Measuring Spontaneous Phage Induction in Lysogenic Bacteria Over Time

Take an overnight culture of a lysogenic bacterium, where most cells harbor dormant phage DNA integrated within their genome.

Inoculate it into fresh media and incubate.

Some bacteria undergo spontaneous induction, entering the lytic cycle, where the phage DNA is excised, replicated, and viral proteins are produced.

Newly assembled infective phages release through cell lysis.

Collect culture samples at defined intervals.

To count viable non-induced lysogens, spot serial dilutions of each sample onto an agar plate and incubate.

Count colonies to calculate colony-forming units (CFUs).

To count infective phages, spot the same dilutions onto an antibiotic agar plate pre-seeded with antibiotic-resistant indicator host cells. Since the lysogens are antibiotic-sensitive, only resistant hosts can grow.

Phages infect and lyse cells, forming clear plaques.

Count plaques to calculate plaque-forming units (PFUs). Lower PFUs indicate fewer spontaneous induction events.

Plot CFUs and PFUs over time. During the early exponential phase of bacterial growth, PFUs are lowest relative to CFUs, indicating minimal spontaneous phage induction.

To begin set up fresh lysogen and indicator host cultures by inoculating