Exercise 6-1: Yeast UV sensitivity
Expose fission yeast cells to UV light
Each team has been assigned a different fission yeast strain. You will determine
whether the strain you have been assigned is sensitive to ultraviolet light (UV-sensitive).
In addition, if the strain you work with is UV-sensitive, you will determine what UV
dosage results in 50% killing.
Objectives: Learn to
1) spread yeast cells on agar in Petri dish
2) make serial dilutions
3) determine cell density of cultures by using a hemacytometer
4) collect, analyze, and report experimental data
Materials needed
Fission yeast cells (grown overnight at 25°C in YES, then diluted to OD595 = 0.5)
YES agar plates (2 plates per team)
YES media (25 mL)
Pipetman and pipet tips
1.5 ml microfuge tubes
1-mL and 5-mL glass pipets
sterile glass culture tubes
Extra-fine point Sharpie
Aluminum Foil
Glass beads
UV Crosslinker
Black construction paper squares
Tally counter
Hemacytometer with cover slips
Glass bead waste beakers
Pipet tip waste beakers
Preparing the YES agar plates
1.
Using a extra fine-point Sharpie, label 10 YES agar plates with the following
information (remember to label along the inside edge of the bottom plate). Make
sure you keep the writing along the INSIDE EDGE OF THE BOTTOM OF THE
PLATE—keep most of the bottom clear of any markings (except for label below).
Plate 1.
Strain Number
0
date your team name
Plate 2.
Strain Number
0
date your team name
Plate 3.
Strain Number
50
date your team name
Plate 4.
Strain Number
50
date your team name
Plate 5.
Strain Number
100 date your team name
Plate 6.
Strain Number
100 date your team name
Plate 7.
Strain Number
150 date your team name
Plate 8.
Strain Number
150 date your team name
Plate 9.
Strain Number
200 date your team name
Plate 10.
Strain Number
200 date your team name
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Making serial dilutions
1.
Label three 5 mL culture tubes:
1.
10-1
2.
10-2
3.
10-3
2.
Using a 5 mL pipet, carefully pipet 4.5 mL YES media into each culture tube, recapping the tubes.
NOTE: Before taking yeast cells out of a tube or flask, remember to mix the tube.
Yeast cells sink to the bottom of the tube or flask when the tube sits undisturbed.
ADDITIONAL NOTE: If the pipetman you are using does not fit through the neck of the
tube, use a glass pipet to transfer a small volume (1.5 ml) to a sterile 1.5 ml microfuge
tube.
3.
Add 500 µL of your fission yeast strain to Tube 1 (10-1). This is a 10-fold dilution
of your fission yeast culture. Mix the cells.
4.
Transfer 500 µL of the cells in Tube 1 into Tube 2 (10-2). This is a 100-fold
dilution of your fission yeast culture. Mix the cells.
5.
Transfer 500 µL of the cells in Tube 2 into Tube 3 (10-3). This is a 1000-fold
dilution of your fission yeast culture. Mix the cells.
Determine cell density using the hemacytometer
1.
Make sure the hemacytometer and cover slip are CLEAN.
2.
Set the hemacytometer carefully on the benchtop and place the cover slip over
the counting surface. Note that the hemacytometer has TWO counting surfaces.
Figure 1. Typical hemacytometer grid
Add cell volume to
these “triangleshape” troughs on
each edge of the
counting surface
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3.
4.
5.
6.
7.
Select two of the yeast dilutions you will be counting and suspend the cells by
swirling or vortexing. Remember, yeast cells will sink to the bottom of the tube
over time.
Draw up 10 µL of cells from one dilution and dispense the volume to one
hemacytometer counting surface (see Figure 1).
Draw up 10 µl of cells from the other dilution and dispense the volume to the
second hemacytometer counting surface (see Figure 1).
Carefully place the hemacytometer on the microscope stage and focus on one of
the counting surfaces.
Count the number of cells found in the 4 x 4 grid (sometimes 5 x 5 grid on some
hemacytometers) labeled 1, 2, 3, and 4 shown in Figure 1. If there are too many
cells to count, use a higher dilution. For the number to be significant, you total
count for the 4 grid areas must be a minimum ~150 -200 cells.
Table 1: Record of hemacytometer counts
Dilution
Grid 1
Grid 2
Grid 3
Grid 4
TOTAL cells
(n)
Tube 1 (10-1)
Tube 2 (10-2)
Tube 3 (10-3)
Calculation of cell density
Total cells in 4 grids counted = n
The density of your undiluted cell culture in cells/ ml is:
= [n]/4 x 104 x DILUTION FACTOR
Based on your cell density calculation, select the appropriate dilution (Tube 1, 2, or 3)
that is approximately 2000 cells/ml. Alternatively, you can make a fresh dilution in 5 ml
YES media that is 2 x 103 cells/ml. You will be plating cells from this dilution for this
exercise.
Which dilution have you selected to use for this exercise? What is the cell
density of this dilution? Alternatively, if you made a fresh dilution, explain how
you made this dilution. What is the cell density of this dilution?
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Plating yeast cells
Lift the plate lid briefly to add cells or glass beads. Minimize the amount of time that
your plate is left open.
1.
Transfer 200 µL of the 2 x 103 dilution yeast cells onto Plates 1 through 10.
2.
CAREFULLY, add 6 – 10 sterile glass beads to each plate.
3.
Spread cells on plate by moving the beads on the plate in back and forth pattern
to cover the whole surface of the agar. Do not swirl the plate in a circular pattern
only or your cells will only go the perimeter.
4.
Spread the cells until the liquid is absorbed.
5.
CAREFULLY, tip the glass bead to one end of the dish and then pour the beads
into the USED GLASS BEAD container.
Exposing the cells to UV light (Two teams work together for this step)
1.
Place two plates into the UV Crosslinker bottom-side down (each team places
two plates in crosslinker)
2.
Remove the plate lids and place next to the plates.
3.
Turn on the UV crosslinker to expose the cells to UV light.
Plates 3 – 4 Energy setting 50 J/m2
Plates 5 – 6 Energy setting 100 J/m2
Plates 7 – 8 Energy setting 150 J/m2
Plates 9 – 10 Energy setting 200 J/m2
4.
Replace the lids on each plate.
5.
Quickly remove the four plates, tape the 4 plates together (make sure they are all
oriented in the same direction), and immediately wrap the plates with aluminum
foil. Note: tape together plates 1 and 2 and wrap in foil also. Label the stack of
plates with your team name and the date.
6.
Incubate the plates, inverted, at 25°C for several days to allow colony formation.
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