Table 2.

Properties that are often tested in characterizing phages but are not essential for phage therapy.

Property	Description (Reference for Methodology)	References (Examples)
Efficiency of plating (EOP)	Number of plaques or lysis measurements are compared to a reference phage/host combination for relative EOP. Number of plaques compared to the number of phage particles (as determined by a non-culture method such as epifluoresence microscopy counting) used for infection is the absolute EOP. EOP between any phage pair may vary on different hosts. Often performed when multiple phages have been isolated to establish which are more or less virulent, especially when planning to combine phages to make a cocktail. EOP of each cocktail component prevents use of phages with very low killing efficiency [60].	[157]
Phage morphology by electron microscopy	Until the advent of next-generation sequencing of phage genomes, morphology was considered essential for classifying novel phages and phages of different morphologies were often used to make a broader, more diverse phage cocktail. Classification is now more commonly done by genome comparisons but knowing the morphology is still useful for quickly and easily showing diversity in a phage mixture [158].	[159,160]
Whole genome sequence	If not determined earlier, this can confirm lack of toxin genes and ability to form a lysogen. Also somewhat useful for identifying related phages for inferences of some gene functions, for example [135]. For overviews of sequencing methods see [62,135,161,162,163,164]. Phage taxonomic classification is still under development so using sequence data for this is potentially limited [165,166].	[167,168]
One step growth curve	Phages with long latent periods may be less useful as therapeutic phages. Burst size can also be determined if the treatment will be with fewer phage than can immediately infect all the bacteria infecting the patient. As discussed elsewhere in this paper, it is also important to remember that growth rates in the laboratory may not reflect growth rates in patients, in biofilms, and in other potential phage application locations [60,169,170].	[171,172]
Pulse-field gel electrophoresis (PFGE)	PFGE is a modification of conventional agarose gel electrophoresis which uses a variable electric voltage to drive DNA migration instead of the standard continuous voltage. This allows for the separation of much larger pieces of DNA. It can be used to directly measure the size of a phage genome. It can also be used for restriction fragment length polymorphism (RFLP) analysis when multiple phages have been isolated to show that each phage is different by comparing restriction enzyme digested genomic DNA although depending on the size of the phage genome, this differentiation can also be done using conventional gel electrophoresis. [173]	[174,175]