HUBS 2206 Cells Practical: Cell Counting, Culture Techniques

This practical assignment focuses on cell counting and culture techniques using a haemocytometer and two different cell lines: Jurkat and OI fibroblasts. The experiment involves determining viable cell counts and percentages, comparing the morphology of Jurkat and OI cells, and explaining the detachment and reattachment processes during cell culture. The report includes calculations of viable cell concentration, analysis of cell viability based on blue/non-blue staining, and observations on cell behavior after different incubation periods. The results indicate differences in cell viability between the Jurkat and OI cell lines and highlight the importance of proper cell handling techniques. Desklib provides access to similar solved assignments and study resources for students.

Running Head: CELLS PRACTICAL
Cells Practical
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1CELLS PRACTICAL
1. Haemocytometer is a type of specialised slide filled with liquid of known volume and
contain a counting chamber for cell, which contains grid of perpendicular lines and of
specific dimension. These grid helps in counting the number of cells in a solution of
specific volume. Number of cells present per millilitre of solution is counted
(LeGresley, and Georgina).
2. Jurkat cells lines are immortalized cell line that are derived from blood and OI cell are
derived from fibroblast. Both Jurkat cell and the OI cell line appear differently in to
the culture flask when observed under microscope. Jurkat cells appear spherical and
have a number of projection out from the main part of the cell when under the
microscope whereas OI cell appear flattened. OI cell from skin appears flattened
because it is attached to the surface of the culture flask through the secretion of
extracellular matrix protein, but the Jurkat cells are not attached to the surface of the
culture flask and remain suspended in the solution (Żuryń, et al.,).
3. The cells are detached by treating the cells with an enzyme trypsin. Trypsin is a
protease which will help in breaking the protein which is responsible for the
attachment of cell to the cells culture flask. If the trypsinization is done for long time,
it can lead to cell damage. Hence to stop this process DMEM cell culture media is
added (Shin, et al).
4. The OI cells derived from skin were detached from the culture flask using the trypsin.
When the cell was transferred in to the new flask it again reattaches to the cell surface
through the secretion of extracellular matrix protein as the effect of trypsin is
neutralized using DMEM (Shin, et al).

2CELLS PRACTICAL
18 live cells
1 dead cells
17 live cells
1 dead cells
16 live cells
1 dead cells
20 live cells
2 dead cells
Total cells- 76
Total viable cells- 71
Total dead cells – 5
 Percentage of viable cells- number of viable cells
total number of c ells *100
= 71/ 76*100
= 93.421%
 Percentage of non- viable cells = number of non viable cells
total number of cells *100
= 5/76*100
= 6.57
 Average of viable cells - Totalnumber of viable cells
Total number of squares
= 71/4
= 17.75
 Dilution factor- Final volume
Volume of cells
= 4

3CELLS PRACTICAL
 Viable cell concentration (viable cells per millilitre)
= (average of cells/ ml)*(dilution factor)* 104
= 17.75*4*104
= 7,10,000
5. Blue cells are the dead cells and the non-blue cells are the viable cells (Cadena-
Herrera, et al.,). Proportion of blue to Non-blue cells in Jurkat cell counting done in
the 1st week is 11/61 (0.18). Proportion of blue to Non-blue cells in OI cells derived
from skin done in the 2nd week is 5/71 (0.074). From the result it can be stated that
proportion of blue to non-blue cells is more in Jurkat cell as compared to the OI cell,
which means in jurkat cell more number of dead cell are observed.
From the result it can be explained, OI cells are handled more carefully and
bought to healthy state as compared to Jurkat cell line. After overnight incubation
more number of cells are observed than before.
Figure: Cells after 30 minutes Figure: Cells after overnight incubation
6. The cells when transferred to the new flask after initially seeding it was detached from
surface of the culture flask and appears spherical. After incubating for 30 minutes it
was observed that the cell is again reattached to the surface of the culture flask and
appears flattened as well the solution appear clearer (Cadena-Herrera, et al.,).

4CELLS PRACTICAL
After overnight incubation of the culture, it was observed that the colour of the
media is changed and appear yellowish an along with that more number of cell are
observed. Initially the colour of the media is light blue and also less number of cell
observed.
Figure: Cells after 30 minutes Figure: Cells after overnight incubation

5CELLS PRACTICAL
References
Cadena-Herrera, Daniela, et al. "Validation of three viable-cell counting methods: manual,
semi-automated, and automated." Biotechnology Reports 7 (2015): 9-16.
LeGresley, Murielle, and Georgina McDermott. "Counting chamber methods for quantitative
phytoplankton analysis—haemocytometer, Palmer-Maloney cell and Sedgewick-Rafter cell."
UNESCO (IOC Manuals and Guides) (2010): 25-30.
Shin, Tae Hwan, et al. "Quality and freshness of human bone marrow-derived mesenchymal
stem cells decrease over time after trypsinization and storage in phosphate-buffered saline."
Scientific reports 7.1 (2017): 1106.
Żuryń, Agnieszka, et al. "The influence of arsenic trioxide on the cell cycle, apoptosis and
expression of cyclin D1 in the Jurkat cell line." Acta histochemica 116.8 (2014): 1350-1358.