Figuring we were not getting all the water out of the tissue nor getting our resin into the tissue we have tried the following procedures:
1. Cut our root tip sample even smaller.
2. We obtained some Z-6040 which has been mentioned several times on this
list for helping to infiltrate impervious biological specimens. We used it
at 1% as advised by Virginia Lindley's article. But we also continued its
use in the LR White resin as recommended by Stacie Kirsch at EMS.
3. We found we had to extend our Abs. ETOH times to over night and several
days of changes of LR White. We switched to LR White as recommended in
Lindley's article.
At this point, we had some success, but still had some root tips
that wanted to float (even under vacuum). I have not worked with root tips
or mycorrhiza before, but I may be doing more in the future. Do we need to
use acetone or propylene oxide to remove all the water?
Thank-you for any comments or helpful suggestions. If you should
like respond to me at my local address, I shall summarize for the listserver.
Marilee Sellers
E-mail marilee.sellers@nau.edu
>Marilee, A microwave protocol is especially well suited for your work. I >recommend checking the recent article by Giberson RT, Demaree RS, Jr. Microwave >fixation: understanding the variables to achieve rapid reproducible results. >Microsc Res Tech 1995;32: 246-54. > >Gary R. Login, Beth Israel Hospital, Pathology >617-667-2034; glogin@bih.harvard.edu Gary, This is one alternative I hadn't even considered. Thank-you. I do not have a research microwave. But will see if ours would do. Marilee
Marilee Sellers
E-mail marilee.sellers@nau.edu
>Marilee, > >A year or so ago, we were working on a similar sounding project looking >at mycorrhizal associations in pine roots (different pine). We were >interested mainly in the composition of some inclusions but prepared material >for general morphology too. We used a "standard glut/Fa" primary fix and an Os >tet secondary followed by acetone dehydration and embedment in Spurr'Us or >our own variant thereof (VCD and Quetol). We had no problem with infiltration >or sectioning. > >Here's my twenty questions: > >1. Do you use vacuum during fixation? I generally bounce the tissue >during primary fixation by cycling the vacuum until the tissue stays >submerged up to the boiling pressure of the fixative. We have not used vacuum during fixation. Most of the time the tissue did not float until after the Abs. Etoh. We shall include this. >2. Did you try to embed any of the floating material? I generally don't >try to embed "floaters". My thinking is that if they have enough air in them >to float after Os tet density increase, they've got too much air for good >transfer of any thing into or out of the tissue. We did embed some floaters. And they were poorly infiltrated. >3. When do you notice the problem in infiltration? i.e. during >sectioning, or after observation. I've had occasional problems with LR white >bonding to cell walls. Usually we can get enough to hold still for a picture >if we carbon coat them after labeling and staining and before viewing. The >stuff is supposed to be able to handle some water as an impurity, but I'm not >sure I believe it. >4. What kind of knife do you use? I used glass since our root samples came >from the real world and had entrained some sand as well as air. If yours don't >have anything hard in them (or you'Uve got lots of money;-)) might use a >diamond if youUre ot now. I cut a lot of hydroponically grown roots for class >and generally get O.K. sections in regular resin with my diamond knife. Even >they hydroponically grown tips have considerable exhaustible air in them. We notice the problems immediately upon sectioning. These root tips are from sandy soil, and the tips are cleaned as much as possible before processing. As a precaution, we use glass knives to cut them.
John Heckman
heckman@pilot.msu.edu