BVM Cell culture Assignment

COLLEGE OF VETERINARY MEDICINE AND BIOMEDICAL SCIENCES

DEPARTMENT OF MICROBIOLOGY, PARASITOLOGY AND BIOTECHNOLOGY

BLS 201: TISSUE AND CELL CULTURE TECHNIQUES

BVM Y4

GROUP ASSIGNMENT – MEDIA AND CELL CULTURE 2024

Instructions

1. Each group must attempt all questions
2. Each group will be given one or two questions to present during class hours
3. The presenter will be selected randomly during the day of presentation
4. The presentation will be scored

 

Question 1: Cell culture media and reagents

Discuss the types, composition, and functions of cell culture media and reagents used in virology. Explain how cell culture media is prepared and highlight its significance in viral diagnostics and research in veterinary medicine.

Question 2: Cell culture techniques

Discuss the principles, techniques, and applications of cell and tissue culture in biological research.

Question 3: Cell Count

HELA cells attached to a cell culture flask at 80% confluency were trypsinized, centrifuged, and resuspended in 10 mL of RPMI-1640 containing fetal bovine serum to prepare a stock cell suspension. A 100 µL aliquot of this stock suspension was added to 1900 µL of RPMI-1640 to make a working solution. From this working solution, 300 µL were mixed with 100 µL of trypan blue, and the mixture was loaded onto a hemocytometer.

Using the hemocytometer, the following cell counts (number of cells per square) were observed in four quadrants: 120, 135, 150, and 140 cells.

• a) Calculate the average cell count per square.
• b) Determine the cell concentration in the diluted suspension (cells/mL).
• c) Calculate the total number of cells in the stock suspension.

Question 4: Total Cell Count

A researcher has a T-75 flask of fibroblast cells that are 90% confluent. The cells were trypsinized, and the resulting cell suspension was resuspended in 20 mL of complete DMEM medium to make a stock suspension. A 50 µL aliquot of this stock suspension was diluted in 450 µL of PBS to prepare a 1:10 dilution. From the dilution, 100 µL was mixed with 100 µL of trypan blue and loaded onto a hemocytometer. The following cell counts (number of cells per square) were observed in four quadrants: 85, 90, 88, and 92 cells. Based on this data:

• a) Calculate the average cell count per square.
• b) Determine the cell concentration in the diluted suspension (cells/mL).
• c) Calculate the cell concentration in the stock suspension (cells/mL).
• d) Determine the total number of cells in the T-75 flask.

Question 5: Cell Viability

After preparing a cell suspension from a confluent monolayer of Vero cells, you mix 100 µL of the suspension with 100 µL of trypan blue. The mixture is loaded onto a hemocytometer for counting. The following cell counts are recorded in four quadrants of the hemocytometer:

• Live cells: 100, 120, 110, 130
• Dead cells: 10, 15, 20, 12

The total volume of the cell suspension is 5 mL.

• a) Calculate the percentage of cell viability.
• b) Determine the total number of live cells in the suspension.

Question 6: Cell Seeding Density

A researcher wants to seed a 75 cm² tissue culture flask with a cell density of 5×10⁴ cells/cm². The researcher has a cell suspension with a concentration of 2×10⁶ cells/mL.

• a) How many cells are required to seed the flask at the desired density?
• b) What volume of the cell suspension is needed to seed the flask?
• c) If the researcher only has a 10 mL stock of the cell suspension, will they have enough to seed the flask?

Question 7: Splitting and passaging of cells

You have a cell culture with a concentration of 6×10⁵cells/mL in a 10mL volume. You plan to split this culture at a 1:4 ratio into a new flask.

Calculate:

• a) The number of cells you will transfer into the new flask.
• b) The volume of fresh medium needed to achieve the desired 1:4 split.

Question 8: Multiplicity of Infection (MOI)

You are performing an experiment to infect Vero cells with a virus. The following details are provided:

• • The number of Vero cells in the culture is 5×10⁶.
  • The virus stock has a concentration of 2×10⁸ virions/mL.
  • You wish to infect the culture at a Multiplicity of Infection (MOI) of 1.
• a) Calculate the total number of viral particles required to achieve an MOI of 1.
• b) Determine the volume of the virus stock (in mL) that needs to be added to the cell culture to achieve this MOI.

Question 9: Determining Viral Titer After Infection

A researcher infects a cell culture of 2×10⁶ cells with a virus at an MOI of 0.5. After 24 hours, the supernatant is collected, and a plaque assay is performed to determine the viral titer. The plaque assay reveals that there are 1.2×10⁶ plaque-forming units (PFU) in 1 mL of the supernatant.

• a) Calculate the number of viral particles required to infect the cell culture at the given MOI.
• b) Determine the viral titer in the supernatant in PFU/mL.
• c) Calculate the total number of viral particles that were released into the supernatant.

Question 10: Design of Cell Culture Laboratory

How does the design of a cell culture laboratory impact the efficiency, reproducibility, and overall success of cellular experiments? Discuss key considerations in laboratory layout, equipment selection, and workflow optimization, highlighting the importance of creating an environment that fosters cell viability, experimental consistency, and researcher safety.

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