Determining Phage Titer

Materials

• Top Agar (LB-Miller + 0.8% agar)
• LB-MIller (for diluting phage)

• Small test tubes
• Heat block @ 60°C that can hold individual small test tubes

Procedure

1. Microwave flask of Top Agar to melt. Be sure to loosen the cap! For 250 ml of top agar ~15 min at power level 2 works well without any danger of boiling over.
2. Allow agar to cool until handleable, then prepare 4 ml aliquots in small test tubes for as many samples as you will be handling. Place tubes in a heat block or water bath set at 60°C.
3. Prepare dilutions of the phage stock in LB-Miller. Typically a dilution series from 10⁰; to 10¹⁰ in 10² increments will give one plate with a countable number of plaques. These dilutions can be easily prepared by making 1.7 ml Eppendorf tubes with 990 µl LB and transferring 10 µl from the higher stock, mixing, and transferring to the next sample.

1. Optional: Incubate unshaken at 37°C for 30 minutes.
2. Add each sample to a top agar aliquot. Quickly but thoroughly mix by vortexing. You can vortex at full speed so the whirlpool reaches the bottom of the tube without it spilling out of the top. Pour the entire contents of test tube on top of an LB plate and rapidly rotate the plate around so that the agar covers the entire surface evenly before it cools.
3. Let the plates cool ~15-30 minutes at room temperature and then transfer to an incubator at 30°C or 37°C.

Expected Results

	Phage T6 plaques after overnight growth at 37°C of a 106 dilution of lysate. Plaques for most E. coli T-even phages resemble these. They reach a maximum size and then stop spreading. Notice that the top agar was not spread to all places on top of this plate.
	Phage T7 plaques after overnight growth at 37°C of a 104 dilution of a freezer stock. T7 plaques continue to grow. Incubate the plates at 30°C if you want to slow plaque growth.
	Phage T7 plaques after overnight growth at 37°C of a 102 dilution of a freezer stock. The phage has lysed all bacteria in this plate. The remaining colonies (small if they are embedded in the top agar and large if they reach the surface) are derived from mutant bacterial cells that have become resistant to the phage.

Troubleshooting

• Top-agar layer is grainy or clumpy.
  It likely cooled too much before you added phage+cells and poured it.

Example of Calculating Phage Titer

Determining Phage Titer

Materials

• Phage lysate or freezer stock (Procedure: Phage Lysate Preparation)
• Top Agar (LB-Miller + 0.8% agar)
• LB-MIller (for diluting phage)
• Small test tubes
• Heat block @ 60°C that can hold individual small test tubes

Procedure

1. Microwave flask of Top Agar to melt. Be sure to loosen the cap! For 250 ml of top agar ~15 min at power level 2 works well without any danger of boiling over.
2. Allow agar to cool until handleable, then prepare 4 ml aliquots in small test tubes for as many samples as you will be handling. Place tubes in a heat block or water bath set at 60°C.
3. Prepare dilutions of the phage stock in LB-Miller. Typically a dilution series from 10⁰; to 10¹⁰ in 10² increments will give one plate with a countable number of plaques. These dilutions can be easily prepared by making 1.7 ml Eppendorf tubes with 990 µl LB and transferring 10 µl from the higher stock, mixing, and transferring to the next sample.
4. Combine 100 µl of exponentially growing host cells and 100 µl of phage dilution in a fresh Eppendorf. Use exponential phase cells of a sensitive E. coli host strain prepared as described in the Phage Lysate Protocol.
5. Optional: Incubate unshaken at 37°C for 30 minutes.
6. Add each sample to a top agar aliquot. Quickly but thoroughly mix by vortexing. You can vortex at full speed so the whirlpool reaches the bottom of the tube without it spilling out of the top. Pour the entire contents of test tube on top of an LB plate and rapidly rotate the plate around so that the agar covers the entire surface evenly before it cools.
7. Let the plates cool ~15-30 minutes at room temperature and then transfer to an incubator at 30°C or 37°C.

Expected Results

	Phage T6 plaques after overnight growth at 37°C of a 106 dilution of lysate. Plaques for most E. coli T-even phages resemble these. They reach a maximum size and then stop spreading. Notice that the top agar was not spread to all places on top of this plate.
	Phage T7 plaques after overnight growth at 37°C of a 104 dilution of a freezer stock. T7 plaques continue to grow. Incubate the plates at 30°C if you want to slow plaque growth.
	Phage T7 plaques after overnight growth at 37°C of a 102 dilution of a freezer stock. The phage has lysed all bacteria in this plate. The remaining colonies (small if they are embedded in the top agar and large if they reach the surface) are derived from mutant bacterial cells that have become resistant to the phage.

