Oct 31, 2025
Isolating bacteriophage from environmental samples
- Madison Altieri1
- 1Bowling Green State University
Protocol Citation: Madison Altieri 2025. Isolating bacteriophage from environmental samples. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmb19bg3p/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: October 30, 2025
Last Modified: October 31, 2025
Protocol Integer ID: 231184
Keywords: isolating bacteriophage, bacteriophage from environmental sample, lytic bacteriophage, environmental sample, method, dirty method, right method
Abstract
There are two methods a quick and dirty method, and the technically right method that selects for lytic bacteriophage.
Troubleshooting
Quick and dirty isolation
2d 0h 5m
Collecting environmental samples
Collecting samples does not have to be in a sterile container. Often I will wash old water bottles or use Ziploc bags to collect samples.
The more sample collected, the better. You can shake to homegenate the sample
Incubating to grow phage
5mL broth (typically LB or TSA)
1mL bacterial strain (the strain you would like to isolate phage against)
1mL environmental sample
Incubation
overnight room temperature 24hrs
The incubation time and temperature will be dependent on bacterial type
1d
Filtration of phage from sample
Syringe filter (5-10mL syringe with leur lock) with 0.22µm filter (25mm diameter is fine)
Remove the plunger from the syringe
Attach 0.22µm filter
Dump or pipette incubated sample into syringe
Carefully push the plunger down. Do not let the plunger go backwards
If resistance is met, it is best to stop and change the filter. The filter can be removed and a new filter can be screwed on.
5m
Spot the samples on plates with bacterial strain
I will edit this to have the spot protocol linked in as soon as it's published.
1d
The technically right method for isolating phage
6d 0h 15m
This protocol is usually to isolate predominantly lytic phage. This process usually selects for the predominant lytic phage that infects the host bacteria. This is also very close to the SEA-PHAGE protocol from the manual.
Collecting environmental samples
Collecting samples does not have to be in a sterile container. Often I will wash old water bottles or use Ziploc bags to collect samples.
The more sample collected, the better. You can shake to homegenate the sample
Filter out the phage from an environmental sample
Filtration through filter
Push desired amount of environmental sample through a 0.22µm filter
The more sample you use, the more organisms inside, as well as phage! Therefore, you can increase your chances of getting phage.
Filtration through centrifugation
Centrifuge the desired amount of environmental sample 5,000rpm 10 minutes 4˚C
Pipette off the supernatant into the container you want to incubate the sample in
You can also do both centrifugation and filtration with a 0.22µm filter
10m
Incubate to grow phage
Try to add a significant amount of nutrient broth to the environmental sample.
25mL broth
10mL environmental sample
500-1,000µL bacterial strain (you can also use a bacterial strain cocktail such as various E. coli strains).
Incubation
Incubate at the temperature of the bacteria shaking for 3-5 days.
Check each day
Because phage kill bacteria, you won't actually be looking for cloudy growth, but for more transparent growth. You can wait the whole 5 days, or if you see transparent growth, you can pull the sample.
5d
Filter the sample
Filter your sample with a 0.22µm filter
You can filter the entire thing with a larger filter, or you can syringe filter a portion (~5-10mL) of the sample.
5m
Spot or plaque assay
I will edit this to have the spot or plaque assay protocol linked in as soon as it's published.
1d