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I certify that the attached work is entirely my own (or where submitted to meet the requirements
of an approved group assignment is the work of the group), except where work quoted or
paraphrased is acknowledged in the text. I also certify that it has not been submitted for assessment in any other
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I agree that Deakin University may make and retain copies of this work for the purposes of marking and review
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Student Name:___Nguyen My Ha LE Student ID:___220437751____

Student Signature: _____Nguyen My Ha LE____________________Date:_____13/8/2021____________

Demonstrator use only Marks: _______/29

Practical 3 WORKSHEET: Tissue Culture
Answer all the question below.
Activity 1: Micropipetting
1.1 Label pipette diagram using the following: (1.5 marks)

Tip ejector, Shaft, Volumeter display, Push button, Disposable tip (location), Thumbwheel
Push button

Thumbwee
Shaf

Volumeter display

Tip ejector

Disposable
tip
1.2 What is the maximum volume the pictured pipette can transfer? (0.5 marks) 1000µL

1.3 If a 20 – 200 µL micropipette is set to

0 a) How many µL is it set to measure? (0.5 marks) 67 µL
6
7 b) How many mL is this equivalent to? (0.5 marks). 0.067L

1.4 Why should you avoid touching the micropipette tips? (0.5 marks)
To keep it in aseptic condition, not to be contaminated.

1.5 When aspirating liquid, what are the two main factors that influence accurate liquid uptake? (1 mark)

- Using pipette that match volume. The accuracy of pipette will decrease when the volume of discharge is close
to the smallest volume that pipette can tolerate.

- Using the largest volume possible. The use of pipette in large volumes always volume results that are more
accurate than small volumes.

HMM103 T2 Practical #3 Worksheet Page 1

1.6 What happens if you push the plunger to the second stop before drawing up the liquid? (1 mark)

When push the plunger to the second stop before drawing up the liquid, it will put out all
the liquid

Activity 2: Sub-culturing, Cell counting and Calculating

2.1 What is the main reason to have good aseptic techniques when doing cell culturing? (1 mark)

During cell culture, aseptic method or sterile technique is utilised to avoid contamination of sterile media and
equipment.

2.2 What are the main elements in aseptic technique? (2 marks)

Barriers, patient equipment and preparation, environmental controls, contact guidelines

2.3 If you were splitting your cells in the culture flask at a 1:8 dilution, what does this mean? (2 marks)

Cells ready for harvesting 1 to 8 days

2.4 You are provided with 3 images of SKOV3 cells grown at different cell concentrations. Estimate the confluency of the
cells present in each image. (1.5 marks)

Image A B C
Confluency: 90% 50% 10%

2.5 You are given a cell suspension and asked to do a cell count. Use the image below to calculate the cell
concentration. (2 marks)

a) Average number of cells in the grid square:

77

b) Cell concentration:

77 x 2 x 10^3 = 154 x 10^3 = 1.54 x 10^6 cells/ml

HMM103 T2 Practical #3 Worksheet Page 2

 You now need to plate 1 x 103 cells on a glass slide to perform a cell smear.
c) What volume would you need to collect from your cell suspension to plate on the slide?
Volume = 1000/ (1.54 x10^6) = 0.00065 ml = 0.65 µl

2.6 You are now asked to do a cell viability test with a new cell suspension. Cell viability will help determine the
percentage of live and dead cells in a cell suspension. To do a cell viability, you mix equal volumes of the cell
suspension with a blue dye known as Trypan blue. Trypan blue dye stains dead cells blue, while leaving the
live/healthy cells clear.

You have 10 µL of cell suspension to which you add 10 µL of Trypan blue.

Use the image below to answer the questions (3 marks)

a) Average number of cells in the grid square:

54 + 11 = 65 cells

b) Cell concentration:

65 x 2 x 10^4 = 130 x 10^4 = 1.3 x 10^6 cells/ml

c) Percentage of live cells:

54/65 x100 = 83.08%

Activity 3: Cell Smear Staining

Below are images of blood smears, one from a healthy person and the other from a patient that has sickle cell
anaemia. Describe the morphology of the cells you see in the two stains. (4 marks)

Health Sickle cell

anaemia

Red blood cells in a healthy person are consistent in size, measuring around 7-8 m in diameter and lacking a
nucleus. They have a hollow in the middle and are spherical and flat . After dyeing the blood smear, the RBCs
look pink to crimson in hue with a pale core due to the hemoglobin inside.
Because of a mutation in the beta globin chain of haemoglobin, RBCs in sickle cell anemia take on a sickle or
cresent shape rather than being spherical. These cells are unable to transport oxygen. Sickle cell anemia causes
bodies to appear on a blood smear. These bodies are DNA that stains the red blood cell a dark purple color.

HMM103 T2 Practical #3 Worksheet Page 3

Morphology of Different Cell Types
When working with different cell lines, such as different cancer cell line, you need to be able to differentiate
between normal and cancerous cell lines. Use the images below to describe your observation between the
normal cell types and the cancerous cells. (8 marks total)
Cell Type Observations

- Normal breast cells: cells of comparable size are noticed,

and cells are connected to one another, indicating
communication and signal transmission.
- Breast cancer cells: cells of various sizes have been
observed. It indicates that cell development is erratic. There
is no such thing as cell transdiuction. Damaged cells can be
seen ( no appoptosis of cells). Because of the huge size of the
Normal breast cells Breast cancer cells
nucleus, it is possible that cacer cells have a high copy of
chromosome due to mutations.

- Normal colon cells: All cells grow at the same rate and are
of identical size.
- Colon cancer cells: Cell growth is irregular, and as a result
of this irregular growth and the lack of apoptosis, cell
proliferation accelerates, forming tumors , and in the case of
colon cancer, polyps are visible in the colon

Normal colon cells Colon cancer cells

END of Practical #3 Worksheet

HMM103 T2 Practical #3 Worksheet Page 4