BLS 201_Prob 2010

BLS 201: Cell and Tissue Culture Techniques
Probation/First sitting, September 2010

Question 1:
Swine testicle (ST) cells attached to a cell culture flask at 80% confluency were split
using trypsin and versene in 50 ml cell culture medium. Typsinized cells were
centrifuged at 1500 rpm and the cell pellet was diluted in 10 ml of RPMI-1640 containing
foetal bovine serum to obtain a stock cell suspension. 100 μl of the stock cell suspension
was then added onto 1900 μl of RPMI-1640. From the cell-RPMI-1640 mixture, 300 μl
were added onto 100 μl of trypan blue and cells were then added onto a haemocytometer.
The picture below was seen under the light microscope.

a) Calculate the total number of cells in the stock cell suspension (8 marks)
b) Calculate the volume (in μl) of the stock cell suspension required to make 20 ml
of cell suspension at 5 × 105 cells/ml (8 marks)
c) Explain the function(s) of each of the following reagents used during cell
splitting:
i. Trypsin (1 marks)
ii. Versene (1 marks)
iii. Trypan blue (1 marks)
iv. Foetal bovine serum (1 marks)

Question 2:
You are provided with the following antibodies: FITC-conjugated anti-mouse swine
antibodies, horse raddish peroxidase-conjugated anti-mouse swine antibodies, antiporcine
circovirus mouse antibodies, anti-porcine parvovirus mouse antibodies, FITCconjugated
anti-porcine mouse antibodies, horse raddish peroxidase-conjugated antiporcine
mouse antibodies and streptavidin-conjugated anti-mouse swine antibodies. You
are also given a tissue sample suspension from a pig suspected to be infected with
porcine circovirus, PK-15 cells which supports porcine circovirus replication grown in a
tissue culture flask and a 96-well microtitre plate. Describe how you will perform an
immunoperoxidase monolayer assay (IPMA) to diagnose porcine circovirus infection
assuming that the sample is porcine circovirus positive (20 marks).

Question 3:
Provide the function(s) of the following equipments and reagents in cell culture;
(i) Biosafety cabinet type II, (ii) water bath, (iii) ultracentrifuge, (iv) freezer, (v) liquid
nitrogen container, (vi) autoclave, (vii) light microscope, (viii) microtitre plate, (ix) petri
dish, (x) Nonidet 40, (xi) DMEM, (xii) Sodium pyruvate, (xiii) EGTA, (xiv) HEPES,
(xv) Hoechst, (xvi) Alexa-Fluor 594, (xvii) cell scrapper, (xviii) confocal microscope,
(xix) DMSO and (xx) autoclave (20 marks)

Question 4:
a) Write short notes on the detection of virus infection in cell cultures using direct
immunofluorescence assay (10 marks)
b) List five components of a cell culture medium, and describe the function of each
of the component (10 marks)

Question 5:
a) Provide brief explanation of the following terms as applied in virus titration by
using cell culture systems:
i. Tissue culture infective dose 50 (TCID50) (5 marks)
ii. Plaque forming units per ml (PFU/ml) (5 marks)
b) The following data were obtained during virus titration after inoculating the
monolayer of chicken embryo fibroblasts (CEF) with 0.01ml of the virus:

By using the Reed and Muench method, calculate the TCID₅₀ of the virus (10 marks)