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 the overnight cultures in 100 milliliters of LB at a ratio of one to 100. Incubate at 37 degrees Celsius with shaking at 180 RPM.

To monitor lysogen growth, collect a one milliliter sample every hour from the point of inoculation for eight hours. Serially, dilute the culture by adding 100 microliters into 900 microliters of the LB medium. Vortex at the maximum speed, and continue the dilution series from 10 to the minus one to 10 to the minus nine.

Spot 10 microliters of the required dilution onto an LB agar plate. Allow to dry and incubate at 30 degrees Celsius for 18 to 24 hours. After incubation, count the number of colonies and calculate the number of viable bacterial cells using the given formula.

Next, to enumerate the infective phage particles in the temporal sample, add 100 microliters of log phase rifampicin resistant indicator host cells to the molten top agar, along with rifampicin. Spot 10 microliters of serially diluted lysogen culture onto the inoculated top agar layer. Allowed to dry, and incubate at 37 degrees Celsius for 18 to 24 hours.

After incubation, count the number of plaques and calculate the infective phage particles using the given formula.