NetNotes
Edited by Thomas E. Phillips University of Missouri
phillipst@missouri.edu
Selected postings from the Microscopy Listserver from July 1, 2011 to August 31, 2011. 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: bacteria for SEM I am having some diffi culty with dehydration of my specimens,
and help I could get would be much appreciated. I am working with a Gram-negative, motile, alpha-proteobacteria. My general protocol is as follows: 1) Fix for 15 min. at room temperature with 2.5% glutaraldehyde in a 0.1 M Sodium Cacodylate solution (pH ~7). 2) Dehydrate with an ethanol series—30%, 50%, 70%, 100% (×3), then critical point dry. Unfortunately, my bacteria are coming out wrinkled, shriveled, and just generally improperly dehydrated. I have tried several permutations of this protocol including fi xing at in the fridge for an hour, post-fi xing with 1% osmium tetroxide for 30 min. before the dehydration process, but I have not found anything that has really helped. Does anyone have advice/experience with this sort of preparation? Benjamin Brezler
benjamin.brezler@
gmail.com Mon Jul 11 When I have problems, I buckle down and do what Dennis
Kunkel does (Google his stuff to see beautiful microbes): Your fi x is OK, wash well, postfi x 1% OsO4, wash with cacodylate again, then (I think this is the trick) dehydrate in ethanol, 10%, 20%, 30%, 50%, 70%, 85%, 95%, two times each dilution; fi rst time for 5 min, second time for 15 min. Don’t rush this part. When in 70% ethanol, transfer to fi lter holder (or smooth lens tissue origami packets). Dehydrate in 100% ethanol 3 × 10 min, then critical point dry. Tina (Weatherby) Carvalho
tina@pbrc.hawaii.edu Mon Jul 11 Your fi x seems short to me—I generally use 30–60 minutes in
1% to 1.25% glut in buff er. T e dehydration defi nitely needs expand- ing—especially the lack of 90% and 95% ethanol. What do you mean by “critical point dry”? T at is, what is your exact procedure? You have to think of the CPD in the same way as you do the dehydration. T e ethanol must be replaced within the cells with liquid CO2, which requires fl ushing and soaking cycles, analogous to the dehydration steps used to substitute H2O with ethanol. I generally use 5 × 5 minute soaks when doing bacteria on membranes. If I’m doing cells on agar, I treat them as tissue—the agar has to be dried properly to maintain the relationships between the cells (they’re a community, not just a bunch of bugs). T e time and number of steps varies with the cells, the community (how many diff erent types, what kind of biofi lm, etc.), and is pretty much empirical. Have you tried drying from HMDS (hexamethyldisilazane)? T at can work very nicely. And some bacteria will air dry from 100% ethanol or acetone just fi ne. Phil Oshel
oshel1pe@cmich.edu Tue Jul 12 I do an extended fi xation in glutaraldehyde (1 day) and osmium
(1–2 days), followed by dehydration in ethanol like Tina describes. T en, a long exchange of CO2 in the CPD device, like Phil mentions. John J. Bozzola
bozzola@siu.edu Tue Jul 12 Joining in the chorus of excellent responses you’ve gotten
so far, here are a couple of thoughts: In my hands, short (<4 hrs @ room temperature) fi xations of bacteria can cause later shrinkage problems. Admittedly I usually work with Gram-positives, but even
54
Gram-negative bugs oſt en need at least an overnight fi x. If you’re trying to maintain any extracellular matrix, this problem becomes even more acute—16–24 hrs at room temperature is where I usually start. T ough not usually an issue with single bacterial cells, the osmolality of your fi x is pretty low, too. As other commentators have noted, this ethanol series is short and pretty abrupt: I’ve moved toward more steps rather than fewer (I typically do eight gradations—25% 50% 70% 85% 95% × 2 100% × 2). Even this may not be enough: I'm considering moving to 10 stages (adding an early (~10%) and a late (~90%)) for my most morphologically important specimens. Tina also suggested lengthening the transition times—while it hasn't helped my specifi c preps, it is an excellent point. Overall, I think you may be trying to rush your preps. Good SEM prep takes lots of time (and fi guring out the most effi cient prep for your particular sample oſt en takes even longer). Aaron Barnes
barnesa@umn.edu Tue Jul 12
Specimen Preparation: freezing glutaraldehyde We routinely store small ampoules of our glutaraldehyde in the
freezer. Someone noticed that 4 out of the 20 or so ampoules did not freeze even aſt er 4 months. We fi rst thought they must be diff erent concentrations, but they are all 8%. Anyone know why this might occur? John J. Bozzola
bozzola@siu.edu Wed Jul 20 I do not have the answer but will share a similar observation
with Mowiol mounting medium. I freeze 2 mL aliquots of a batch and store them all in one rack in my -20 C freezer. Invariably some freeze and others don’t. It doesn’t appear to be related to where they are stored in the freezer. Initially I blamed this on technicians who failed to adequately mix the ingredients. But then I made a batch and stirred it for a ridiculous amount of time and still observed the variability. Perhaps the answer to John’s question will also solve my mystery. Tom Phillips
phillipst@missouri.edu Wed Jul 20 Have you checked to see if you have a non-frosting freezer? Some
freezers routinely switch on a thawing cycle to remove ice from the chamber. You can easily tell of you have one or not—if your freezer is fi lled with ice, you don’t have one. If there is no ice build-up, it is because the machine is thawing it on a time schedule. Paul Webster
pwebster@hei.org Wed Jul 20 T is is a good sign! Obviously your solution in the state of a
super-cooled fl uid and is very clean (pure) and without any particles (serving as starters for freezing of ice crystals) just tap hardly on the vial immediately aſt er taking from the freezer and usually it should freeze (get solid) within a second. Peter Heimann peter.heimann@
uni-bielefeld.de T u Jul 21
Specimen Preparation: sodium ethoxide I need to etch some Epon from my blocks, and I intended using sodium ethoxide. Unfortunately, I can’t fi nd the detailed protocol that
doi:10.1017/S1551929511001258
www.microscopy-today.com • 2011 November
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