Practical Method for Isolation of Phage Deletion Mutants

The growing concern about multi-drug resistant pathogenic bacteria has led to a renewed interest in the study of bacteriophages as antimicrobials and as therapeutic agents against infectious diseases (phage therapy). Phages to be used for this purpose have to be subjected to in-depth genomic characterization. It is essential to ascribe specific functions to phage genes, which will give information to unravel phage biology and to ensure the lack of undesirable genes, such as virulence and antibiotic resistance genes. Here, we describe a simple protocol for the selection of phage mutants carrying random deletions along the phage genome. Theoretically, any DNA region might be removed with the only requirement that the phage particle viability remains unaffected. This technique is based on the instability of phage particles in the presence of chelating compounds. A fraction of the phage population naturally lacking DNA segments will survive the treatment. Within the context of phages as antimicrobials, this protocol is useful to select lytic variants from temperate phages. In terms of phage efficiency, virulent phages are preferred over temperate ones to remove undesirable bacteria. This protocol has been used to obtain gene mutations that are involved in the lysogenic cycle of phages infecting Gram-positive bacteria (Staphylococcus and Lactobacillus).


Introduction
The isolation and characterization of phage deletion mutants is a widely used tool for the analysis of gene function and regulation. In this regard, homologous recombination using a removable plasmid is a common technique to delete or exchange a target genome sequence in temperate phages. The recombination process is performed while the phage is inserted in the host bacterial genome as a prophage, and then, the induction of the lytic cycle by an external signal will release the mutated viral particles. However, methods to select mutant particles of virulent phages are scarce, and most of them involve the use of UV radiation or chemicals [1,2]. More recently, molecular biology techniques based on CRISPR-Cas (clustered, regularly interspaced, short palindromic repeats technology for generating RNA-guided nucleases with customizable specificities) systems have allowed accurately removing and swapping DNA fragments of some virulent phages [3][4][5][6]. One main disadvantage of these techniques is the need of previous knowledge regarding the phage genome sequence, making these methodologies time-consuming. In addition, the complete protocol is not available for all bacterial species and their phages.
In this article, we report an easy and rapid way to isolate phage deletion mutants based on the instability of phage particles in the presence of chelating agents, such as EDTA (Ethylenediaminetetraacetic acid), sodium citrate, or sodium pyrophosphate. Chelating agents can react with the cations attached to the phage structure, breaking the bonds that maintain particle conformation [7]. Moreover, this destabilizing effect increases with temperature [8]. However,

