BSc. BIOTECHNOLOGY AND LABORATORY SCIENCES
BLS201: CELL AND TISSUE CULTURE TECHNIQUES
TEST 22nd 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 |
- 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)