LIFE 212 Laboratory Report 12 Name: _____________________________
Due: _____________________________
Section: _____________________________
The Hemocytometer
Determining Density and Viability of Cells in Suspension
50 pts total plus Notebook Conclusion (4 pts)
Understanding the cell counting procedure is important for cell culture and virology studies. Accurate cell densities must be determined for successful cell culture of mammalian, plant and insect cell culture. In modern research labs, automated cell counters and/or flow cytometry can be used to measure cell density. This exercise will give you experience in the thinking processes required for simple experimental design and for manual cell counting. Read over the Kendall-Hunt background material. Features of a good experimental hypothesis-
Clearly stated
Testable
Specific
Easily understood
Directions.
1.
Experimental goal: Determine the best treatment that could be used to kill about 50% of mouse 3T3
cells in suspension.
2.
Lightly vortex the cells to mix.
3.
Control cells-
a.
Pipette 20 ul cells into a new tube. b.
Add 20ul trypan blue to the cells.
c.
Set up the hemacytometer with the special rectangular cover slip and add 10 µL of the stain/cell suspension into each groove for loading into both counting chambers. d.
Count 5 squares per counting chamber in both counting chambers. Record the number of clear and blue cells in each square in the table below.
e.
Use the equation below to determine the # cells per square.
4.
Experimental treatment
a.
Choose an experimental treatment. The GTA can advise you on the best approach to take.
i.
Lightly vortex cells. Pipette 100ul cells into new tube.
ii.
Treat with adverse substance expected to kill ~50% of cells.
iii.
Record treatment type in Table below.
iv.
Return to your bench.
b.
After treating your cells-
i.
Pipette 20 ul treated cells into a new tube. ii.
Add 20ul trypan blue to the cells.
iii.
Set up the hemacytometer with the special rectangular cover slip and add 10 µL of the stain/cell suspension into each groove for loading into both counting chambers.
iv.
*** If the treatment did not kill at least 30% of the cells, repeat the treatment or alter the conditions. *** If the treatment killed over 80% of the cells check with the GTA before proceeding.
v.
Once you’ve identified an appropriate treatment, count 5 squares per counting chamber in both counting chambers. Record the number of clear and blue cells in each square in the table below.
c.
Use the equation below to determine the # cells per square.