Experimental Design
The protocol described here has been optimized for the selection of staphylococcal phage mutants with a "clear lysis plaque" phenotype, by using sodium pyrophosphate as a chelating agent. Thus, all the protocols, materials and methods are reliable for temperate phages infecting staphylococcal species; nevertheless, this protocol was also used in temperate bacteriophages infecting Lactobacillus [10]. However, it must be noted that the protocol may have to be adapted for each specific bacterium-phage pair in order to ensure successful results ( Figure 1). Furthermore, this protocol can also be used for the selection of mutant variants of virulent phages in which phage viability is not compromised. To note, all the steps performed in this protocol should be perform under sterile conditions using autoclaved material and a laminar airflow cabinet.
Before starting the protocol, it is necessary to determine the best conditions for growing the host bacterial culture. To do that, it is recommended to perform a growth curve at 37 • C (or optimum temperature for the phage host bacterium) in a shaker incubator during 8 h, measuring at different sampling times the absorbance, or optical density at 600 nm (OD 600 ) in an "Eppendorf Biophotometer" or similar, and the bacterial concentration (CFU/mL) by plating onto solid medium plates and further incubation. For this study, the temperate phage phiH5 and the host strain S. aureus Sa9 were selected; although any other phage susceptible strain could be used for this purpose. The following steps consist of propagation and purification of the phage on the selected host strain. Although these steps are not mandatory, it is advisable to purify the phage before obtaining the phage mutants, in order to ensure the success of the subsequent sodium pyrophosphate treatment. Thus, the presence of contaminants in the phage stock might entrap the sodium pyrophosphate molecules and decrease their activity, requiring a higher concentration of the compound to achieve the same effect. Phage propagation should be performed in two steps: first, in small volumes using the double-layer technique, followed by an up-scaled propagation in 1 liter of liquid medium. Prior to purification, the bacteriophage lysate should be filtered and concentrated by precipitation with NaCl and PEG 8000. Then, purification should be carried out by a conventional CsCl gradient using a ultracentrifuge. As mentioned previously, phage purification is recommended, but is not essential. However, skipping this step may lead to a lower efficiency of the protocol. The chelating agent treatment is performed using a purified and concentrated phage stock and different concentrations of sodium pyrophosphate. Exposure of the phage to the chelating agent for 30 min at 37 • C will cause a dramatic decline in phage survival (ideally phage titers should decrease between 90 and 99% after treatment). From this treatment, phages were selected for the following rounds of treatment until obtaining a phage survival rate of 100%, even at high concentrations of the chelating agent. The concentrations of sodium pyrophosphate may vary depending on the specific phage.
After reaching a survival rate of 100%, the putative phage deletion mutants are isolated and propagated for further studies that are intended to characterize the mutations. Characterization is often carried out by DNA restriction analysis or PCR. For phages with a known genome sequence, PCR reactions can be designed to amplify regions along the genome and easily identify the deletion. For phages with an unknown genome sequence, DNA deletions should be identified by DNA extraction and further restriction analysis. This method will also provide an estimate of the deletion size.
Moreover, the selection of "clear lysis plaque" phage deletion mutants is also performed by collecting the surviving phages, isolating single lysis plaques, and testing their ability to produce turbid or transparent halos by the drop assay. Surviving bacteria inside the halo will be finally tested for susceptibility to the phage.

•
S. aureus Sa9 [11], isolated from a mastitic milk sample was used as the host strain of the phage. • Phage phiH5, isolated from raw milk was used to select "clear lysis plaques" deletion mutants [11] where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD 600 in the y-axis and the time of incubation of the culture in the x-axis.
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in
where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol.

CRITICAL STEP
The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in
where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).
Methods Protoc. 2018, 1, 6 5 of 14 Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). • Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis.
CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days •
Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock. • Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate. where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step.

•
In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 • C for 18 h. Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). • Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis.
CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days •
Inoculate one S. aureus single colony to obtain an o/n culture (see above). CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 • C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL.

Bacterial Growth Conditions and Phage Titration. 4 Days
• Streak the S. aureus host strain onto a TSA (TSB supplemented with 2% w/v agar) plate using a sterile tip and incubate the plate at 37 °C for 18 h to obtain isolated colonies. OPTIONAL STEP Supplement the growth medium with 10 mM CaCl 2 and 10 mM MgSO 4 . Addition of these cations is recommended for most phages, although it is not always strictly necessary, as some phages can be propagated efficiently without them. • Take 1 mL sample after 1 h incubation and measure the OD 600 . Continue incubating the culture at 37 • C with shaking until it reaches the exponential growth phase.

PAUSE STEP
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol.

CRITICAL STEP
The bacterial cells should be in the exponential growing state to ensure proper phage propagation.
• Transfer the exponential growing bacteria to four sterile 50 mL tubes (10 mL in each tube). One tube will be the control and the other three tubes will be used for phage propagation with three different MOIs (0.1, 1 and 10). To calculate the volume (V 0 ) of the phage that should be added to the different tubes follow the equation: where V 1 is the final volume of the mixture (in this case 10 mL); C 0 is the concentration of the previously titrated phage stock (≥10 9 PFU/mL), and C 1 is the desired final phage concentration. For calculating C 1 follow the equation: where C b is the concentration of the bacterial culture at an exponential growing phase (expressed as CFU/mL) and MOI is the multiplicity of infection defined as PFU/CFU.
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube.
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix CRITICAL STEP Incubation should proceed until complete lysis of the culture is observed (transparent tube compared to the turbid control tube). This can take more or less time depending on the MOI, the phage and the host strain used.
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above).  Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above).
where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol.

