“CELL FRACTIONATION: EXTRACTION, HOMOGENIZATION AND

CENTRIFUGATION”

I. Introduction

Cell fractionation is the process of producing relatively pure fractions of

cellular components. The process involves two basic steps: disruption of the tissue
and lysis of the cells, followed by centrifugation. Although electron microscopy has
allowed the appearance of cell organelles to be studied, elucidation of their function
required a method for separating the different organelles from the remaining cell
components so that their biochemical properties could be studied.

Extraction it is the first step toward isolating any sub-cellular structures. In order
to maintain the biological activity of organelles and bio-molecules, they must be
extracted in mild conditions called cell-free systems. For these, the cells or tissues
are suspended in a solution of appropriate pH and salt content, usually isotonic
sucrose (0.25 mol/L) at0-40°C.

The separation (fractionation) of various components of the homogenate is

carried out by a series of centrifugations in an instrument called preparative
ultracentrifuge. The ultracentrifuge has a metal rotor containing cylindrical holes to
accommodate centrifuge tubes and a motor that spin the rotor at high speed to
generate centrifugal forces. Theodor Svedberg (1926) first developed die
ultracentrifuge which he used to estimate the molecular weight of hemoglobin.

OBJECTIVES:
  Study the cellular structures and organelles of this sample that we’ve used.

MATERIALS

 Jatropha Curcas (sample we’ve used)

 4mL sucrose
 Dropper
 Microscope
 Glass slide
 Cover slip
 Mortar and Pestle
 Paper tissue
 Ice cubes
 Centrifuge
 Plastic test tube

II. METHODOLOGY

First, before we conduct this exercise, the instructor explained the procedure
to easily understand the exercise that we perform and she also explained the
materials that we needed and how we used it in this experiment. After the she
explained the procedure, we do this experiment to be able to seen the organelles
of the sample (Jatropha Curcas) that we’ve used through microscope.

III. RESULT AND DISCUSSION

Chloroplast
Figure 1. Homogenate before
centrifugation
 In the figure 1 shown above the sample of homogenate before centrifugation, in
the 400x high power lens Magnification we’ve seen the chloroplast the green colour in
the picture above.

Tissue Debris

Nuclei

Intact cell

Chloroplast

Figure 2. after 10 minutes of Centrifugation (Nuclear fraction)

In the picture shown below, the homogenate sample undergo centrifugation for
10 minutes, under the 400X high power lens magnification we’ve seen under the
microscope the chloroplast, intact cell, nuclei and tissue debris.
 Lysosome

Mitochondria

Figure 3. after 20 minutes of centrifugation ( Mitochondrial Fraction)

In the picture shown above, the homogenate sample undergo centrifugation for
20 minutes, under the 400x high power lens magnification, we’ve seen under the
microscope in Mitochondrial Fraction only the mitochondria and the Lysosome.

Plasma
membrane

Ribosome
Figure 4. after 60 minutes of centrifugation ( Microsomal Fraction)

In the figure 4 shown, the homogenate sample undergo the centrifugation for 60
minutes, under the 400X high power lens magnification, we’ve seen under the
microscope in Microsomal Fraction is only ribosomes and Plasma membrane.

IV. ANALYSIS AND INTERPRETATION

In the pictures above which is the figure 1 to 4, the activity we do, it was
successful and not totally easy but slight hard. We observe that in the figure 1 which is
the homogenate sample we only have seen the chloroplast under the microscope, in
figure 2, we’ve only seen under the microscope the chloroplast, intact cell, nuclei and
tissue debris with magnification of 400X high power lens, in the figure 3, we’ve seen
under the microscope in Mitochondrial Fraction only the mitochondria and the
Lysosome and the figure 4, we’ve seen under the microscope in Microsomal Fraction is
only ribosomes and Plasma membrane.

IV. CONCLUSION

In this experiment, was successfully done and not easy to do. In all figures above as
you can see in the pictures above all samples homogenate and put in the centrifuge as
we can observe through microscope we also seen in our naked eye the organelles from
the sample we’ve used and easy to recognize the organelles through the help of module
or the procedures

. VI. REFERRENCES

https://www.rachitscellanalogy.weebly.com/ribosomes.html

https://en.m.wikipedia.org/wiki/History_of_cell_membrane_theory