BLS 201: Cell and Tissue Culture Techniques 03^rd February, 2011 Continuous Assessment Test

Question 1:

Swine testicle (ST) cells attached to a cell culture flask at 80% confluency were split using trypsin and versene in 100 ml cell culture medium. Typsinized cells were centrifuged at 800 g and the cell pellet was diluted in 5 ml of RPMI-1640 containing foetal bovine serum to obtain a stock cell suspension. 500 µl of the stock cell suspension was then added onto 4500 µl of RPMI-1640. From the cell-RPMI-1640 mixture, 400 µl were added onto 200 µl of trypan blue and cells were then added onto a cell counting chamber. The picture below was seen under the light microscope.

a) Calculate the total number of cells in the stock cell suspension (10 marks)
b) Calculate the volume (in µl) of the stock cell suspension made after trypsinization required to make 20 ml of cell suspension at 5 × 105 cells/ml (12 marks)
c) Explain the function(s) of each of the following reagents used during cell splitting:
i. Trypsin (2 marks)
ii. Versene (2 marks)
iii. Trypan blue (2 marks)
iv. Foetal bovine serum (2 marks)

Question 2:
You are provided with the following antibodies: FITC-conjugated anti-mouse swine antibodies, horse raddish peroxidase-conjugated anti-mouse swine antibodies, anti- porcine circovirus mouse antibodies, anti-porcine parvovirus mouse antibodies, FITC-

conjugated anti-porcine mouse antibodies, horse raddish peroxidase-conjugated anti- porcine mouse antibodies and streptavidin-conjugated anti-mouse swine antibodies. You are also given a 1 ml cryovial containing PK-15 cells which supports porcine circovirus replication from a liquid nitrogen and porcine circovirus virus stock from an ultralow deep freezer. Describe how you will perform an immunoperoxidase monolayer assay (IPMA) in a 96-well microtitre plate (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) inverted 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) cryoware marker (30 marks)

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