Pacini at unisi.it
Mon Jan 26 07:15:36 EST 1998
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This is my suggestions.
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here there are two methods that I frequently use for pollen counts in
the anther. The method you have to use depends on the number of pollen
grains per anther. To have an idea you can simply squash a mature anther
in oil and then observe under the microscope. If the pollen grains are
in the order of few hundreds or less (in some cases there are few tens
per anther), you may squash a mature anther in Carlberla's solution and
then directly count all the pollen grains. This is the recipe for
a)5 ml of glicerin
b)10 ml EtOH 95%
c)15 ml distilled water
d) basic fucsin 1% in water
Mix a, b and c; add drop by drop d untill the solution is slightly pink
(DON MAKE THE SOLUTION RED!!, add some drops of glicerine jel. Squash
the mature anther in a drop of Carlberla's solution and count the pollen
grains under the microscope. The exine has a great affinity for basic
fucsin, so pollen grains are well red coloured and the other anther cells
If pollen grains are in the order of thousands you can use always
Carlberla's solution (with the addition of some drops of a detergent) but you
have to operate in an another way.
Put a known volume (V) of the solution in an eppendorf vial and squash a mature
anther with a needle (or better with a glass rod of appropriate size) in the vial.
Sonicate for 1 min. Shake well. Repeat these treatments until no more grains are inside
the anther (the anther shoud be clear, also you can check for pollen grains
under the microscope). When you are sure that no pollen grains are in the
anther, take few µl (v) (5-10) of the solution and put it on a slide and count
the pollen grains. Repeat 2 or three times. If the number of pollen grains
is quite costant, all is OK, otherwise you must be sure that there is an
omogeneus dispersion of pollen grains in the solution. Knowing the number (n)
of pollen grains in this small volume you can calculate the total number
of grains: V/v x n.
Sometimes the problem is to have an appropriate concentration of pollen
grains in the vial, that is to say haw much solution (V) you have to use.
You have only to try!
I hope that this could be usefull for you. Bye.
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