2. Methods

2.1 Equipment 

Note: All equipment used in this study must be sterilised to prevent contamination.

No.

Equipments

Quantity

1.
UV-sensitive yeast strain (mutant in several DNA repair pathways)

2.
Yeast medium, yeast extract + dextrose (YPD)

3.
Sterile dilution tubes
5
4.
Petri dishes
8
5.
Sterile distilled water
1∫
6.
Micro pipettes (1ml)
6
7.
L-shaped spreaders
8
8.
Disposable gloves
1 pack
9.
Inoculating loops
8
10.
Microwave oven
1
11.
Permanent marker (black)
1
12.
Aluminium foil
1 pack
13.
Stopwatch
1


2.2 Procedure


Phase 1: Pouring the yeast extract peptone dextrose (YPD) Plates


1. Loosen the cap of the YPD agar bottle.
2. Heat the bottle in a microwave until the contents are completely melted.
2.1 Stop the microwave to swirl the bottle every minute to keep the contents from boiling over.

3. Pour the melted YPD agar into 10 petri dishes.
3.1 Cover them immediately after pouring so the dishes remain sterile 
3.2 Always protect your hands with heat-resistant gloves
3.3 Pour agar and ensure the amount is just enough to cover the bottom of each plate
3.4 Let the agar harden at room temperature overnight

Figure 1.1

Phase 2: Streaking the Master Plate


  1. To make sure your work area is clean, wipe the table with 70% alcohol.
  2. Use a pair of disposable gloves to keep the inoculating loops sterile
  3. Touch the loop on an area where you can see yeast growing in the tube.

3.1 The yeast is shipped in a tube containing agar and nutrients
  1. Glide the inoculating loop in a zigzag pattern across one of the YPD plates that was poured previously.
  2. Use a fresh loop (without yeast) to make another zigzag pattern through the first zigzag pattern. The idea is to get separate yeast cells that will grow into well-separated colonies.
  3. Label the plate "Master Plate" with a permanent marker. Mark the date on the plate.
  4. Wrap the plate in aluminium foil to protect it from light.
  5. Allow the yeast to grow for two days at 27° C - 32° C.
Figure 1.2

Phase 3: Labeling the Tubes and Making a Yeast Cell Suspension

  1. Label the five tubes, as follows:
  • 1
  • 1:10
  • 1:100
  • 1:1000
  • 1:10 000
  1. After the two days, put on a pair of disposable gloves and use an inoculating loop to collect a mass of yeast from the master plate. The yeast should be approximately 1 mm in diameter.
  2. Smear the yeast inside the tube labeled "1" toward the bottom on the side of the tube.
  3. Use a 5-m∫ bulb pipette to add 5 m∫ of sterile water to the tube with the yeast.
  4. Shake the tube until the yeast are suspended.

Phase 4: Making Serial Dilutions

Use a new pipette for each transfer below to prevent any contamination.

  1. Use the 3.5m∫ sterile pipette to add 2.25m∫ of sterile water into each of the tubes labeled 1:10, 1:100, 1:1,000 and 1:10,000.
  2. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1 to tube labeled 1:10 and mix thoroughly.
  3. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1:10 to tube labeled 1:100.
  4. Mix thoroughly to ensure the yeasts are evenly spread out in the suspension.
  5. Use a clean 1m∫ pipette to transfer 0.250m∫ of yeast from the tube labeled 1:100 to tube labeled 1:1,000.
  6. Mix thoroughly.
  7. Use a clean 1m∫ pipette to transfer 0.250 m∫ of yeast from the tube labeled 1:1,000 to tube labeled 1:10,000.

Figure 1.3

Phase 5: Spreading the Yeast onto Agar Plates

1. Label the bottoms of four agar plates with the following to prevent mixing up the agar plates when the lids are removed.

  • 1:1000/control
  • 1:1000/exposed
  • 1:10 000/control
  • 1:10 000/exposed
Figure 1.4

Phase 6: Exposing the yeast to UV light

1. Cover all plates with black paper to prevent any UV rays from entering it.
2. Expose the plates labeled "Exposed" to the sunlight.
3. Wrap the plates labeled "Control" in aluminium foil to protect the yeast from light.
4. The time for which the yeast are exposed depends on the time of day. The plates should be positioned so that the sunlight hits the yeast from directly above. Use the guide below:


  • Morning: 3 - 4 minutes
  • Noon: 2 - 3 minutes
  • Mid afternoon: 3 - 4 minutes

5. After exposing the yeast, cover them back with the lids.
6. Wrap the plates in aluminium foil to protect them from light and let them sit at room temperature for two days.

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