CRITICAL STEP
The titer of the concentrated phage stock is usually 100 times higher than the one obtained in a liquid propagation. • OPTIONAL STEP Perform phage purification.

Phage Purification. 1 Day (OPTIONAL STEP)
• Using the concentrated phage stock previously obtained, add CsCl to a final concentration of 0.75 g/mL, and mix vigorously.   Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). • Repeat the experiment two extra times to obtain three biological replicates.
• Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days •
Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock. • Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate. • After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix CRITICAL STEP The dilution step is carried out in order to obtain a phage stock of 10 9 PFU/mL to proceed with the chelating agent treatment. Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). • Repeat the experiment two extra times to obtain three biological replicates.
• Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days •
Inoculate one S. aureus single colony to obtain an o/n culture (see above). CRITICAL STEP For some phages, the destabilizing effect might be higher using other chelating agents such as EDTA or sodium citrate, but the procedure to follow in these cases is identical to that using sodium pyrophosphate. Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). • Repeat the experiment two extra times to obtain three biological replicates.
• Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days •
Inoculate one S. aureus single colony to obtain an o/n culture (see above).

CRITICAL STEP
The sodium pyrophosphate treatment causes a decrease in the initial phage titer that should be around 90-99%. Some phages have a very stable particle structure; in these Methods and Protoc. 2018, 1, 6 8 of 14 cases, a higher concentration of the chelating agent or an increase in the incubation temperature might be necessary.

•
Collect the surviving phages from the plate in which the sodium pyrophosphate treatment led to a survival rate of about 10% of the phage population ( Figure 2). To do this, add 1 mL of SM buffer to the plate corresponding to the phage dilution 10 • and incubate 1 h with slight shaking (60 rpm) at room temperature. Afterwards, collect the liquid and centrifuge 10 min at 13,600× g, at 4 • C.
Keep the supernatant containing the surviving phages.

Bacterial Growth Conditions and Phage Titration. 4 Days
• Streak the S. aureus host strain onto a TSA (TSB supplemented with 2% w/v agar) plate using a sterile tip and incubate the plate at 37 °C for 18 h to obtain isolated colonies. PAUSE STEP After the colonies are grown, plates can be stored at 4 °C for up to 1 month. • Inoculate one S. aureus single colony from the TSA plate into a sterile 10 mL tube containing 5 mL of sterile TSB). Incubate at 37 °C with shaking (250 rpm) for 18 h to obtain an overnight (o/n) culture. OPTIONAL STEP Take 1 mL of the o/n culture and add glycerol to a final concentration of 20% in order to prepare a frozen stock of the host strain. Store this stock at −80 °C.

•
In order to perform a bacterial growth curve, inoculate 50 mL of TSB with 1% (v/v) of the S. aureus o/n culture into a 100 mL sterile bottle and incubate at 37 °C with shaking. • Take 2 mL samples at time points during incubation (0, 1, 2, 3, 4, 5, 6, 7, 8 and 24 h). Transfer 1 mL sample to a plastic cuvette and measure the optical density at an absorbance wavelength of 600 nm (OD600). At the same time, perform 1:10 serial dilutions of the sample into PBS buffer.

PAUSE STEP
The phage stock can be kept at 4 • C for 1 month before continuing with the protocol.
Methods Protoc. 2018, 1, 6 5 of 14 Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol. CRITICAL STEP Repeat the pyrophosphate treatment from 3 to 5 rounds until phage survival reaches a plateau of 100% even at high concentrations of the chelating agent ( Figure 2).

•
Pick isolated lysis plaques with a sterile tip from the sample treated with the highest concentration of sodium pyrophosphate, and suspend each lysis plaque in 100 µL of SM buffer.
• Sterile aerosol filter pipet tips (VWR, Radnor, PA, USA). Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation:

Bacterial Growth Conditions and
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above).
where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol.
CRITICAL STEP The number of lysis plaques to be tested cannot be calculated in advance, but it is recommended to select about 10-20 lysis plaques for an initial screening for the desired mutant.
OPTIONAL STEP Propagate the putative phage deletion mutants following previous Sections 3.1 and 3.2.
• For temperate phages, the protocol can be extended to select virulent phages with "clear lysis plaque" phenotype (following section). Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation: where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure. where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step.

