T ere you could probably fi nd someone close-by that can recover the silver from the fi lms. T e process is also described in several places on the forum for anyone interested. Göran Axelsson
axelsson@acc.umu.se Wed Jan 7
doi: 10.1017/S1551929515000188
www.microscopy-today.com • 2015 May
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