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Preparation of Hemocytometer

This document discusses the technique of hemocytometry, which uses a hemocytometer to count and characterize cells. A hemocytometer is a specialized glass slide with an engraved grid used to count cells under a microscope. The document describes how to prepare a hemocytometer, introduce cell culture samples, count cells within the grid squares, and calculate cell concentration. It also explains how trypan blue vital stain can be used with a hemocytometer to determine cell viability by distinguishing between living and dead cells. The goal is to use hemocytometry to determine the concentration and viability of sample cell cultures.

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Jo Fernandez
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69 views

Preparation of Hemocytometer

This document discusses the technique of hemocytometry, which uses a hemocytometer to count and characterize cells. A hemocytometer is a specialized glass slide with an engraved grid used to count cells under a microscope. The document describes how to prepare a hemocytometer, introduce cell culture samples, count cells within the grid squares, and calculate cell concentration. It also explains how trypan blue vital stain can be used with a hemocytometer to determine cell viability by distinguishing between living and dead cells. The goal is to use hemocytometry to determine the concentration and viability of sample cell cultures.

Uploaded by

Jo Fernandez
Copyright
© © All Rights Reserved
Available Formats
Download as DOCX, PDF, TXT or read online on Scribd
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Introduction

All living things are made up of cells. They


make up the bodies of the simplest to the most
complex organisms including us humans. It is
basically through the specialization of cells that
we are able to function. Cells, therefore, are
fundamental units of life. Study of their
structure, function, and behavior is nothing but
necessary in order to understand the basic ways
of life. (Alberts, et al.,2014) With this need of
understanding, humans developed ways to study
the so-called basic units of life. Microscopes that
were once very simple are now more advanced
hence allowing a wider range of applications.
Different laboratory techniques have also been
developed resulting to inventions that have
helped make life activities simpler.
Hemocytometry is one of the few methods
developed today that help in the quantitation,
identification, and characterization of cells.
(Atala and Lanza, 2002) Particularly, it is a
technique that uses an instrument called
hemocytometer for counting cells. A
hemocytometer is a much thicker modified glass
slide engraved with counting grids of known
areas. Usually, it has cover slip that is also
thicker than the common cover slip to help
easily overcome the surface tension of the cell
culture drop. (Atala and Lanza, 2002) Often
times, a hemocytometer is also used in
determining cell viability using the dye
exclusion method a test that relies on the
ability of living cells to exclude certain stains
from entering the cell membrane. Dead cells will
take up the stain and will therefore be differently
colored from the living ones. (Celis, 2006)
In this experiment, hemocytometry will be
utilized to determine cell concentration and cell
viability of sample cell cultures. It is expected
that by the end of the experiment, knowledge
and understanding of the whole method will be
gained. Proper usage of the hemocytometer,
proper counting techniques, and other laboratory
techniques are also hoped to be obtained.
Materials and Methods
Preparation of hemocytometer
The surface of the hemocytometer and the
coverslip were first cleaned by using 70%
ethanol and Kimwipes. This was done to make
sure that the cells to be introduced will flow
smoothly in the hemocytometer and have an
equal distribution. Extra care was observed since
the cover slip is very fragile. The mounting
supports for the coverslip were then moistened
to prevent the coverslip from sliding and
moving. After pressing both sides of the
mounted coverslip, cell culture was prepared.
Fifteen L of the culture was first obtained using
a micropipette and was then introduced into one
of the counting chambers of the hemocytometer.
Such volume was used to ensure that no liquid
will overflow and that the grid will be instantly
covered through capillary action.
Obtaining cell concentration
The hemocytometer was put on the stage of the
light microscope. The counting grid was focused
at a low power magnification then the four
corner squares located to view the 16 smaller
squares found within each corner square. Cells
within each corner square were later counted.
Cells that were on or were touching the left and
top lines were counted while those on or
touching the right and bottom lines were
ignored. This was done to avoid doubling or
overestimation of count. After manually
counting the cells on the four corner squares,
cell concentration per milliliter was determined
using the following equation:



Determining cell growth with vital stain
As for the next part of the exercise, two flasks
(one treated, the other not) labeled Flask B and
Flask C were compared. One hundred and fifty
microliters of the cell cultrure from each flask
were pipetted and placed in two separate
microcentrifuge tubes. Seventy five microliters
of the vital stain Trypan Blue were later added to
each of the microcentrifuge tubes. Fifteen L of
both solutions were introduced into a
hemocytometer for cell counting. Due to the
presence of the vital stain, two kinds of cells
were observed. Some were colored blue while
some remained colorless suggesting difference
in viability. The number of viable cells and non-
vialble cells in the two solutions were then
compared. Percent viabilty and non-viability of
each flask were later determined through data
manipulation.

References
Alberts, B., Bray, D., Hopkin, K., Johnson, A.,
Lewis, J., Raff, M., . . . Walter, P.
(2014).Essential Cell Biology (4th ed.). NY:
Garland Science. pp. 1-2
Atala, A., & Lanza, R. P. (2002). Methods of
tissue engineering. San Diego, CA: Academic
Press. pp. 55-56
Celis, J. E. (2006). Cell biology: A laboratory
handbook. Amsterdam: Elsevier Academic. pp.
21-22

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