Aug 07, 2025
Amplifying Bacteriophage
- Madison Altieri1
- 1Bowling Green State University
Protocol Citation: Madison Altieri 2025. Amplifying Bacteriophage. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqpjdovk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 07, 2025
Last Modified: August 07, 2025
Protocol Integer ID: 224266
Keywords: amplifying bacteriophage, amplification phage, phage, liquid culture, spot test
Abstract
This is for the amplification phage by making webbed plates (Path A; ~3days) or by liquid culture (~2days). This protocol also details how to titer phage through a spot test.
Materials
Phage Buffer
- 100mL ddHO
- 2mL 100mM MgSO4 (or 200µL 1M)
- 1mL 500mCaCl2
- 1mL 1M Tris-Cl
- filter sterilize 0.22µm
T-top
- 10g Tryptone
- 8g NaCl
- 1L ddH2O
- Mix and aliquot 100mL into bottles
- Into bottles, put 0.75g agar
- autoclave
T-broth
- 10g Tryptone
- 8g NaCl
- 1L ddH2O
T-plate
- 10g Tryptone
- 8g NaCl
- 15g Agar
- 1L ddH2O
Troubleshooting
Day 1 Path A: Plating Bacteria and Phage to make a webbed plate
15m
Incubate Bacteria and Phage
100µL of overnight culture
100µL of bacteriophage culture
You will need to adjust this accordingly based on the phage titer.
Time of incubation will also be dependent on the phage: ~00:10:00-00:30:00
15m
Making Top agar (With nutrient deficient T-plates)
Make melted agar from T-top, T-broth, and 1M CaCl2
Each plate should have a total of 3mL top agar
- 1:1 ratio of T-top and T-broth and for every 10mL 100µL of CaCl2
- Mix 1M CaCl2 and T-broth
- Add melted T-top
- Ex.) if you were making 4 plates (4 * 3 = 12mL). Meaning you would add 6mL T-top, 6mL T-broth, and ~100µL CaCl2
Take the 200µL phage and bacterial mix and mix with 3mL top agar - pour immediately onto T-plate
Sit at room temperature to solidify
Typically 2-3 plates will give a good titer
Note: make sure that the top agar is not too hot that it will immediately kill the bacteria around 40-60˚C depending on bacteria
Webbed Plate
1d
Day 2 Path A: Scrape Method
15m
Prepare items
- Beaker with ethanol and metal scraping device
- Conical tube with 5mL phage buffer
- 0.22µm filter and syringe
- Sterile conical tube for final filtrate
Scraping
Sterilize the scraper with ethanol and bunsen burner - wait to cool
Physically scrape off the top bacteria and phage from the T-plate and place in the 5mL of phage buffer. If you have more than one plate combine all scrapings into the same 5mL phage buffer
Centrifuge 4˚C 00:10:00 5,000 RPM
- Filter supernatant with 0.22µm
- Store phage at 4˚C
- To freeze phage in -80˚C: 1mL phage + 1mL 50% Glycerol
15m
Day 1 Path B: Liquid Amplification of phage
4h 45m
Liquid Culture
In a test tube:
5mL of desired broth (usually LB)
100µL Bacteria
10µL 1M CaCl2
Incubate until log phase (E. coli ~00:45:00)
45m
Bacteriophage
Add Bacteriophage: the amount you add will depend on your titer. For E. coli lytic phage, 10µL will result in amplified phage.
Note: incubation times after adding bacteriophage vary depending on host bacteria. For E. coli ~04:00:00 will give you titers of 108+ phage.
4h
Filter
Filter the amplified phage with a 0.22µm filter into a sterile conical tube
- Store phage at 4˚C
- To freeze phage in -80˚C: 1mL phage + 1mL 50% Glycerol
Checking Phage Titer
1d
Making Top agar (With nutrient deficient T-plates)
Make melted agar from T-top, T-broth, and 1M CaCl2
Each plate should have a total of 3mL top agar
- 1:1 ratio of T-top and T-broth and for every 10mL 100µL of CaCl2
- Mix 1M CaCl2 and T-broth
- Add melted T-top
- Ex.) if you were making 4 plates (4 * 3 = 12mL). Meaning you would add 6mL T-top, 6mL T-broth, and ~100µL CaCl2
- Take the 100µL host bacteria and mix with 3mL top agar - pour immediately onto T-plate
Sit at room temperature to solidify
Serial Dilution
Perform 10-fold serial dilution of filtered phage out to -8 to -12
Spot 5µL of each serial dilution onto the solidified T-plate
Incubate overnight
Note: if you want to have a more accurate titer, serial dilute and mix onto an entire plate with 100µL host bacteria and 3mL top agar
1d