GEN 213

CELL BIOLOGY LABORATORY

Experiment 1

CELL COUNTING

SUBMITTED BY ŞEVVAL SARGIN

Course instructor: Dr. ESRA AYDEMİR

Submitted to: ŞEVVAL SARGIN

EXPERIMENT DATE : 03.12.2020

SUBMISSION DATE : 05.12.2020

Biruni University
Istanbul
2020
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TABLE OF CONTENTS

1. OBJECTIVE........................................................................................................................3
2. THEORY.............................................................................................................................3
3. MATERIALS......................................................................................................................5
4. METHOD............................................................................................................................6
5. DISCUSSION......................................................................................................................8

REFERENCES......................................................................................................................10
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1.OBJECTIVE

The aim of the experiment is to distinguish live cells among the suspension cells using the
hemocytometer method and count. Cell counting is done in order to determine an accurate
number of viable cells in a given cell suspension.

2.THEORY
2.1. Cell Counting
This unit presents protocols for counting cells using either a hemacytometer or electronically
using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination
of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject
to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and
easily performed using an electronic counter, but live‐dead discrimination is unreliable. Cell
populations containing large numbers of dead cells and/or cell clumps are difficult to count
accurately. In addition, electronic counting requires resetting of the instrument for cell
populations of different sizes; heterogeneous populations can give rise to inaccurate counts,
and resting and activated cells may require counting at separate settings. In general, electronic
cell counting is best performed on fresh peripheral blood cells.[1]

2.2 Counting Chamber (Hemocytometer)

It provides an overview of hemocytometer counting. Enumeration of cells propagated in vitro

may be conveniently determined by using well-dispersed cell or nuclei suspensions in a
standard hemocytometer chamber. In the method described in the chapter, thick glass
chambers are divided into sections of calibrated area and depth. The total number of cells in a
suspension is then easily calculated from the counts of cells in the hemocytometer chamber.
For the preparation and counting of cell suspensions, cell monolayers are gently dispersed
with enzyme and re-suspended in tissue culture medium. If the resulting cell suspension is too
concentrated, a dilution should be made prior to filling the hemocytometer with the sample.
For the ease and accuracy of counting, chambers should be filled with cell suspensions
containing approximately 30–45 cells/mm2 on the ruled hemocytometer chamber. Dilutions
may be made in a test tube or in a blood dilution pipette. If determination of the viability of
cells is desired, a vital stain may be incorporated into the diluting fluid allowing simultaneous
enumeration of total cells and percentage of viable cells in the suspension.[2] Currently, the
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most widely used method of direct cell count is trypan blue staining followed by microscopic
measurement using a hemocytometer. This technique involves trypsinizing adherent cells,
removing cells from culture, centrifuging and resuspending, staining with trypan blue, and
cell counting. Some cells may not survive the trypsinization step due to degradation of
chromatin by trypsin. The loss of any number of cells is a source of underestimated cell
numbers and makes it difficult to reach a satisfactory cell number. The reliability of the trypan
blue method depends on a uniform, single cell suspension that does not contain clusters,
which will certainly compromise the accuracy of counting.[3]

Figure 1

Figure 2
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Figure 3

3. MATERIALS

 Incubator
 Medium
 Flask
 Tripsin
 T75 Flask
 Distile Water
 Falcon Tube
 Centrifuge
 Trypan Blue
 Hemocytometer
 Micropipette
 Microscope

4. METHOD

Firstly, take the T98G vial containing a brain tumor cell line (a type of adherent cell) from the
incubator and prepare it for observation.After remove dirty media.Wash your vial (cells) with
1X PBS. Shake your bottle and then remove the cleaning residue.Use 1 ml of trypsin
(protease) to cut the bonds between adhering cells and the dish surface (T75).Put the bottle
filled with trypsin into the incubator. Take it after 5 minutes.Wash the vial with 9-10 ml of
medium to deactivate the trypsin.Take the liquid filled with the collection of cells and
medium; then put in a 15 ml falcon tube.Centrifuge under 4 centrifuges at 1500 RPM for 5
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minutes to obtain the cell pellet.After centrifugation, remove some pellet until 3-4 ml remain
to obtain a denser pellet.Use trypan blue for 20 µl and 20 µl cell mix solution to achieve a 1: 2
ratio.Put these two in the hemocytometer for 10 µl - the maximum value of the
hemocytometer.Take the hemocytometer for microscopy. And arrange the microscope to
observe the cells. While observing the cell, consider that the blue stained cells are dead and
not counted.

5. DISCUSSION

There are several sources of inaccuracy;The presence of air bubbles and debris in the
chamber.Overfilling the chamber such that sample runs into the channels or the other
chamber. Incomplete filling of the chamber Cells not evenly distributed throughout the
chamber. Too few cells to count. This can be overcome by centrifuging the cells, re-
suspending in a smaller volume and recounting.Too many cells to count. This can be
overcome by using a higher dilution factor in trypan blue e.g. 1:10.
Also, the suspension should be mixed with unknown. The hemocytometer chamber should be
very clean, do not pay attention to its sterility. Include the cells by touching the upper and left
middle lines. Cells at the bottom and right that touch the middle line are not counted. These
rules should be observed when counting.

REFERENCES

1. Phelan, M. C., & Lawler, G. (1997). Cell counting. Current Protocols in Cytometry,
(1), A-3A.

2. Absher, M. (1973). Hemocytometer counting. In Tissue culture (pp. 395-397).

Academic Press.

3. Felice, D. L., Sun, J., & Liu, R. H. (2009). A modified methylene blue assay for
accurate cell counting. Journal of Functional Foods, 1(1), 109-118.

4. Butler M., Spearman M. (2007) Hücre Sayımı ve Canlılık Ölçümleri. İçinde: Pörtner R.
(eds) Animal Cell Biotechnology. Biyoteknolojide Yöntemler, cilt 24. Humana Press.
https://doi.org/10.1007/978-1-59745-399-8_8 (FIGURE 3)

5. https://rsscience.com/how-to-use-a-hemocytometer-to-count-cells/ (FIGURE 1)