• Top-agar layer is grainy or clumpy.
  It likely cooled too much before you added phage+cells and poured it.

Example of Calculating Phage Titer

Since you are plating 100 µl of each stock you should multiply by 10 and also by the dilution of the tube that gave a countable number of plaques to determine the PFU/ml in the original lysate. So, if there were 25 plaques in the 100 µl you mixed in of the 10⁶ dilution, then the lysate would have 2.5×10⁸ PFU/ml.

Determining Phage Titer

Materials

• Phage lysate or freezer stock (Procedure: Phage Lysate Preparation)
• Top Agar (LB-Miller + 0.8% agar)
• LB-MIller (for diluting phage)
• Small test tubes

Procedure

1. Prepare dilutions of the phage stock in LB-Miller. Typically a dilution series from 10⁰; to 10¹⁰ in 10² increments will give one plate with a countable number of plaques. These dilutions can be easily prepared by making 1.7 ml Eppendorf tubes with 990 µl LB and transferring 10 µl from the higher stock, mixing, and transferring to the next sample.

1. Optional: Incubate unshaken at 37°C for 30 minutes.

1. Let the plates cool ~15-30 minutes at room temperature and then transfer to an incubator at 30°C or 37°C.

Expected Results

Example of Calculating Phage Titer

Since you are plating 100 µl of each stock you should multiply by 10 and also by the dilution of the tube that gave a countable number of plaques to determine the PFU/ml in the original lysate. So, if there were 25 plaques in the 100 µl you mixed in of the 10⁶ dilution, then the lysate would have 2.5×10⁸ PFU/ml.

Determining Phage Titer

Materials

• Phage lysate or freezer stock (Procedure: Phage Lysate Preparation)
• Top Agar (LB-Miller + 0.8% agar)
• LB-MIller (for diluting phage)
• Small test tubes
• Heat block @ 55°C that can hold individual small test tubes

Procedure

1. Microwave flask of Top Agar to melt. Allow to cool until handleable, then prepare 4 ml aliquots in small test tubes for as many samples as you will be handling. Place tubes in a heat block or water bath set at 55-60°C.
2. Prepare dilutions of the phage stock in LB-Miller. Typically a dilution series from 10⁰; to 10¹⁰ in 10² increments will give one plate with a countable number of plaques. These dilutions can be easily prepared by making 1.7 ml Eppendorf tubes with 990 µl LB and transferring 10 µl from the higher stock, mixing, and transferring to the next sample.
3. Combine 100 µl of exponentially growing host cells and 100 µl of phage dilution in a fresh Eppendorf.
4. Optional: Incubate unshaken at 37°C for 30 minutes.
5. Add each sample to a top agar aliquot. Quickly mix by pipetting up and down or gently vortexing. Avoid bubbles. Pour the entire contents of test tube on top of an LB plate and rapidly rotate the plate around so that the agar covers the entire surface evenly before it cools.
6. Let the plates cool ~15-30 minutes at room temperature and then transfer to an incubator at 30°C or 37°C.

Example of Calculating Phage Titer

Since you are plating 100 µl of each stock you should multiply by 10 and also by the dilution of the tube that gave a countable number of plaques to determine the PFU/ml in the original lysate. So, if there were 25 plaques in the 100 µl you mixed in of the 10⁶ dilution, then the lysate would have 2.5×10⁸ PFU/ml.

Determining Phage Titer

Determining Phage Titer

1. Microwave flask of Top Agar (LB + 0.8% agar) to melt. Allow to cool until handleable, then prepare 4 ml aliquots in small test tubes for as many samples as you will be handling. Place tubes in a heat block set at 55-60°C.
2. Prepare dilutions of the phage stock in LB. Typically a dilution series from 10⁰ to 10¹⁰ in 10² increments will give one plate with a countable number of plaques. This can be easily prepared by making 1.7 ml Eppendorf tubes with 990 µl LB and transferring 10 µl from the higher stock, mixing, and transferring to the next sample.
3. Combine 100 µl of exponentially growing host cells and 100 µl of phage dilution. Incubate unshaken at 37°C for 30 minutes.
4. Add each sample to the top agar aliquot. Mix by pipetting up and down to avoid bubbles. Pour entire contents of test tube on top of an LB plate.

-- Main.ColinBrown - 14 Dec 2017