Bacteriophages can be relatively easily isolated from various sources.
The best source of a given phage is material where its specific host is abundant.
Bacteriophages active towards pathogenic microflora are usually acquired from the material obtained from a given patient (urine, faeces, pus, etc.). The other alternative which further gained wide spread is the isolation of virulent phage isolates from sewage waters (normally from the clinic).Thus, for phages infecting the human gut flora, municipal sewage is an excellent source.
Before starting isolation of a Bacteriophage, its bacterial host needs to be isolated and grown in pure culture. During phage isolation, it is generally best to use host bacteria in the exponential growth phase, although some phages also appear able to propagate in bacteria in the stationary growth phase.
The growth conditions (medium, oxygen level, temperature, incubation time, etc.) should be chosen according to the preference of the host bacterium.
Phages can be isolated from various aqueous (e.g., water) and nonaqueous (e.g., soil) sources. If phages are to be isolated from nonaqueous samples, the samples first should be suspended in a medium suitable for the growth of the targeted bacterial species.
To remove solids, indigenous bacteria, and
other organisms, the samples can be “clarified.” This step can be accomplished by centrifugation (1500g, 20 min), which will remove most of the bulk solids, after which the supernatant can be processed for phage isolation.
Filtering the suspended samples through a 0.45 mm or 0.2 mm membrane filter (preferably a tangential flow filter, to avoid rapid clogging) is an alternative approach, but this step may still need to be preceded by a centrifugation step or by pre-filtering the suspensions through larger-pore filters. Sometimes samples can be processed directly for phage isolation without clarification.
Enrichment of the phages in the source material often facilitates phage isolation.
The ratio between source material and added (concentrated) growth medium should be varied to accommodate differences in abundance of phages and the nature of the source material, as should the addition of host bacteria. If one wants to enrich for particularly virulent, broad-spectrum phages that initially constitute a very small fraction of the phages in the sample, the protocol should utilize a larger volume of
source material, several different host bacteria, and several cycles of selection.
If the volume of the sample is large compared to the final culture volume, however, some of the sample’s components may interfere with bacterial growth.
That problem can often be circumvented if the volume of source material is ≤1%–10% of the culture’s final volume.
If the sample constitutes more than 10% of the final volume, one should add enough 10X culture medium to have the final enrichment culture contain 1X culture medium.
There are a lot of protocols, for example:
5oo ml of municipal sewage is mixed with 100 ml of 10X Bacterial growth Broth and with 1X Bacterial culture medium. This sample is incubated overnight at 37°C and after the Phage Titre value
(= Plaque Number*Reciprocal of Diluition*Reciprocal of Volume in ml ) is calculated by Titration of bacteriophage suspensions using the agar overlay method.
Chloroform is often used to break open infected cells, since only cells whose peptidoglycan layer has been weakened by
phageencoded lysozyme or endolysin will be lysed by chloroform. However, it should be omitted from all procedures where phage with lipid-containing envelopes are expected or sought.
Once isolated, phages can be resuspended in phage diluent (TSG or SM), or growth medium.
The best source of a given phage is material where its specific host is abundant.
Bacteriophages active towards pathogenic microflora are usually acquired from the material obtained from a given patient (urine, faeces, pus, etc.). The other alternative which further gained wide spread is the isolation of virulent phage isolates from sewage waters (normally from the clinic).Thus, for phages infecting the human gut flora, municipal sewage is an excellent source.
Before starting isolation of a Bacteriophage, its bacterial host needs to be isolated and grown in pure culture. During phage isolation, it is generally best to use host bacteria in the exponential growth phase, although some phages also appear able to propagate in bacteria in the stationary growth phase.
The growth conditions (medium, oxygen level, temperature, incubation time, etc.) should be chosen according to the preference of the host bacterium.
Phages can be isolated from various aqueous (e.g., water) and nonaqueous (e.g., soil) sources. If phages are to be isolated from nonaqueous samples, the samples first should be suspended in a medium suitable for the growth of the targeted bacterial species.
To remove solids, indigenous bacteria, and
other organisms, the samples can be “clarified.” This step can be accomplished by centrifugation (1500g, 20 min), which will remove most of the bulk solids, after which the supernatant can be processed for phage isolation.
Filtering the suspended samples through a 0.45 mm or 0.2 mm membrane filter (preferably a tangential flow filter, to avoid rapid clogging) is an alternative approach, but this step may still need to be preceded by a centrifugation step or by pre-filtering the suspensions through larger-pore filters. Sometimes samples can be processed directly for phage isolation without clarification.
Enrichment of the Bacteriophages in the source material
Enrichment of the phages in the source material often facilitates phage isolation.
The ratio between source material and added (concentrated) growth medium should be varied to accommodate differences in abundance of phages and the nature of the source material, as should the addition of host bacteria. If one wants to enrich for particularly virulent, broad-spectrum phages that initially constitute a very small fraction of the phages in the sample, the protocol should utilize a larger volume of
source material, several different host bacteria, and several cycles of selection.
If the volume of the sample is large compared to the final culture volume, however, some of the sample’s components may interfere with bacterial growth.
That problem can often be circumvented if the volume of source material is ≤1%–10% of the culture’s final volume.
If the sample constitutes more than 10% of the final volume, one should add enough 10X culture medium to have the final enrichment culture contain 1X culture medium.
There are a lot of protocols, for example:
5oo ml of municipal sewage is mixed with 100 ml of 10X Bacterial growth Broth and with 1X Bacterial culture medium. This sample is incubated overnight at 37°C and after the Phage Titre value
(= Plaque Number*Reciprocal of Diluition*Reciprocal of Volume in ml ) is calculated by Titration of bacteriophage suspensions using the agar overlay method.
Chloroform is often used to break open infected cells, since only cells whose peptidoglycan layer has been weakened by
phageencoded lysozyme or endolysin will be lysed by chloroform. However, it should be omitted from all procedures where phage with lipid-containing envelopes are expected or sought.
Once isolated, phages can be resuspended in phage diluent (TSG or SM), or growth medium.
