BLS 201_CAT_2011

 

BSc. BIOTECHNOLOGY AND LABORATORY SCIENCES

BLS201: CELL AND TISSUE CULTURE TECHNIQUES

TEST     22^nd December, 2011 TIME: 1 Hour

Q1. List the differences between primary cells and continuous cell lines under the following criteria (12 marks):

 

Criteria	Primary cells	Continuous cell lines
Examples	1.	1.
	2.	2.
	3.	3.
Lifespan or passages
Degree of differentiation
Source of cells in the laboratory

 

Q2. List TWO functions of the following in cell culture (12 marks):

Item	Functions
Trypsin	1.
	2.
Foetal bovine serum	1.
	2.
0.45 µm syringe filter	1.
	2.
Water bath	1.
	2.
-20 °C freezer	1.
	2.
Methanol	1.
	2.

 

Q3. An 100 ml cell stock suspension was obtained after trypsinization of confluent cells. 200 μl of the stock cell suspension was then added onto 800 μl of RPMI-1640. From the cell-RPMI-1640 mixture, 400 μl were added onto 100 μl of trypan blue and cells were then added onto a hemocytometer. A total 200 cells (of which 50 stained blue with trypan blue) were seen in three hemocytometer counting grids.

a) Determine the number of cells per ml in the stock cell suspension (5 marks).

b) Determine the cell viability in percentage hemocytometer (5 marks).

c) Determine the number of cells left in the stock cell suspension (5 marks).

d)Determine the volume (in μl) of the stock cell suspension made after trypsinization required to make 10 ml of cell suspension at 1 × 10⁵ cells/ml ((5 marks).

 

Q4. Name seven antibiotics and fungicides that are commonly used in cell culture (7 marks)

 

1
2
3
4
5
6
7

 

5. What would happen if the following mistakes are done while manipulating cells (30 marks)

Cells are split in the presence of foetal bovine serum
Cells are cryopreserved without glycerol
CO2 tank is empty and cell culture medium does not contain HEPES
A nuclear staining is required while cells were fixed using paraformaldehyde
Cells are not mounted with glycerine DABCO after FITC staining
H202 is not included during an IPMA
EDTA is added in cell culture medium
A thermostat is removed from the incubator
A detergent such as Nonidet or Tween 20 is added to cell culture medium
Cells are seeded without counting using a haemocytometer

 

Q6. Briefly explain how you would perform hemadsorption (5 marks)

 

 

Q7. Briefly explain how you would obtain and seed porcine alveolar macrophages (5 marks)

 

 

 

 

 

 

 

Q8. You are provided with the following (7 marks): Anti-human CD4 mouse antibodies

Anti-human CD4 rabbit anibodies

FITC-conjugated goat anti-mouse antibodies HRP-conjugated mouse anti-rabbit antibodies

FITC-conjugated anti-human CD4 mouse antibodies FITC-conjugated anti-mouse CD4 human antibodies Anti-rabbit CD4 mouse antibodies

HRP-conjugated rabbit anti-mouse antibodies Biotinylated anti-mouse CD4 human antibodies Anti-mouse CD4 human antibodies

HRP-conjugated human anti-mouse antibodies Biotinylated anti-human CD4 mouse antibodies Biotinylated anti-rabbit CD4 mouse antibodies

FITC-conjugated human anti-mouse antibodies FITC-conjugated streptavidin Alexa-fluor-conjugated streptavidin

FITC-conjugated biotin

FITC-conjugated mouse anti-goat antibodies

 

Choose the reagents from the list that you would use to stain human CD4+ cells during:

Direct immunofluorescence	1.
Indirect immunofluorescence	1.
	2.
Immunoperoxidase	1.
	2.
Biotin-avidin system	1.
	2.

 

Q9. Briefly explain how would you study attachment of viruses onto cells (2 marks)