The cellular division is a biochemical technique
used to separate the different orgánulos and cellular components
for its study. The technique begins with the homogenization. The
weave is crushed in a homogenizing one so that the cells are compressed
and its content is freed. What soon one takes control of that extract
is to put under it different centrifugalizations from different
speeds and times, so that we obtain enriched fractions of the different
We left from 0.5 grams of liver of chicken and a solution TRIS-HCL
mother 1M. We needed for homogeneizado the 10 mililiter TRIS-HCl
solution 10 mm, reason why we took 10 microliters from the solution
mother and filled up with water distilled until 10ml.
In a piston of potter we along with put the 0.5 grams of liver of
chicken 4 mililiter of solution TRIS-HCL 10mM. We crushed until
homogeneizar without it is left no fragment. Soon we passed 1 mililiter
of the extract to 3 tubes eppendorf marked with the word 3000 nucleus
and centrifuged to rpm during 5 minutes.
After this, we praised/poured off the sobrenadante to 3 clean tubes
and we marked with the word mitocondrias to them. Pellet that has
been denominates nuclear fraction and the resuspendemos in 250 microliters
of saccharose-TRIS. We stored this fraction to - 20ºC for later
tests. And we centrifuged 10000 the remaining sobrenadante to rpm
during 10 minutes.
Once finished the centrifugalization, we praised/poured off the
sobrenadante to 3 new tubes eppendorf marked with the word microsomas.
Pellet that we have had left is the mitocondrial fraction and the
resuspendemos in 250 microliters of solution saccharose-TRIS and
we stored - 20ºC. We centrifuged the sobrenadante to 12000
rpm during 25 minutes.
Finalized the centrifugalization, we praised/poured off the sobrenadante
to 3 tubes eppendorf marked with the word citosol and stored - 20ºC.
What we have left in pellet is the microsomal fraction and the resuspendemos
in 250 microliters of solution saccharose-TRIS and we stored.
Now we will observe the nuclear fraction. For it we do frotis with
a drop of the tube marked with nucleus. We threw hematoxilina and
we left to him during 2 minutes. We washed with hot water, we placed
covers and we observed.
Observation of mitocondrias. We took 200 microliters from the mitoncondrial
fraction of the tube marked with mitocondrias and added to 300 microliters
of plug TRIS-HCL and 500 microliters of green Jano 0.1%, so that
we are diluting to the mitocondrial fraction 5 times. We do frotis
and we observed the optical microscope. We see amount of ovoid spheres
and that must correspond to mitocondrias, lisosomas and peroxisomas.
But it is impossible to be able to differentiate the ultrastructure.
In order to make a count of mitocondrias, we make use of hemocitómetro.
We placed a cubreobjeto on the apparatus. This one has two hollows
to the sides of covers, so that the suspención with colorante
in those hollows is injected that soon happens below covers and
the camera by capillarity fills. And we observed the optical microscope
to make count.
Hematocitómetro has under covers a drawn into squares zone,
so that it makes the count easy of the orgánulos. Each grid
represents 0.1 mm cubical, that is to say, 0.0001 cm to fourth.
Soon, the number of mitocondrias by mililiter is equal to the average
of mitocondrias by grids x dilution factor.
The total number of mitocondrias in the aliquot one that we took
would be the number of mitocondrias by mililiter x volume of aliquot.