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Cellular division

 

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 orgánulos.
Procedure:
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. 1cm cúbico=1ml.
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.

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