6/8/98
Are there any advantages to using cacodylate buffers in fixatives as
opposed to phosphate buffers? Are there any disadvantages in addition to
the toxicity of cacodylate buffers?Specifically, I am working with human
corneas. Thanks for your replies.
Dan Caruso
Medjet Inc.
mdjt-clin@monmouth.com
precipitation of insoluble calcium phosphate.
2. Micro organisms do not grow in cacodylate buffer as they do in
phosphate buffer. So you must prepare fresh phosphate more
frequently...you may also store phosphate buffer in the freezer and thaw
as needed.
Delilah F. Wood
wood@aggie.pw.usda.gov
One would certainly think so. However, I've noticed that 0.2 M caco. buffer,
pH=7.0, after long storage times (I'm looking at a bottle of 100 ml mixed up two
years ago) accumulates a bit of fluffy stuff at the bottom that is a mid-tone
grey in color, but not a solid black. It kinda looks like fungal stuff, like the
stuff that will grow in phosphate buffer after extended times, but I've not
checked into it. I do not use such contaminated buffer in any preps I do.
Has anybody else noticed this, and if so got any idea what it is? Should I add
0.02% sodium azide as a preservative?
Thanks for any insight you can give,
Gib Ahlstrand
There is a short article published in Stain Technology, vol. 55 #3,1980, pp 191-192, that states that the fungus is Penicillium stoloniferum, a common labatory contaminant.
Doug Bray
bray@HG.ULETH.CA
there is an excellent review of the "buffer in EM"-problem in G. Griffith's
book "Fine structure in Immunocytochemistry", pp.57 Springer-Verlag, Berlin,
1993.
Hope that helps.
Heinz
hefeh@Rcs1.urz.tu-dresden.de
it gives a finer grain than phosphate.
Do remember that if you use uranyl acetate en bloc, you need to wash out
the cacodyoate or the phosphate before adding the uranium because it is
not soluble, and small precipitates will make the image very grainy. We
use uranyl acetate in veronal acetate buffer; thus, we wash once in
phosphate buffer after osmium, then twice (15-20 min each wash) with
veronal buffer before adding the stain. Some folks use aqueus uranyl
acetate.
Sara E. Miller, Ph. D.
P. O. Box 3020
Duke University Medical Center
Durham, NC 27710
Ph: 919 684-3452
FAX: 919 684-8735
saram@acpub.duke.edu
minute pinch of sodium azide to prevent unwanted growth in any buffer.
We just throw in a few grains into 1 liter--like about a 1 mm pile on the
end of a small spatula. If you need to measure to check your pile the
first time, the azide should be about 0.002 M.
Remember this stuff is toxic to people as well as bugs, don't eat it!
Sara E. Miller, Ph. D.
P. O. Box 3020
Duke University Medical Center
Durham, NC 27710
Ph: 919 684-3452
FAX: 919 684-8735
saram@acpub.duke.edu
Sometimes, however, no hyphae may be encountered but simply amorphous
material suggesting some chemical reaction (oxidation/reduction) taking
place with the arsenate buffer.
I would hesitate to add azide as it is a potent poison and may affect
ultrastructure - especially of mitochondria. Also, therte are the dangers
of pouring azide down the drain (formation of explosive lead azide with
plumbing solder).
John J. Bozzola
Cacodylate is probably one of the most cytoplasmic extracting
buffers where as pbs or phosphate is better but must be replaced before
osmication. My rule is PBS or phosphate buffers for immuno-EM and
cacodylate for standard fixations. Check out Hyat or Glauret's text
series on fixation/buffers.
MICHAEL DELANNOY
delannoy@welchlink.welch.jhu.edu