BLS 201_CAT 1_2009

SOKOINE UNIVERSITY OF AGRICULTURE
BLS 201: CELL AND TISSUE CULTURE TECHNIQUES TEST, 2009

Instructor: Time 2 hours. Attempt all questions

Question 1: Cell counting (20 marks)
PK‐15 cells attached to a cell culture flask at 100% confluency were split using trypsin. 50 ml of the
cells suspension after trypsinization was centrifuged at 1500 rpm and the cell pellet was diluted in
10 ml of RPMI‐1640 to obtain a stock cell suspension. 200 μl of the stock cell suspension was then
added onto 1800 μl of RPMI‐1640, and from this cell mixture 300 μl were added onto 100 μl of
trypan blue. The mixture of cells and trypan blue was then added onto a haemocytometer and the
picture below was seen under the light microscope. Calculate the total number of cells in the stock
cell suspension (use all the five haemocytometer chambers in counting).

Question 2: Immunoperoxidase monolayer assay (IPMA) (30 marks)
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 tissue sample suspension from a pig
suspected to be infected with porcine circovirus, PK‐15 cell line which supports porcine circovirus
replication grown in a tissue culture flask and a 96‐well microtitre plate. Describe how you will
perform an IPMA to diagnose porcine circovirus infection assuming that the sample is positive.

Question 3: Cell culture equipment and reagents(15 marks)
Provide the function of the following equipment 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) foetal bovine
serum, (xi) Nonidet 40, (xii) DMEM, (xiii) Sodium pyruvate, (xiv) EGTA and (xv)

Question 4: Fixation (5 marks)
Write short notes on how cells can be fixed.

Question 5: Virus detection (15 marks)
Write short notes on how the following techniques can be used to detect virus infection in cell
cultures; (i) indirect immunofluorescence assay (10 marks) and (ii) hemaglutination assay (5 marks).

Question 6: Cell culture medium (5 marks)
List the components of a cell culture medium, and describe the function of each of the component.

Question 7: Quiz (10 marks)