Microscopy

Question 1

what does the diaphragm do on a microscope? revolving nose piece?

Answer 1

diaphragm: modifies intensity of light
revolving nose piece: holds objective lens

Question 2

what is kohler illumination?

Answer 2

• provide EVEN illumination of the field of view
• Reduces artifacts (one corner being dark)
• Involves adjusting the field diaphragm while viewing field of view under different objectives/eyepieces

Question 3

which cells do you count on haemocytometer?

Answer 3

4 corner quadrants and center on both chambers (10 total)
- cells on right an top of line ARE counted
- blue cells are dead
- clear cells are live

Question 4

what does trypan blue do?

Answer 4

dyes dead cells

Question 5

how do you calculate cell count?

Answer 5

record number of cells in all 4 chambers and average them

multiply by 250 (to convert to cells/mL) and multiply by 2 (dilution factor bc trypan blue)

this is done separately from dead and live cells

Question 6

how do you calculate cell viability?

Answer 6

1-(# blue cells/#total cells)

Question 7

why do we multiple cell viability by 250?

Answer 7

volume of chamber = 0.004mm^3/cell=0.004mL/cell

take inverse for cells/mL = 250