Practical methods for determining phage growth parameters.

Bacteriophage growth may be differentiated into sequential steps: (i) phage collision with an adsorption-susceptible bacterium, (ii) virion attachment, (iii) virion nucleic acid uptake, (iv) an eclipse period during which infections synthesize phage proteins and nucleic acid, (v) a "post-eclipse" period during which virions mature, (vi) a virion release step, and (vii) a diffusion-delimited period of virion extracellular search for bacteria to adsorb (1). The latent period begins at the point of virion attachment (ii) and/or nucleic acid uptake (iii) and ends with infection termination, spanning both the eclipse (iv) and the post-eclipse maturation (v) periods. For lytic phages, latent-period termination occurs at lysis, i.e., at the point of phage-progeny release (vi). A second compound step is phage adsorption, which, depending upon one's perspective, can begin with virion release (vi), may include the virion extracellular search (vii), certainly involves virion collision with (i) and then attachment to (ii) a bacterium, and ends either with irreversible virion attachment to bacteria (ii) or with phage nucleic acid uptake into cytoplasm (iii). Thus, the phage life cycle, particularly for virulent phages, consists of an adsorption period, virion attachment/nucleic acid uptake, a latent period, and virion release ((2), p. 13, citing d'Herelle). The duration of these steps together define the phage generation time and help to define rates of phage population growth. Also controlling rates of phage population growth is the number of phage progeny produced per infection: the phage burst size. In this chapter we present protocols for determining phage growth parameters, particularly phage rate of adsorption, latent period, eclipse period, and burst size.