Chromosome counts
Dear all,
I have been trying to get chromosome numbers for a number of
scleractinian coral species, but in each of my preparations I obtain a wide
range of chromosome counts, typically ranging from 24 to 30. The mode of the
distribution seem to indicate in most cases a basic number of 28, but as this
mode represent less than 50% of the observations it is far from being totally
convincing. Here is the protocol I've been using: 10-11 hours old coral embryos
were cultured with colchicine 0.02% in seawater for 2 hours, followed by a
20 minutes 65:35 seawater:tapwater hypotonic treatment. Treated embryos
were then fixed in three changes of freshly mixed, absolute ethanol:50% glacial
acetic acid (1/1, v/v), and refrigerated during storage. To remove lipids prior
to staining, fixed embryos were soaked in diethyl ether for 4-6 hours, then
briefly returned to fixative. Embryos were stained with 2% lacto-aceto-orcein on
a glass slide for 15 minutes, gently rinsed with tapwater, and squashed under a
cover slip. Dehydratation of the quash preparations was retarded for several
days by sealing the cover slip edges with clear nail polish. (from Kenyon,
Evolution 51(3), 1997, pp. 756-767).
I found in the litterature that the same problem occured with
sea anemones (e.g., Fukui, J.mar.biol.Ass. U.K. (1993), 73, 971-973) using
Meredith's method for mammalian tissue (Chromosoma 26 (1969), 254) and in hydra
(Rahat et al, Experientia 41 (1985), 282-283) using also this method
(Pieces of tissue were transfered into a Dryer tube containing 0.5ml 60% acetic
acid. Within 5 minutes the tissue loses its coherence, and a cell suspension
could be formed by aspiration with a finely-drawn pasteur pipette. A drop of
cell suspension was then transferred onto a microscope slide on a hot plate at
about 60oC and immediately withdrawn. The same drop of cell suspension was
applied to the slide in this manner 5-10 times before discarding. The procedure
was repeated until all the fixed material was applied unto the slide. The slides
were places vertically for 10 minutes in a 5% aqueous solution of Giemsa stain,
rinsed with tap water and air-dried.).
I wonder if anybody out there has experienced this kind of
problem doing chromosome counts in any species ; any suggestion is welcome! I
can also send some pictures of the preparations I obtain if it can help to
indentify the problem. I am not an histologist, but a graduate student with a
background in biochemistry trying to get my protocols working with no technical
expertise available in my university...
Thanks in advance for your help!
Jean-Francois Flot
Department of Chemistry, Biology and Marine
Science
University of the Ryukyus
Okinawa, Japan
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