Phage Titration and Propagation in
• In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the CRITICAL STEP For some phages, it is necessary to incubate the plates for a longer period in order to get a more evident transparent phenotype. where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using CRITICAL STEP The number of bacteria from the wild-type phage halo is expected to be high, while none or a low number of colonies should be present in the "clear lysis plaque" halo. Virulent phages with "clear lysis plaque" phenotype are not able to lysogenize their host; therefore, no cells are expected to survive after infection. By contrast, a high number of lysogenic cells (resistant to phage infection) will be generated inside the halo of the wild-type temperate phage.

•
Confirm the lytic phenotype. Pick up 3 isolated colonies from the plate that comes from the wild-type phage halo and all the colonies from the "clear lysis plaque" mutant halo. Introduce these colonies into sterile tubes containing 3 mL TSB and incubate with shaking (250 rpm) for 18 h at 37 • C (Figures 3 and 4). Add 100 µL of the desired dilution to a TSA plate and spread using a sterile L-shape spreader until liquid dries. Incubate the plate at 37 °C for 18 h. • Count the number of colonies in the plate containing 30-300 colony forming units (CFU). Calculate the viable cell counts of the culture using the equation:

PAUSE STEP
where N is the number of colonies counted on the plate (expressed as CFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL).

•
Repeat the experiment two extra times to obtain three biological replicates. • Represent graphically the mean ± standard deviation of the CFU/mL in a logarithmic scale or the OD600 in the y-axis and the time of incubation of the culture in the x-axis. CRITICAL STEP The number of CFU/mL should be calculated for each host strain, but generally speaking, it is very similar among strains belonging to the same species. The calculation of the CFU/mL in the exponential culture (Cb) is necessary to allow for the accurate determination of the multiplicity of infection (MOI). Calculation of this parameter is decisive in order to achieve an optimum yield in the phage propagation procedure.

Phage Titration and Propagation in Small Volume. 2-5 Days
• Inoculate one S. aureus single colony to obtain an o/n culture (see above). • Make 1:10 serial dilutions of the initial phage stock.

•
Determine the number of infective phage particles by using the double-layer plaque assay. Take 100 µL of a S. aureus o/n culture and mix with 100 µL of the desired phage dilution into a 15 mL tube. Add 3 mL of semisolid TSA (0.7% agar) and pour the mixture onto a TSA plate. Let the soft agar medium solidify, and then, incubate the plate at 37 °C for 18 h. This experiment must be performed in triplicate.

•
After incubation, count the number of plaque forming units (PFU) in those plates where the number is between 30-300 PFU. Calculate the phage titer of the original phage sample by using the equation: where N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the sample volume poured onto the plate (expressed in mL). CRITICAL STEP If the titer of the phage suspension is ≥10 9 PFU/mL, then the phage propagation and concentration protocol can be followed from Section 3.3. If not, propagation in solid medium should be performed before proceeding to the next step. • In order to carry out the propagation in solid medium, take 100 µL of diluted phage stock mix with 100 µL of a S. aureus o/n culture into a 15 mL tube. Add 3 mL of TSA 0.7% and pour the mixture onto a TSA plate. Repeat the procedure to get 5 plates. Let the medium solidify and then, incubate the plates at 37 °C for 18 h. CRITICAL STEP After incubation, the overlaid plate should be completely transparent reflecting the occurrence of confluent lysis. The minimum concentration of phages that should be plated to ensure confluent lysis varies between 10 4 and 10 6 PFU/plate. Add 1 mL of SM buffer to each plate and incubate with gentle shaking (60 rpm) at 20 °C for 1 h. Recover the SM buffer from the five plates and transfer it to a 15 mL tube. Centrifuge 10 min at 13,600 × g. Transfer the supernatant containing the phages to a new sterile tube and perform phage titration again. If it is needed, solid propagation can be repeated again with this new phage stock in order to obtain a concentration ≥10 9 PFU/mL. PAUSE STEP Phage stock solutions can be kept at 4 °C for up to three months after filtration using a 0.45 µm cellulose acetate membrane filter. For longer periods of time, a phage stock can be kept at −80 °C with 20% of glycerol. CRITICAL STEP After the incubation, two phenotypes can be observed: the presence of a clear halo, which means that the strain is sensitive to the phage, or complete growth of the bacteria inside the halo which indicates resistance to the phage. The presence of a transparent halo on the surviving bacterial lawn is indicative that the putative "clear lysis plaque" phage mutant is a real virulent phage.

