> ---------- Forwarded message ----------
> Date: 4 Jan 1995 16:17:29 -0800
> From: S. Hua <u9114317 at MUSS.CIS.MCMASTER.CA>
> To: urodeles at net.bio.net> Subject: axolotl problems
>> Well, to all the fans of the axolotl, I must admit that this is indeed an
> interesting breed. Unfortunately, however, I am experiencing
> difficulties in handling the embryos.
> I am looking for some advice on handling the embryos after I have removed
> the vitelline membrane. I am working in different concentrations of NAM
> and have found that by using this medium, I am having more success in
> removing the membrane without puncturing the actual embryo. This was a
> great improvement over working in the solution that the embryos came
> in. One problem still exists. The ectoderm of the embryo in contact
> with the plastic petri dish is deteriorating, leaving an embryo with a
> yolky bottom and a perfectly good animal hemisphere. This has happened
> over and over again, and I'm wondering whether it's the medium that I'm
> working in.
Some aspect of the typical petri dish's plastic causes the embryo to adhere
in that spot and everytime you try to move them, or over the period they
develop, you get this sticking and then this "mushifying" you described
follows the sticking.
I have three suggestions that have worked very well in our lab.
1) Line your petri dish with sterilized beeswax and make a tiny
depression with the end of a small glass ball the same diameter (but less
than half the height) of the embryo. Dr. Pieter Nieuwkoop taught us this
wonderful classical technique.It works beautifully with no adhering of
the embryo to the dish because of the wax lining. You should be able to
get information and descriptions of this technique from any good
experimental embryology text, especially the older ones. Make sure you
use real beeswax and not parafin as it is too hard. You can get beeswax
at any good art supply store.
2) Child's plasticene clay can also be used to line the dish and
provide a small depression. We have used this extensively and done careful
toxicological work on the black color to ensure it has no effect on the
embryo.If you would like details I'd be happy to forward them.
3) Line your dish with Agar.Agar is somewhat softer than beeswax and is
recommended for culturing pre tail bud stage frog embryos in
"A Manual of Experimental Embryology" by Viktor Hamburger,
Chicago University Press 1960
The main advantage we found to agar is that our microbiology department
upstairs routinely prepares sterile agar plates by the dozens so it is
easy and cheap to get. Use a heated glass rod to melt the depression in
agar or else you'll get jagged edges.
> I've tried using antibiotics (gentamicin), thinking that the
> problem might lie bacteria growing on the plastic petri dish. But this
> proved to not be the problem either.
> My suspicion is that the embryo is faced with the problem of becoming a
> flat glob of cells without the protective vitelline membrane.
Until the neural plate has become visible you do need the vitelline
membrane (as you have observed) to support the embryo's vegetal half.
You should find the problem is solved by providing a cup to sit the
embryo in instead. Good luck and feel free to e-mail me
for more information if you need it.
Natalie K Bjorklund
> I am
> working on lineage studies of cilia and would like to see my embryos
> making it past stage 26-28 (according to Detlaff's table). Please give
> feel free to give me some advice. It would be greatly appreciated, as I
> am feeling really bad about these wasted embryos.
>> Steve Hua (the guy who switched from Necturus to the ever popular
> Ambystoma mexicanum.)