Expected Results
The protocol described here has been used for the selection of "clear lysis plaque" phage mutants from phage phiH5 [12]. Initially, a growth curve of the host bacterium S. aureus Sa9 was performed at 37 • C with shaking, in order to determine the number of bacterial cells present in the exponential growing phase. The results showed that log phase started at OD 600 = 0.1, which corresponds to 10 8 CFU/mL. The stationary phase began after 8 h of incubation, when a total number of bacteria of 10 9 CFU/mL and an absorbance of~2 were reached. To propagate phage phiH5 in solid medium, 100 µL of an o/n culture of the host bacterium was infected with 100 µL of a phage stock with a titer of 10 7 PFU/mL, using the double layer technique. After this first propagation step, the new phage stock (10 8 PFU/mL) was used to repeat propagation in solid medium. In this way, the volume of phage stock was increased to 5 mL, with a concentration of 10 9 PFU/mL. Next, propagation in liquid medium was carried out in 5 mL of exponentially growing bacteria (OD 600 nm = 0.1 which corresponds to 10 8 CFU/mL), using three different MOIs (0.1, 1 and 10). All of the cultures lysed completely after 3 h of incubation at 37 • C; however, the phage titer (10 9 PFU/mL) obtained after infection with a MOI of 1 was higher than that obtained with the other two MOIs (10 8 PFU/mL). Thus, a MOI of 1 seems to be optimal for the propagation of phiH5 on S. aureus Sa9. Then, the protocol was scaled to 50 mL, 100 mL, and 1 liter; obtaining a phage titer~10 9 PFU/mL after each round of propagation. Finally, the phage was purified using a CsCl gradient and dialyzed, resulting in a purified phage stock of about 10 11 PFU/mL.
The dialyzed and purified phiH5 stock was treated with increasing concentrations of sodium pyrophosphate in six different rounds, until a plateau of 100% of surviving phages was reached ( Figure 5).
Once phiH5 was exposed to six rounds of pyrophosphate treatment, the surviving phages from the sixth round (sample exposed to 200 mM sodium pyrophosphate) were collected and diluted to observe isolated lysis plaques. The opacity of the resulting plaques was compared with the ones that were obtained from the wild-type phage stock. The most transparent lysis plaques (21 transparent lysis plaques, Figure 6), so-called "clear lysis plaques" were picked up and suspended in SM buffer for further analysis. A drop of each phage suspension and a drop of the wild-type phage were poured onto a S. aureus Sa9 lawn. After the incubation of these plates, the colonies growing inside the halo of phage lysis were scratched and streaked on agar plates to obtain single colonies. From the 21 scratched halos, only one resulted in no surviving bacteria, since no colonies were recovered. This was confirmed as a real "clear lysis plaque" phage mutant. From the remaining 20 halos and from the wild-type phage halo, three colonies were picked up to confirm their phenotype. All of these colonies resulted resistant to phage infection, indicating their lysogenic status. The putative phage mutant with "clear lysis plaque" phenotype (named phiIPLA88) was characterized by DNA restriction analysis. Since no changes in the restriction pattern were observed (data not shown), the complete genome sequence of phiIPLA88 was obtained and compared with that of the wild-type phage phiH5. A point mutation (1-base replacement) leading to the loss of the start codon was found in the repressor-encoding gene. As a consequence of this mutation, phage phiIPLA88 is an obligate virulent phage [12]. It is worth noting that mutant virulent phages can spontaneously occur in a temperate phage population, therefore, it might be possible to isolate them directly. However, the frequency of spontaneous mutation in phages is generally very low (>10 −8 ) [13], and this protocol facilitates their selection.